U.S. patent application number 11/824672 was filed with the patent office on 2008-09-18 for novel morphogenic protein compositions of matter.
This patent application is currently assigned to Stryker Corporation. Invention is credited to William K. Jones, Thangavel Kuberasampath, Hermann Oppermann, Engin Ozkaynak, David C. Rueger, Ronald F. Tucker.
Application Number | 20080227956 11/824672 |
Document ID | / |
Family ID | 27578075 |
Filed Date | 2008-09-18 |
United States Patent
Application |
20080227956 |
Kind Code |
A1 |
Jones; William K. ; et
al. |
September 18, 2008 |
Novel morphogenic protein compositions of matter
Abstract
Disclosed are novel compositions of morphogenic proteins
constituting soluble forms of these proteins, antibodies that
distinguish between soluble and mature forms, and method for
producing these morphogenic proteins and antibodies.
Inventors: |
Jones; William K.;
(Brookline, MA) ; Tucker; Ronald F.; (Holliston,
MA) ; Rueger; David C.; (Hopkinton, MA) ;
Oppermann; Hermann; (Medway, MA) ; Ozkaynak;
Engin; (Milford, MA) ; Kuberasampath; Thangavel;
(Medway, MA) |
Correspondence
Address: |
Kirkpatrick & Lockhart Preston Gates Ellis LLP;(FORMERLY KIRKPATRICK &
LOCKHART NICHOLSON GRAHAM)
STATE STREET FINANCIAL CENTER, One Lincoln Street
BOSTON
MA
02111-2950
US
|
Assignee: |
Stryker Corporation
Kalamazoo
MI
|
Family ID: |
27578075 |
Appl. No.: |
11/824672 |
Filed: |
July 2, 2007 |
Related U.S. Patent Documents
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7268208 |
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11824672 |
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08402542 |
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10122026 |
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08040510 |
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08402542 |
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08029335 |
Mar 4, 1993 |
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08040510 |
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07971091 |
Nov 3, 1992 |
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08029335 |
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07946235 |
Sep 16, 1992 |
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07971091 |
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07938336 |
Aug 28, 1992 |
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07946235 |
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07923780 |
Jul 31, 1992 |
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07938336 |
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07752857 |
Aug 30, 1991 |
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07923780 |
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07752764 |
Aug 30, 1991 |
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07752857 |
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07667274 |
Mar 11, 1991 |
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07752764 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
A23L 33/19 20160801;
C07K 16/22 20130101; A61L 27/24 20130101; Y10S 435/975 20130101;
C08L 89/00 20130101; C08L 89/00 20130101; C07K 14/495 20130101;
A61K 2300/00 20130101; Y10S 436/815 20130101; A61K 48/00 20130101;
A61K 38/1875 20130101; G01N 33/6893 20130101; A61K 6/20 20200101;
A61K 6/20 20200101; A61K 38/1875 20130101; G01N 2500/10 20130101;
A23C 9/20 20130101; Y10S 436/811 20130101; A61K 38/1703 20130101;
A61L 27/227 20130101; A61K 38/17 20130101; A61F 2310/00365
20130101; C07K 14/51 20130101; A61K 6/20 20200101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 14/435 20060101
C07K014/435 |
Claims
1-25. (canceled)
26. An isolated peptide comprising residues 30-290 of SEQ ID NO:
17, wherein said peptide solubilizes a morphogen under
physiological conditions when said morphogen is mixed with said
peptide.
27. The isolated peptide of claim 26, wherein said morphogen is
BMP-3.
28. A method for solubilizing a morphogen comprising the step of
mixing the isolated peptide of claim 26 with a morphogen, wherein
said morphogen is BMP-3.
29. An isolated peptide comprising residues 30-374 of SEQ ID NO:
16, wherein said peptide solubilizes a morphogen under
physiological conditions when said morphogen is mixed with said
peptide.
30. The isolated peptide of claim 29, wherein said morphogen is
BMP-6.
31. A method for solubilizing a morphogen comprising the step of
mixing the isolated peptide of claim 29 with a morphogen, wherein
said morphogen is BMP-6.
32. An isolated peptide comprising residues 30-316 of SEQ ID NO:
18, wherein said peptide solubilizes a morphogen under
physiological conditions when said morphogen is mixed with said
peptide.
33. The isolated peptide of claim 32, wherein said morphogen is
BMP-5.
34. A method for solubilizing a morphogen comprising the step of
mixing the isolated peptide of claim 32 with a morphogen, wherein
said morphogen is BMP-5.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of (1) U.S. Ser.
No. 08/029,335, filed Mar. 4, 1993, (2) U.S. Ser. No. 07/971,091,
filed Nov. 3, 1992; (3) U.S. Ser. No. 07/946,235, filed Sep. 16,
1992; (4) U.S. Ser. No. 07/938,336, filed Aug. 28, 1992; (5) U.S.
Ser. No. 07/923,780, filed Jul. 31, 1992, which is a
continuation-in-part of U.S. Ser. No. 07/752,857, filed Aug. 30,
1991, now abandoned; and (6) U.S. Ser. No. 07/752,764, filed Aug.
30, 1991, a continuation-in-part of U.S. Pat. No. 667,274, filed
Mar. 11, 1991, now abandoned. The disclosures of these applications
is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to morphogenic
proteins and, more particularly, to compositions having improved
solubility in aqueous solvents.
BACKGROUND OF THE INVENTION
[0003] Morphogenic proteins ("morphogens") are well known and
described in the art. See, for example, U.S. Pat. Nos. 4,968,590;
5,011,691; 5,018,753; PCT US92/01968 and PCT US92/07432; as well as
various articles published in the scientific literature, including
Ozkaynak et al. (1992) J. Biol. Chem. 267:25220-25227 and Ozkaynak
et al. (1991) Biochem. Biophys. Res. Comm. 179:116-123. The art has
described how to isolate morphogenic proteins from bone, how to
identify genes encoding these proteins and how to express them
using recombinant DNA technology. The morphogenic proteins are
capable of inducing endochondral bone formation and other tissue
formation in a mammal when they are properly folded, dimerized and
disulfide bonded to produce a dimeric species having the
appropriate three dimensional conformation. The proteins have
utility in therapeutic applications, either by direct or systemic
administration. Where bone induction is desired, for example, the
morphogen typically is provided to the desired site for bone
formation in a mammal in association with a suitable matrix having
the appropriate conformation to allow the infiltration,
proliferation and differentiation of migrating progenitor cells.
The morphogenic protein adsorbed to the surfaces of a suitable
matrix is generally referred to in the art as an osteogenic device.
The proteins can be isolated from bone or, preferably, the gene
encoding the protein is produced recombinantly in a suitable host
cell.
[0004] The morphogen precursor polypeptide chains share a common
structural motif, including a N-terminal signal sequence and pro
region, both of which are cleaved to produce a mature sequence,
capable of disulfide bonding and comprising an N-terminal extension
and a C-terminal domain whose amino acid sequence is highly
conserved among members of the family. In their mature dimeric
forms, the morphogens typically are fairly insoluble under
physiological conditions. Increasing the solubility of these
proteins has significant medical utility as it would enhance
systemic administration of morphogens as therapeutics. Various
carrier proteins, including serum albumin and casein are known to
increase the solubility of morphogens (see, for example, PCT
US92/07432). PCT US92/05309 (WO 93/00050) discusses the use of
various solubilizing agents, including various amino acids and
methyl esters thereof, as well as guanidine, sodium chloride and
heparin, to increase the solubility of mature dimeric BMP2.
[0005] Improved methods for the recombinant expression of
morphogenic proteins is an ongoing effort in the art. It is an
object of this invention to provide an improvement in the methods
for producing and purifying morphogenic proteins having high
specific activity, and for formulating compositions and osteogenic
devices comprising these proteins. Another object is to provide
soluble forms of morphogenic proteins consisting essentially of
amino acid sequences derived from morphogenic proteins. Another
object is to provide formulations which stabilize the soluble
complex of morphogenic proteins. Still another object is to provide
means for distinguishing between soluble forms of the protein and
the mature morphogenic species, to provide means for quantitating
the amounts of these proteins in a fluid, including a body fluid,
such as serum, cerebro-sprinal fluid or peritoneal fluid, and to
provide polyclonal and monoclonal antibodies capable of
distinguishing between these various species.
[0006] Another object is to provide antibodies and biological
diagnostic assays for monitoring the concentration of morphogens
and endogenous anti-morphogen antibodies present in a body fluid
and to provide assays for detecting fluctuations in the
concentrations of these proteins in a body fluid. U.S. Pat. No.
4,857,456 and Urist et al. (1984) Proc. Soc. Exp. Biol. Med.
176:472-475 describe a serum assay for detecting a protein
purported to be a bone morphogenetic protein. The protein is not a
member of the morphogen family of proteins described herein,
differing in molecular weight, structural characteristics and
solubility from these proteins.
SUMMARY OF THE INVENTION
[0007] It has now been discovered that morphogenic protein secreted
into cultured medium from mammalian cells contains as a significant
fraction of the secreted protein a soluble form of the protein, and
that this soluble form comprises the mature dimeric species,
including truncated forms thereof, noncovalently associated with at
least one, and preferably two pro domains. It further has been
discovered that antibodies can be used to discriminate between
these two forms of the protein. These antibodies may be used as
part of a purification scheme to selectively isolate the mature or
the soluble form of morphogenic protein, as well as to quantitate
the amount of mature and soluble forms produced. These antibodies
also may be used as part of diagnostic treatments to monitor the
concentration of morphogenic proteins in solution in a body and to
detect fluctuations in the concentration of the proteins in their
various forms. The antibodies and proteins also may be used in
diagnostic assays to detect and monitor concentrations of
endogenous anti-morphogen antibodies to the various forms of these
proteins in the body.
[0008] An important embodiment of the invention is a dimeric
protein comprising a pair of polypeptide subunits associated to
define a dimeric structure having morphogenic activity. As defined
herein and in parent, related applications, morphogens generally
are capable of all of the following biological functions in a
morphogenically permissive environment: stimulating proliferation
of progenitor cells; stimulating the differentiation of progenitor
cells; stimulating the proliferation of differentiated cells, and
supporting the growth and maintenance of differentiated cells.
[0009] Each of the subunits of the dimeric morphogenic protein
comprises at least the 100 amino acid peptide sequence having the
pattern of seven or more cysteine residues characteristic of the
morphogen family. Preferably, at least one of the subunits
comprises the mature form of a subunit of a member of the morphogen
family, or an allelic, species, mutant or chimeric variant thereof,
noncovalently complexed with a peptide comprising part or all of a
pro region of a member of the morphogen family, or an allelic,
species, mutant or chimeric variant thereof. The pair of subunits
and one or, preferably, two pro region peptides, together form a
complex which is more soluble in aqueous solvents than the
uncomplexed pair of subunits.
[0010] Preferably, both subunits comprise a mature form of a
subunit of a member of the morphogen family or an allelic, species,
or mutant, including chimeric, variant thereof, and both subunits
are noncovalently complexed with a pro region comprising peptide,
or a fragment thereof. Most preferably, each subunit is the mature
form of human OP-1, or a species, allelic or other mutant variant
thereof, and the pro region is the entire or partial sequence of
the pro region of human OP-1, or a species, allelic or other mutant
variant thereof. Preferred pro regions are full length forms of the
pro region. Pro region fragments preferably include the first 18
amino acids of the pro sequence. Other useful pro region fragments
are truncated sequences of the intact pro region sequence, the
truncation occurring at the proteolytic cleavage site
Arg-Xaa-Xaa-Arg.
[0011] As used herein, the mature form of a morphogen protein
subunit includes the intact C-terminal domain and intact or
truncated forms of the N-terminal extensions. For example, useful
mature forms of OP-1 include dimeric species defined by residues
293-431 of Seq ID No. 1, as well as truncated sequences thereof,
including sequences defined by residues 300-431, 313-431, 315-431,
316-431 and 318-431. Note that this last sequence retains only
about the last 10 residues of the N-terminal extension sequence.
FIG. 2 presents the N-terminal extensions for a number of preferred
morphogen sequences. Canonical Arg-Xaa-Xaa-Arg cleavage sites where
truncation may occur are boxed or underlined in the figure. As will
be appreciated by those skilled in the art, mature dimeric species
may include subunit combinations having different N-terminal
truncations.
[0012] Other soluble forms of morphogens include dimers of the
uncleaved pro forms of these proteins (see below), as well as
"hemi-dimers" wherein one subunit of the dimer is an uncleaved pro
form of the protein, and the other subunit comprises the mature
form of the protein, including truncated forms thereof, preferably
noncovalently associated with a cleaved pro domain.
[0013] The soluble proteins of this invention are useful in the
formation of therapeutic compositions for administration to a
mammal, particularly a human, and for the development of biological
assays for monitoring the concentration of these proteins and
endogenous antibodies to these proteins in body fluids, including,
but not limited to, serum, cerebrospinal fluid and peritoneal
fluid.
[0014] The foregoing and other objects, features and advantages of
the present invention will be made more apparent from the following
detailed description of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a schematic representation of a morphogen
polypeptide chain as expressed from a nucleic acid encoding the
sequence, wherein the cross-hatched region represents the signal
sequence; the stippled region represents the pro domain; the
hatched region represents the N-terminus ("N-terminal extension")
of the mature protein sequence; and the open region represents the
C-terminal region of the mature protein sequence defining the
conserved seven cysteine domain, the conserved cysteines being
indicated by vertical hatched lines;
[0016] FIG. 2 lists the sequences of the N-terminal extensions of
the mature forms of various morphogens; and
[0017] FIG. 3 is a gel filtration column elution profile of a
soluble morphogen (OP-1) produced and purified from a mammalian
cell culture by IMAC, S-Sepharose and S-200HR chromatography in TBS
(Tris-buffered saline), wherein V.sub.O is the void volume, ADH is
alcohol dehydrogenase (MW 150 kDa), BSA is bovine serum albumin (MW
67 kDa), CA is carbonic anhydrase (MW 29 kDa) and CytC is
cytochrome C (MW 12.5 kDa).
DETAILED DESCRIPTION
[0018] A soluble form of morphogenic proteins now has been
discovered wherein the proteins consist essentially of the amino
acid sequence of the protein. The soluble form is a non-covalently
associated complex comprising the pro domain or a fragment thereof,
noncovalently associated or complexed with a dimeric protein
species having morphogenic activity, each polypeptide of the dimer
having less than 200 amino acids and comprising at least the
C-terminal six, and preferably seven cysteine skeleton defined by
residues 330-431 and 335-431, respectively, of Seq. ID No. 1.
Preferably, the polypeptide chains of the dimeric species comprise
the mature forms of these sequences, or truncated forms thereof.
Preferred truncated forms comprise the intact C-terminal domain and
at least 10 amino acids of the N-terminal extension sequence. The
soluble forms of these morphogenic proteins may be isolated from
cultured cell medium, a mammalian body fluid, or may be formulated
in vitro.
[0019] In vivo, under physiological conditions, the pro domain may
serve to enhance the transportability of the proteins, and/or to
protect the proteins from proteases and scavenger molecules,
including antibodies. The pro domains also may aid in targeting the
proteins to a particular tissue and/or to present the morphogen to
a morphogen cell surface receptor by interaction with a co-receptor
molecule. The isolated proteins may be used in therapeutic
formulations, particularly for oral or parenteral administration,
and in the development of diagnostic assays to monitor the level of
endogenous morphogens and endogenous anti-morphogen antibodies.
[0020] Detailed descriptions of the utility of these morphogens in
therapies to regenerate lost or damaged tissues and/or to inhibit
the tissue destructive effects of tissue disorders or diseases, are
provided in co-pending U.S. patent application Ser. No. 07/752,764,
filed Aug. 31, 1991; 07/938,336, filed Aug. 28, 1992; 07/923,780,
filed Jul. 31, 1992; 07/945,292, filed Sep. 15, 1992; 07/945,285,
filed Sep. 15, 1992; 07/938,337, filed Aug. 28, 1992; 07/922,813,
filed Jul. 31, 1992; 07/946,235, filed Sep. 16, 1992; 07/946,238,
filed Sep. 16, 1992; 07/945,286, filed Sep. 15, 1992; and
07/971,071, filed Nov. 3, 1992, the disclosures of which are
incorporated herein by reference. Morphogens, including the soluble
morphogen complexes of this invention, are envisioned to have
particular utility as part of therapies for regenerating lost or
damaged bone, dentin, periodontal, liver, cardiac, lung and nerve
tissue, as well as for protecting these tissues from the tissue
destructive effects associated with an immunological response. The
proteins also are anticipated to provide a tissue protective effect
in the treatment of metabolic bone disorders, such as osteoporosis,
osteomalacia and osteosarcoma; in the treatment of liver disorders,
including cirrhosis, hepatitis, alcohol liver disease and hepatic
encephalopathy; and in the treatment or prevention of ischemia
reperfusion-associated tissue damage, particularly to nerve or
cardiac tissue.
[0021] Presented below are detailed descriptions of useful soluble
morphogen complexes of this invention, as well as how to make and
use them.
I. Useful Soluble Morphogen Complexes--Protein Considerations
[0022] Among the morphogens useful in this invention are proteins
originally identified as osteogenic proteins, such as the OP-1,
OP-2 and CBMP2 proteins, as well as amino acid sequence-related
proteins such as DPP (from Drosophila), Vgl (from Xenopus), Vgr-1
(from mouse, see U.S. Pat. No. 5,011,691 to Oppermann et al.),
GDF-1 (from mouse, see Lee (1991) PNAS 88:4250-4254), 60A protein
(from Drosophila, Seq. ID No. 24, see Wharton et al. (1991) PNAS
88:9214-9218), and the recently identified OP-3.
[0023] The members of this family, which are a subclass of the
TGF-.beta. super-family of proteins, share characteristic
structural features, represented-schematically in FIG. 1, as well
as substantial amino acid sequence homology in their C-terminal
domains, including a conserved seven cysteine structure. As
illustrated in the figure, the proteins are translated as a
precursor polypeptide sequence 10, having an N-terminal signal
peptide sequence 12, (the "pre pro" region, indicated in the figure
by cross-hatching), typically less than about 30 residues, followed
by a "pro" region 14, indicated in the figure by stippling, and
which is cleaved to yield the mature sequence 16. The mature
sequence comprises both the conserved C-terminal seven cysteine
domain 20, and an N-terminal sequence 18, referred to herein as an
N-terminal extension, and which varies significantly in sequence
between the various morphogens. Cysteines are represented in the
figure by vertical hatched lines 22. The polypeptide chains
dimerize and these dimers typically are stabilized by at least one
interchain disulfide bond linking the two polypeptide chain
subunits.
[0024] The signal peptide is cleaved rapidly upon translation, at a
cleavage site that can be predicted in a given sequence using the
method of Von Heijne ((1986) Nucleic Acids Research 14:4683-4691.)
The "pro" form of the protein subunit, 24, in FIG. 1, includes both
the pro domain and the mature domain, peptide bonded together.
Typically, this pro form is cleaved while the protein is still
within the cell, and the pro domain remains noncovalently
associated with the mature form of the subunit to form a soluble
species that appears to be the primary form secreted from cultured
mammalian cells. Typically, previous purification techniques
utilized denaturing conditions that disassociated the complex.
[0025] Other soluble forms of morphogens secreted from mammalian
cells include dimers of the pro forms of these proteins, wherein
the pro region is not cleaved from the mature domain, and
"hemi-dimers", wherein one subunit comprises a pro form of the
polypeptide chain subunit and the other subunit comprises the
cleaved mature form of the polypeptide chain subunit (including
truncated forms thereof), preferably noncovalently associated with
a cleaved pro domain.
[0026] The isolated pro domain typically has a substantial
hydrophobic character, as determined both by analysis of the
sequence and by characterization of its properties in solution. The
isolated pro regions alone typically are not significantly soluble
in aqueous solutions, and require the presence of denaturants,
e.g., detergents, urea, guanidine HCl, and the like, and/or one or
more carrier proteins. Accordingly, without being limited to any
given theory, the non-covalent association of the cleaved pro
region with the mature morphogen dimeric species likely involves
interaction of a hydrophobic portion of the pro region with a
corresponding hydrophobic region on the dimeric species, the
interaction of which effectively protects or "hides" an otherwise
exposed hydrophobic region of the mature dimer from exposure to
aqueous environments, enhancing the affinity of the mature dimer
species for aqueous solutions.
[0027] Morphogens comprise a subfamily of proteins within the
TGF-.beta. superfamily of structurally related proteins. Like the
morphogens described herein, TGF-.beta. also has a pro region which
associates non-covalently with the mature TGF-.beta. protein form.
However, unlike the morphogens, the TGF-.beta. pro region contains
numerous cysteines and forms disulfide bonds with a specific
binding protein. The TGF-.beta.1 pro domain also is phosphorylated
at one or more mannose residues, while the morphogen pro regions
typically are not.
[0028] Useful pro domains include the full length pro regions
described below, as well as various truncated forms hereof,
particularly truncated forms cleaved at proteolytic Arg-Xaa-Xaa-Arg
cleavage sites. For example, in OP-1, possible pro sequences
include sequences defined by residues 30-292 (full length form);
48-292; and 158-292. Soluble OP-1 complex stability is enhanced
when the pro region comprises the full length form rather than a
truncated form, such as the 48-292 truncated form, in that residues
30-47 show sequence homology to the N-terminal portions of other
morphogens, and are believed to have particular utility in
enhancing complex stability for all morphogens. Accordingly,
currently preferred pro sequences are those encoding the full
length form of the pro region for a given morphogen (see below).
Other pro sequences contemplated to have utility include
biosynthetic pro sequences, particularly those that incorporate a
sequence derived from the N-terminal portion of one or more
morphogen pro sequences.
[0029] Table I, below, describes the various preferred morphogens
identified to date, including their nomenclature as used herein,
the sequences defining the various regions of the subunit
sequences, their Seq. ID references, and publication sources for
their nucleic acid and amino acid sequences. The disclosure of
these publications is incorporated herein by reference. The mature
protein sequences defined are the longest anticipated forms of
these sequences. As described above, shorter, truncated forms of
these sequences also are contemplated. Preferably, truncated mature
sequences include at least 10 amino acids of the N-terminal
extension. FIG. 2 lists the N-terminal extensions for a number of
the preferred morphogen sequences described below. Arg-Xaa-Xaa-Arg
cleavage sites that may yield truncated sequences of the mature
subunit form are boxed or underlined in the figure.
TABLE-US-00001 TABLE I "OP-1" Refers generically to the group of
morphogenically active proteins expressed from part or all of a DNA
sequence encoding OP-1 protein, including allelic and species
variants thereof, e.g., human OP-1 ("hOP-1"), or mouse OP-1
("mOP-1".) The cDNA sequences and the amino acids encoding the full
length proteins are provided in Seq. Id Nos. 1 and 2 (hOP1) and
Seq. ID Nos. 3 and 4 (mOP1.) The mature proteins are defined by
residues 293-431 (hOP1) and 292-430 (mOP1), wherein the conserved
seven cysteine skeleton is defined by residues 330-431 and 329-430,
respectively, and the N-terminal extensions are defined by residues
293-329 and 292-329, respectively. The "pro" regions of the
proteins, cleaved to yield the mature, morphogenically active
proteins, are defined essentially by residues 30-292 (hOP1) and
residues 30-291 (mOP1). "OP-2" refers generically to the group of
active proteins expressed from part or all of a DNA sequence
encoding OP-2 protein, including allelic and species variants
thereof, e.g., human OP-2 ("hOP-2") or mouse OP-2 ("mOP-2".) The
full length proteins are provided in Seq. ID Nos. 5 and 6 (hOP2)
and Seq. ID Nos. 7 and 8 (mOP2.) The mature proteins are defined
essentially by residues 264-402 (hOP2) and 261-399 (mOP2), wherein
the conserved seven cysteine skeleton is defined by residues
301-402 and 298-399, respectively, and the N-terminal extensions
are defined by residues 264-300 and 261-297, respectively. The
"pro" regions of the proteins, cleaved to yield the mature,
morphogenically active proteins likely are defined essentially by
residues 18-263 (hOP2) and residues 18-260 (mOP2). (Another
cleavage site also occurs 21 residues upstream for both OP-2
proteins.) "OP-3" refers generically to the group of active
proteins expressed from part or all of a DNA sequence encoding OP-3
protein, including allelic and species variants thereof, e.g.,
mouse OP-3 ("mOP-3".) The full length protein is provided in Seq.
ID No. 9. The mature protein is defined essentially by residues
261-399 or 264-399, wherein the conserved seven cysteine skeleton
is defined by residues 298-399 and the N-terminal extension is
defined by residues 264-297 or 261-297. The "pro" region of the
protein, cleaved to yield the mature, morphogenically active
proteins likely is defined essentially by residues 20-262.
"BMP2/BMP4" refers to protein sequences encoded by the human BMP2
and BMP4 genes. The amino acid sequence for the full length
proteins, referred to in the literature as BMP2A and BMP2B, or BMP2
and BMP4 , appear in Seq. ID Nos. 10 and 11, respectively, and in
Wozney, et al. (1988) Science 242: 1528- 1534. The pro domain for
BMP2 (BMP2A) likely includes residues 25-248 or 25-282; the mature
protein, residues 249-396 or 283-396, of which residues
249-296/283-296 define the N-terminal extension and 295- 396 define
the C-terminal domain. The pro domain for BMP4 (BMP2B) likely
includes residues 25-256 or 25-292; the mature protein, residues
257-408 or 293-408, of which 257-307/293-307 define the N- terminal
extension, and 308-408 define the C-terminal domain. "DPP" refers
to protein sequences encoded by the Drosophila DPP gene. The amino
acid sequence for the full length protein, including the mature
form and the pro region, appears in Seq. ID No. 12 and in Padgett,
et al (1987) Nature 325: 81-84. The pro domain likely extends from
the signal peptide cleavage site to residue 456; the mature protein
likely is defined by residues 457-588, where residues 457- 586
define the N-terminal extension and 487-588 define the C-terminal
domain. "Vgl" refers to protein sequences encoded by the Xenopus
Vgl gene. The amino acid sequence for the full length protein,
including the mature form and the pro region, appears in Seq. ID
No. 13 and in Weeks (1987) Cell 51: 861-867. The pro domain likely
extends from the signal peptide cleavage site to residue 246; the
mature protein likely is defined by residues 247-360, where
residues 247-258 define the N-terminal extension, and residues
259-360 define the C-terminal domain. "Vgr-1" refers to protein
sequences encoded by the murine Vgr-1 gene. The amino acid sequence
for the full length protein, including the mature form and the pro
region, appears in Seq. ID No. 14 and in Lyons, et al, (1989) PNAS
86: 4554-4558. The pro domain likely extends from the signal
peptide cleavage site to residue 299; the mature protein likely is
defined by residues 300-438, where residues 300-336 define the
N-terminal extension and residues 337-438 define the C-terminus.
"GDF-1" refers to protein sequences encoded by the human GDF-1
gene. The cDNA and encoded amino sequence for the full length
protein is provided in Seq. ID. No. 15 and Lee (1991) PNAS 88:
4250-4254. The pro domain likely extends from the signal peptide
cleavage site to residue 214; the mature protein likely is defined
by residues 215- 372, where residues 215-256 define the N- terminal
extension and residues 257-372 define the C-terminus. "60A" refers
to protein sequences encoded by the Drosophila 60A gene. The amino
acid sequence for the full length protein appears in Seq. ID No. 16
and in Wharton et al. (1991) PNAS 88: 9214-9218) The pro domain
likely extends from the signal peptide cleavage site to residue
324; the mature protein likely is defined by residues 325-455,
wherein residues 325-353 define the N-terminal extension and
residues 354-455 define the C-terminus. "BMP3" refers to protein
sequences encoded by the human BMP3 gene. The amino acid sequence
for the full length protein, including the mature form and the pro
region, appears in Seq. ID No. 17 and in Wozney et al. (1988)
Science 242: 1528-1534. The pro domain likely extends from the
signal peptide cleavage site to residue 290; the mature protein
likely is defined by residues 291- 472, wherein residues 291-370
define the N-terminal extension and residues 371-472 define the
C-terminus. "BMP5" refers to protein sequences encoded by the human
BMP5 gene. The amino acid sequence for the full length protein,
including the mature form and the pro region, appears in Seq. ID
No. 18 and in Celeste, et al. (1990) PNAS 87: 9843-9847. The pro
domain likely extends from the signal peptide cleavage site to
residue 316; the mature protein likely is defined by residues
317-454, where residues 317-352 define the N-terminus and residues
352-454 define the C-terminus. "BMP6" refers to protein sequences
encoded by the human BMP6 gene. The amino acid sequence for the
full length protein, including the mature form and the pro region,
appears in Seq. ID No. 16 and in Celeste, et al. (1990) PNAS 87:
9843-5847. The pro domain likely includes extends from the signal
peptide cleavage site to residue 374; the mature sequence likely
includes residues 375-513, where residues 375-411 define the
N-terminus and residues 412-513 define the C-terminus.
[0030] Note that the OP-2 and OP-3 proteins have an additional
cysteine residue in the C-terminal region (e.g., see residue 338 in
these sequences), in addition to the conserved cysteine skeleton in
common with the other proteins in this family. The GDF-1 protein
has a four amino acid insert within the conserved skeleton
("Gly-Gly-Pro-Pro") but this insert likely does not interfere with
the relationship of the cysteines in the folded structure. In
addition, the CBMP2 proteins are missing one amino acid residue
within the cysteine skeleton.
[0031] The dimeric morphogen species are inactive when reduced, but
are active as oxidized homodimers and when oxidized in combination
with other morphogens of this invention. Thus, as defined herein, a
morphogen useful in a soluble morphogen complex is a dimeric
protein comprising a pair of polypeptide chains, wherein each
polypeptide chain has less than 200 amino acids and comprises at
least the C-terminal six, preferably seven cysteine skeleton
defined by residues 335-431 of Seq. ID No. 1, including
functionally equivalent arrangements of these cysteines (e.g.,
amino acid insertions or deletions which alter the linear
arrangement of the cysteines in the sequence but not their
relationship in the folded structure), such that, when the
polypeptide chains are folded, the dimeric protein species
comprising the pair of polypeptide chains has the appropriate
three-dimensional structure, including the appropriate intra- or
inter-chain disulfide bonds such that the protein is capable of
acting as a morphogen as defined herein. The solubility of these
structures is improved when the mature dimeric form of a morphogen,
in accordance with the invention, is complexed with at least one,
and preferably two, pro domains.
[0032] Various generic sequences (Generic Sequence 1-6) defining
preferred C-terminal sequences useful in the soluble morphogens of
this invention are described in U.S. Ser. No. 07/923,780,
incorporated herein above by reference. Two currently preferred
generic sequences are described below.
[0033] Generic Sequence 7 (Seq. ID No. 20) and Generic Sequence 8
(Seq. ID No. 21) disclosed below, accommodate the homologies shared
among preferred morphogen protein family members identified to
date, including OP-1, OP-2, OP-3, CBMP2A, CBMP2B, BMP3, 60A, DPP,
Vgl, BMP5, BMP6, Vrg-1, and GDF-1. The amino acid sequences for
these proteins are described herein (see Sequence Listing) and/or
in the art, as well as in PCT publication U.S. Ser. No. 92/07358,
filed Aug. 28, 1992, for example. The generic sequences include
both the amino acid identity shared by these sequences in the
C-terminal domain, defined by the six and seven cysteine skeletons
(Generic Sequences 7 and 8, respectively), as well as alternative
residues for the variable positions within the sequence. The
generic sequences allow for an additional cysteine at position 41
(Generic Sequence 7) or position 46 (Generic Sequence 8), providing
an appropriate cysteine skeleton where inter- or intramolecular
disulfide bonds can form, and containing certain critical amino
acids which influence the tertiary structure of the proteins.
TABLE-US-00002 Generic Sequence 7 Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa
Gly Trp Xaa Xaa 1 5 10 Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa
Tyr 15 20 Cys Xaa Gly Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa 25 30 35
Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa 40 45 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Cys Cys Xaa Pro Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Val Xaa Leu Xaa 75 80 Xaa Xaa Xaa Xaa Met Xaa Val Xaa Xaa Cys Xaa
Cys 85 90 95 Xaa
wherein each Xaa is independently selected from a group of one or
more specified amino acids defined as follows: "Res." means
"residue" and Xaa at res.2=(Tyr or Lys); Xaa at res.3=Val or Ile);
Xaa at res.4=(Ser, Asp or Glu); Xaa at res.6=(Arg, Gln, Ser, Lys or
Ala); Xaa at res.7=(Asp or Glu); Xaa at res.8=(Leu, Val or Ile);
Xaa at res.11 (Gln, Leu, Asp, His, Asn or Ser); Xaa at res.12=(Asp,
Arg, Asn or Glu); Xaa at res. 13=(Trp or Ser); Xaa at res.14=(Ile
or Val); Xaa at res.15=(Ile or Val); Xaa at res.16 (Ala or Ser);
Xaa at res.18=(Glu, Gln, Leu, Lys, Pro or Arg); Xaa at res.19=(Gly
or Ser); Xaa at res.20-(Tyr or Phe); Xaa at res.21=(Ala, Ser, Asp,
Met, H is, Gln, Leu or Gly); Xaa at res.23=(Tyr, Asn or Phe); Xaa
at res.26=(Glu, H is, Tyr, Asp, Gln, Ala or Ser); Xaa at
res.28=(Glu, Lys, Asp, Gln or Ala); Xaa at res.30=(Ala, Ser, Pro,
Gln, Ile or Asn); Xaa at res.31=(Phe, Leu or Tyr); Xaa at
res.33=(Leu, Val or Met); Xaa at res.34=(Asn, Asp, Ala, Thr or
Pro); Xaa at res.35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at
res.36=(Tyr, Cys, H is, Ser or Ile); Xaa at res.37=(Met, Phe, Gly
or Leu); Xaa at res.38=(Asn, Ser or Lys); Xaa at res.39=(Ala, Ser,
Gly or Pro); Xaa at res.40=(Thr, Leu or Ser); Xaa at res.44=(Ile,
Val or Thr); Xaa at res.45=(Val, Leu, Met or Ile); Xaa at
res.46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at
res.48=(Leu or Ile); Xaa at res.49-(Val or Met); Xaa at res.50=(H
is, Asn or Arg); Xaa at res.51=(Phe, Leu, Asn, Ser, Ala or Val);
Xaa at res.52=(Ile, Met, Asn, Ala, Val, Gly or Leu); Xaa at
res.53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or
Val); Xaa at res.55=(Glu, Asp, Asn, Gly, Val, Pro or Lys); Xaa at
res.56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile or His); Xaa at
res.57=(Val, Ala or Ile); Xaa at res.58=(Pro or Asp); Xaa at
res.59=(Lys, Leu or Glu); Xaa at res.60=(Pro, Val or Ala); Xaa at
res.63=(Ala or Val); Xaa at res.65=(Thr, Ala or Glu); Xaa at
res.66=(Gln, Lys, Arg or Glu); Xaa at res.67=(Leu, Met or Val); Xaa
at res.68=(Asn, Ser, Asp or Gly); Xaa at res.69=(Ala, Pro or Ser);
Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res.71=(Ser, Ala or
Pro); Xaa at res.72=(Val, Leu, Met or Ile); Xaa at res.74=(Tyr or
Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res.76=(Asp, Asn
or Leu); Xaa at res.77=(Asp, Glu, Asn, Arg or Ser); Xaa at
res.78=(Ser, Gln, Asn, Tyr or Asp); Xaa at res.79=(Ser, Asn, Asp,
Glu or Lys); Xaa at res.80=(Asn, Thr or Lys); Xaa at res.82=(Ile,
Val or Asn); Xaa at res.84=(Lys or Arg); Xaa at res.85=(Lys, Asn,
Gln, His, Arg or Val); Xaa at res.86=(Tyr, Glu or His); Xaa at
res.87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu, Trp or
Asp); xaa at res.90=(Val, Thr, Ala or Ile); Xaa at res.92=(Arg,
Lys, Val, Asp, Gln or Glu); Xaa at res.93=(Ala, Gly, Glu or Ser);
Xaa at res.95=(Gly or Ala) and Xaa at res.97=(His or Arg).
[0034] As described above, Generic Sequence 8 (Seq. ID No. 21)
includes all of Generic Sequence 7 and in addition includes the
following sequence at its N-terminus:
TABLE-US-00003 Cys Xaa Xaa Xaa Xaa 1 5
[0035] Accordingly, beginning with residue 7, each "Xaa" in Generic
Seq. 8 is a specified amino acid defined as for Generic Seq. 7,
with the distinction that each residue number described for Generic
Sequence 7 is shifted by five in Generic Seq. 8. Thus, "Xaa at
res.2=(Tyr or Lys)" in Gen. Seq. 7 refers to Xaa at res. 7 in
Generic Seq. 8. In Generic Seq. 8, Xaa at res.2=(Lys, Arg, Ala or
Gln); Xaa at res.3=(Lys, Arg or Met); Xaa at res.4=(His, Arg or
Gln); and Xaa at res.5=(Glu, Ser, His, Gly, Arg, Pro, Thr, or
Tyr).
[0036] Accordingly, other useful sequences defining preferred
C-terminal sequences are those sharing at least 70% amino acid
sequence homology or "similarity", and preferably 80% homology or
similarity with any of the sequences incorporated into Generic Seq.
7 and 8 above. These are anticipated to include allelic, species
and mutant variants, as well as novel members of this morphogenic
family of proteins. As used herein, "amino acid sequence homology"
is understood to mean amino acid sequence similarity, and
homologous sequences share identical or similar amino acids, where
similar amino acids are conserved amino acids as defined by Dayoff
et al., Atlas of Protein Sequence and Structure; vol. 5, Suppl.3,
pp. 345-362 (M. O. Dayoff, ed., Nat'l BioMed. Research Fdn.,
Washington D.C. 1978.) Thus, a candidate sequence sharing 70% amino
acid homology with a reference sequence requires that, following
alignment of the candidate sequence with the reference sequence,
70% of the amino acids in the candidate sequence are identical to
the corresponding amino acid in the reference sequence, or
constitute a conserved amino acid change thereto. "Amino acid
sequence identity" is understood to require identical amino acids
between two aligned sequences. Thus, a candidate sequence sharing
60% amino acid identity with a reference sequence requires that,
following alignment of the candidate sequence with the reference
sequence, 60% of the amino acids in the candidate sequence are
identical to the corresponding amino acid in the reference
sequence.
[0037] As used herein, all homologies and identities calculated use
OP-1 as the reference sequence. Also as used herein, sequences are
aligned for homology and identity calculations using the method of
Needleman et al. (1970) J. Mol. Biol. 48:443-453 and identities
calculated by the Align program (DNAstar, Inc.) In all cases,
internal gaps and amino acid insertions in the candidate sequence
as aligned are ignored when making the homology/identity
calculation.
[0038] Also as used herein, "mutant variant" or "mutant protein
variant" is understood to mean an amino acid variant form of the
morphogen protein, wherein the amino acid change or changes in the
sequence do not alter significantly the morphogenic activity (e.g.,
tissue regeneration activity) of the protein, and the variant
molecule performs substantially the same function in substantially
the same way as the naturally occurring form of the molecule.
Mutant variants may include single or multiple amino acid changes,
and are intended to include chimeric sequences as described below.
The variants may be naturally occurring or may be biosynthetically
induced by using standard recombinant DNA techniques or chemical
protein synthesis methodologies.
[0039] The currently most preferred protein sequences useful in
soluble morphogen complexes in this invention include those having
greater than 60% identity, preferably greater than 65% identity,
with the amino acid sequence defining the conserved six cysteine
skeleton of hOP1 (e.g., residues 335-431 of Seq. ID No. 5). These
most preferred sequences include both allelic and species variants
of the OP-1 and OP-2 proteins, including the Drosophila 60A
protein. Accordingly, in another preferred aspect of the invention,
useful morphogens include active proteins comprising species of
polypeptide chains having the generic amino acid sequence herein
referred to as "OPX", which accommodates the homologies between the
various identified species of OP1 and OP2 (Seq. ID No. 22).
[0040] Useful N-terminal extension sequences are listed in FIG. 2
for use with the C-terminal domains described above. Also as
described above, the full length N-terminal extensions, or
truncated forms thereof, may be used in preferred dimeric species.
The mature dimeric species may be produced from intact DNAs, or
truncated forms thereof. It also is envisioned as an embodiment of
the invention that chimeric morphogen sequences can be used. Thus,
DNAs encoding chimeric morphogens may be constructed using part or
all of N-terminal extension from one morphogen and a C-terminal
domain derived from one or more other morphogens. These chimeric
proteins may be synthesized using standard recombinant DNA
methodology and/or automated chemical nucleic acid synthesis
methodology well described in the art. Other chimeric morphogens
include soluble morphogen complexes where the pro domain is encoded
rom a DNA sequence corresponding to one morphogen, and art or all
of the mature domain is encoded by DNA erived from other, different
morphogen(s). These soluble chimerics may be produced from a single
synthetic DNA as described below, or, alternatively, may be
formulated in vitro from isolated components also as described
herein below.
[0041] Finally, the morphogen pro domains and/or mature form
N-terminal extensions themselves may be useful as tissue targeting
sequences. As described above, the morphogen family members share
significant sequence homology in their C-terminal active domains.
By contrast, the sequences diverge significantly in the sequences
which define the pro domain and the N-terminal 39 amino acids of
the mature protein. Accordingly, the pro domain and/or N-terminal
extension sequence may be morphogen-specific. Accordingly, part or
all of these morphogen-specific sequences may serve as tissue
targeting sequences for the morphogens described herein. For
example, the N-terminal extension and/or pro domains may interact
specifically with one or more molecules at the target tissue to
direct the morphogen associated with the pro domain to that tissue.
Thus, for example, the morphogen-specific sequences of OP-1, BMP2
or BMP4, all of which proteins are found naturally associated with
bone tissue (see, for example, U.S. Pat. No. 5,011,691) may be
particularly useful sequences when the morphogen complex is to be
targeted to bone. Similarly, BMP6 (or Vgr-1) specific sequences may
be used when targeting to lung tissue is desired. Alternatively,
the morphogen-specific sequences of GDF-1 may be used to target
soluble morphogen complexes to nerve tissue, particularly brain
tissue, where GDF-1 appears to be primarily expressed (see, for
example, U.S. Pat. No. 922,813 and Lee, PNAS, 88:4250-4254 (1991),
incorporated herein by reference).
II. Recombinant Production of Soluble Morphogen Complexes
[0042] Soluble morphogen complexes can be produced from eukaryotic
host cells, preferably mammalian cells, using standard recombinant
expression techniques. An exemplary protocol currently preferred,
is provided below, using a particular vector construct and chinese
hamster ovary (CHO) cell line. Those skilled in the art will
appreciate that other expression systems are contemplated to be
useful, including other vectors and other cell systems, and the
invention is not intended to be limited to soluble morphogenic
protein complexes produced only by the method detailed
hereinbelow.Similar results to those described herein have been
observed using recombinant expression systems developed for COS and
BSC cells.
[0043] Morphogen DNA encoding the precursor sequence is subcloned
into an insertion site of a suitable, commercially available
pUC-type vector (e.g., pUC-19, ATCC #37254, Rockville, Md.), along
with a suitable promoter/enhancer sequences and 3' termination
sequences. Useful DNA sequences include the published sequences
encoding these proteins, and/or synthetic constructs. Currently
preferred promoter/enhancer sequences are the CMV promoter (human
cytomegalovirus major intermediate--early promoter) and the mouse
mammary tumor virus promoter (mMTV) boosted by the rous sarcoma
virus LTR enhancer sequence (e.g., from Clontech, Inc., Palo Alto).
Expression also may be further enhanced using transactivating
enhancer sequences. The plasmid also contains DHFR as an
amplifiable marker, under SV40 early promoter control (ATCC
#37148). Transfection, cell culturing, gene amplification and
protein expression conditions are standard conditions, well known
in the art, such as are described, for example in Ausubel et al.,
ed., Current Protocols in Molecular Biology, John Wiley & Sons,
NY (1989). Briefly, transfected cells are cultured in medium
containing 0.1-0.5% dialyzed fetal calf serum (FCS) and stably
transfected high expression cell lines are obtained by subcloning
and evaluated by standard Western or Northern blot. Southern blots
also are used to assess the state of integrated sequences and the
extent of their copy number amplification.
[0044] A currently preferred expression vector contains the DHFR
gene, under SV40 early promoter control, as both a selection marker
and as an inducible gene amplifier. The DNA sequence for DHFR is
well characterized in the art, and is available commercially. For
example, a suitable vector may be generated from pMAM-neo
(Clontech, Inc., Palo Alto, Calif.) by replacing the neo gene
(BamHI: digest) with an SphI-BamHI, or a PvuII-BamHI fragment from
pSV5-DHFR (ATCC #37148), which contains the DHFR gene under SV40
early promoter control. A BamHI site can be engineered at the SphI
or PvuII site using standard techniques (e.g., by linker insertion
or site-directed mutagenesis) to allow insertion of the fragment
into the vector backbone. The morphogen. DNA can be inserted into
the polylinker site downstream of the MMTV-LTR sequence (mouse
mammary tumor virus LTR). The CMV promoter sequence then may be
inserted into the expression vector (e.g., from pCDM8, Invitrogen,
Inc.) The SV40 early promoter, which drives DHFR expression,
preferably is modified in these vectors to reduce the level of DHFR
mRNA produced.
[0045] The currently preferred mammalian cell line is a CHO Chinese
hamster ovary, cell line, and the preferred procedure for
establishing a stable morphogen production cell line with high
expression levels comprises transfecting a stable CHO cell line,
preferably CHO-DXB11, with the expression vector described above,
isolating clones with high morphogen expression levels, and
subjecting these clones to cycles of subcloning using a limited
dilution method described below to obtain a population of high
expression clones. Subcloning preferably is performed in the
absence of MTX to identify stable high expression clones which do
not require addition of MTX to the growth media for morphogen
production.
[0046] In the subcloning protocol cells are seeded on ten 100 mm
petri dishes at a cell density of either 50 or 100 cells per plate,
with or preferably without MTX in the culture media. After 14 days
of growth, clones are isolated using cloning cylinders and standard
procedures, and cultured in 24-well plates. Clones then are
screened for morphogen expression by Western immunoblots using
standard procedures, and morphogen expression levels compared to
parental lines. Cell line stability of high expression subclones
then is determined by monitoring morphogen expression levels over
multiple cell passages (e.g., four or five passages).
III. Isolation of Soluble Morphogen Complex from Conditioned Media
or Body Fluid
[0047] Morphogens are expressed from mammalian cells as soluble
complexes. Typically, however the complex is disassociated during
purification, generally by exposure to denaturants often added to
the purification solutions, such as detergents, alcohols, organic
solvents, chaotropic agents and compounds added to reduce the pH of
the solution. Provided below is a currently preferred protocol for
purifying the soluble proteins from conditioned media (or,
optionally, a body fluid such as serum, cerebro-spinal or
peritoneal fluid), under non-denaturing conditions. The method is
rapid, reproducible and yields isolated soluble morphogen complexes
in substantially pure form.
[0048] Soluble morphogen complexes can be isolated from conditioned
media using a simple, three step chromatographic protocol performed
in the absence of denaturants. The protocol involves running the
media (or body fluid) over an affinity column, followed by ion
exchange and gel filtration chromatographies. The affinity column
described below is a Zn-IMAC column. The present protocol has
general applicability to the purification of a variety of
morphogens, all of which are anticipated to be isolatable using
only minor modifications of the protocol described below. An
alternative protocol also envisioned to have utility an
immunoaffinity column, created using standard procedures and, for
example, using antibody specific for a given morphogen pro domain
(complexed, for example, to a protein A-conjugated Sepharose
column.)
[0049] Protocols for developing immunoaffinity columns are well
described in the art, (see, for example, Guide to Protein
Purification, M. Deutscher, ed., Academic Press, San Diego, 1990,
particularly sections VII and XI.)
[0050] In this experiment OP-1 was expressed in CHO cells as
described above. The CHO cell conditioned media containing 0.5% FBS
was initially purified using Immobilized Metal-Ion Affinity
Chromatography (IMAC). The soluble OP-1 complex from conditioned
media binds very selectively to the Zn-IMAC resin and a high
concentration of imidazole (50 mM imidazole, pH 8.0) is required
for the effective elution of the bound complex. The Zn-IMAC step
separates the soluble OP-1 from the bulk of the contaminating serum
proteins that elute in the flow through and 35 mM imidazole wash
fractions. The Zn-IMAC purified soluble OP-1 is next applied to an
S-Sepharose cation-exchange column equilibrated in 20 mM NaPO.sub.4
(pH 7.0) with 50 mM NaCl. This S-Sepharose step serves to further
purify and concentrate the soluble OP-1 complex in preparation for
the following gel filtration step. The protein was applied to a
Sephacryl S-200HR column equilibrated in TBS. Using substantially
the same protocol, soluble morphogens also may be isolated from one
or more body fluids, including serum, cerebro-spinal fluid or
peritoneal fluid.
[0051] IMAC was performed using Chelating-Sepharose (Pharmacia)
that had been charged with three column volumes of 0.2 M
ZnSO.sub.4. The conditioned media was titrated to pH 7.0 and
applied directly to the ZN-IMAC resin equilibrated in 20 mM HEPES
(pH 7.0) with 500 mM NaCl. The Zn-IMAC resin was loaded with 80 mL
of starting conditioned media per mL of resin. After loading the
column was washed with equilibration buffer and most of the
contaminating proteins were eluted with 35 mM imidazole (pH 7.0) in
equilibration buffer. The soluble OP-1 complex is then eluted with
50 mM imidazole (pH 8.0) in 20 mM HEPES and 500 mM NaCl. The 50 mM
imidazole eluate containing the soluble OP-1 complex was diluted
with nine volumes of 20 mM NaPO.sub.4 (pH 7.0) and applied to an
S-Sepharose (Pharmacia) column equilibrated in 20 mM NaPO.sub.4 (pH
7.0) with 50 mM NaCl. The S-Sepharose resin was loaded with an
equivalent of 800 mL of starting conditioned media per mL of resin.
After loading the S-Sepharose column was washed with equilibration
buffer and eluted with 100 mM NaCl followed by 300 mM and 500 mM
NaCl in 20 mM NaPO.sub.4 (pH 7.0). The 300 mM NaCl pool was further
purified using gel filtration chromatography. Fifty mls of the 300
mm NaCl eluate was applied to a 5.0.times.90 cm Sephacryl S-200HR
(Pharmacia) equilibrated in Tris buffered saline (TBS), 50 mM Tris,
150 mM NaCl (pH 7.4). The column was eluted at a flow rate of 5
mL/minute collecting 10 mL fractions. The apparent molecular of the
soluble OP-1 was determined by comparison to protein molecular
weight standards (alcohol dehydrogenase (ADH, 150 kDa), bovine
serum albumin (BSA, 68 kDa), carbonic anhydrase (CA, 30 kDa) and
cytochrome C (cyt C, 12.5 kDa). (see FIG. 3) The purity of the
S-200 column fractions was determined by separation on standard 15%
polyacrylamide SDS gels stained with coomassie blue. The identity
of the mature OP-1 and the pro-domain was determined by N-terminal
sequence analysis after separation of the mature OP-1 from the
pro-domain using standard reverse phase C18 HPLC.
[0052] FIG. 3 shows the absorbance profile at 280 nm. The soluble
OP-1 complex elutes with an apparent molecular weight of 110 kDa.
This agrees well with the predicted composition of the soluble OP-1
complex with one mature OP-1 dimer (35-36 kDa) associated with two
pro-domains (39 kDa each). Purity of the final complex can be
verified by running the appropriate fraction in a reduced 15%
polyacrylamide gel.
[0053] The complex components can be verified by running the
complex-containing fraction from the S-200 or S-200HR columns over
a reverse phase C18 HPLC column and eluting in an acetonitrile
gradient (in 0.1% TFA), using standard procedures. The complex is
dissociated by this step, and the pro domain and mature species
elute as separate species. These separate species then can be
subjected to N-terminal sequencing using standard procedures (see,
for example, Guide to Protein Purification, M. Deutscher, ed.,
Academic Press, San Diego, 1990, particularly pp. 602-613), and the
identity of the isolated 36 kD, 39 kDa proteins confirmed as mature
morphogen and isolated, cleaved pro domain, respectively.
N-terminal sequencing of the isolated pro domain from mammalian
cell produced OP-1 revealed 2 forms of the pro region, the intact
form (beginning at residue 30 of Seq. ID No. 1) and a truncated
form, (beginning at residue 48 of Seq. ID No. 1.) N-terminal
sequencing of the polypeptide subunit of the isolated mature
species reveals a range of N-termini for the mature sequence,
beginning at residues 293, 300, 313, 315, 316, and 318, of Seq. ID
No. 1, all of which are active as demonstrated by the standard bone
induction assay.
V. In Vitro Soluble Morphogen Complex Formation
[0054] As an alternative to purifying soluble complexes from
culture media or a body fluid, soluble complexes may be formulated
from purified pro domains and mature dimeric species. Successful
complex formation apparently requires association of the components
under denaturing conditions sufficient to relax the folded
structure of these molecules, without affecting disulfide bonds.
Preferably, the denaturing conditions mimic the environment of an
intracellular vesicle sufficiently such that the cleaved pro domain
has an opportunity to associate with the mature dimeric species
under relaxed folding conditions. The concentration of denaturant
in the solution then is decreased in a controlled, preferably
step-wise manner, so as to allow proper refolding of the dimer and
pro regions while maintaining the association of the pro domain
with the dimer. Useful denaturants include 4-6M urea or guanidine
hydrochloride (GuHCl), in buffered solutions of pH 4-10, preferably
pH 6-8. The soluble complex then is formed by controlled dialysis
or dilution into a solution having a final denaturant concentration
of less than 0.1-2M urea or GuHCl, preferably 1-2 M urea of GuHCl,
which then preferably can be diluted into a physiological buffer.
Protein purification/renaturing procedures and considerations are
well described in the art, and details for developing a suitable
renaturing protocol readily can be determined by one having
ordinary skill in the art.
[0055] One useful text one the subject is Guide to Protein
Purification, M. Deutscher, ed., Academic Press, San Diego, 1990,
particularly section V. Complex formation also may be aided by
addition of one or more chaperone proteins.
VI. Stability of Soluble Morphogen Complexes
[0056] The stability of the highly purified soluble morphogen
complex in a physiological buffer, e.g., tris-buffered saline (TBS)
and phosphate-buffered saline (PBS), can be enhanced by any of a
number of means. Currently preferred is by means of a pro region
that comprises at least the first 18 amino acids of the pro
sequence (e.g., residues 30-47 of Seq. ID NO. 1 for OP-1), and
preferably is the full length pro region. Residues 30-47 show
sequence homology to the N-terminal portion of other morphogens and
are believed to have particular utility in enhancing complex
stability for all morphogens. Other useful means for enhancing the
stability of soluble morphogen complexes include three classes of
additive. These additives include basic amino acids (e.g.,
L-arginine, lysine and betaine); nonionic detergents (e.g., Tween
80 or NonIdet P-120); and carrier proteins (e.g., serum albumin and
casein). These additives include 1-100 mM, preferably 10-70 mM,
including 50 mM, basic amino acid; 0.01-1.0%, preferably 0.05-0.2%,
including 0.1% (v/v) nonionic detergent; and 0.01-1.0%, preferably
0.05-0.2%, including 0.1% (w/v) carrier protein.
VII. Activity of Soluble Morphogen Complex
[0057] Association of the pro domain with the mature dimeric
species does not interfere with the morphogenic activity of the
protein in vivo as demonstrated by different activity assays.
Specifically, soluble OP-1 complex provided in a standard rat
osteopenia model induces significant increase in bone growth and
osteocalcin production (see Table II, below), in a mannor analogous
to the results obtained using mature morphogen.
[0058] The assay is analogous to the osteoporosis model described
in USSN 923,780, but uses aged female rats rather than
ovariectomized animals. Briefly, young or aged female rats (Charles
River Labs, 115-145, and 335-460 g body weight, respectively) were
dosed daily for 7 days by intravenous tail injection, with either
20 .mu.g/Kg body weight soluble OP-1, or 100 .mu.g/Kg body weight
soluble OP-1. Control groups of young and aged female rats were
dosed only with tris-buffered saline (TBS). Water and food were
provided to all animals ad libitum. After 14 days, animals were
sacrificed, and new bone growth measured by standard histometric
procedures. Osteocalcin concentrations in serum also were measured.
No detrimental effects of morphogen administration were detected as
determined by changes in animal body or organ weight or by
hematology profiles.
TABLE-US-00004 TABLE II No. Bone Area Osteocalcin Animals Animal
Group (B. Ar/T. Ar) (ng/ml) 4 Control 5.50 .+-. 0.64 11.89 .+-.
4.20 5 Aged female, 7.68 .+-. 0.63** 22.24 .+-. 2.28** 20 .mu.g/Kg
sol. OP-1 5 Aged female, 9.82 .+-. 3.31* 20.87 .+-. 6.14* 100
.mu.g/Kg sol. OP-1 *P < 0.05 **P < 0.01
[0059] Similar experiments performed using soluble OP-1 complex in
the osteoporosis model described in USSN 923,780 and incorporated
hereinabove by reference using ovariectomized rats also show no
detrimental effect using the complex form.
[0060] Both mature and soluble morphogen also can induce CAM (cell
adhesion molecule) expression, as described in copending U.S. Ser.
No. 07/022,813, filed Jul. 31, 1992, the disclosure of which is
incorporated hereinabove by reference.
[0061] Briefly, and as described therein, induction of N-CAM
isoforms (N-CAM-180, N-CAM-140 and N-CAM-120) can be monitored by
reaction with the commercially available antibody mAb H28.123
(Sigma Co., St. Louis) and standard Western blot analysis (see, for
example, Molecular Cloning, A Laboratory Manual, Sambrook et al.
eds. Cold Spring Harbor Press, New York, 1989, particularly Section
18). Incubation of a growing culture of transformed cells of
neuronal origin, NG108-15 cels (ATCC, Rockville, Md.) with either
mature morphogen dimers or soluble morphogen complexes (10-100
ng/ml, preferably at least 40 ng/ml) induces a redifferentiation of
these cells back to a morphology characteristic of untransformed
neurons, including specific induction and/or enhanced expression of
all 3 N-CAM isoforms. In the experiment, cells were subcultured on
poly-L-lysine coated 6-well plates and grown in chemically defined
medium for 2 days before the experiment. Fresh aliquots of
morphogen were added (2.5 .mu.l) daily.
VIII. Antibody Production
[0062] Provided below are standard protocols for polycolonal and
monoclonal antibody production. For antibodies which recognize the
soluble complex only, preferably the isolated pro region is used as
the antigen; where antibodies specific to the mature protein are
desired, the antigen preferably comprises at least the C-terminal
domain or the intact mature sequence.
[0063] Polyclonal antibody may be prepared as follows. Each rabbit
is given a primary immunization of 100 ug/500 .mu.l of antigen, in
0.1% SDS mixed with 500 .mu.l Complete Freund's Adjuvant. The
antigen is injected subcutaneously at multiple sites on the back
and flanks of the animal. The rabbit is boosted after a month in
the same manner using incomplete Freund's Adjuvant. Test bleeds are
taken from the ear vein seven days later. Two additional boosts and
test bleeds are performed at monthly intervals until antibody
against the morphogen antigen is detected in the serum using an
ELISA assay. Then, the rabbit is boosted monthly with 100 .mu.g of
antigen and bled (15 ml per bleed) at days seven and ten after
boosting.
[0064] Monoclonal antibody specific for a given morphogen may be
prepared as follows. A mouse is given two injections of the
morphogen antigen. The protein or protein fragment preferably is
recombinantly produced. The first injection contains 100 .mu.g of
antigen in complete Freund's adjuvant and is given subcutaneously.
The second injection contains 50 .mu.g of antigen in incomplete
adjuvant and is given intraperitoneally. The mouse then receives a
total of 230 .mu.g of OP-3 in four intraperitoneal injections at
various times over an eight month period. One week prior to fusion,
the mouse is boosted intraperitoneally with antigen (e.g., 100
.mu.g) and may be additionally boosted with a peptide fragment
conjugated to bovine serum albumin with a suitable crosslinking
agent. This boost can be repeated five days (IP), four days (IP),
three days (IP) and one day (IV) prior to fusion. The mouse spleen
cells then are fused to commercially available myeloma cells at a
ratio of 1:1 using PEG 1500 (Boeringer Mannheim, Germany), and the
fused cells plated and screened for mature or soluble
morphogen-specific antibodies using the appropriate portion of the
morphogen sequence as antigen. The cell fusion and monoclonal
screening steps readily are performed according to standard
procedures well described in standard texts widely available in the
art.
[0065] Using these standard procedures, anti-pro domain antisera
was prepared from rabbits using the isolated pro domain from OP-1
as the antigen, and monoclonal antibody ("mAb") to the mature
domain was produced in mice, using an E. coli-produced truncated
form of OP-1 as antigen.
[0066] Standard Western blot analysis performed under reducing
conditions demonstrates that the anti-pro domain antisera
("anti-pro") is specific for the pro domain only, while the mAb to
mature OP-1 ("anti-mature OP-1") is specific for the dimer
subunits, that the two antibodies do not cross-react, and that the
antibodies and can be used to distinguish between soluble and
mature protein forms in a sample, e.g., of conditioned media or
serum. A tabular representation of the Western blot results is in
Table III below, where reactivity of mAb to mature OP-1 is
indicated by "yy", and reactivity of the anti-pro antisera is
indicated by "xx".
TABLE-US-00005 TABLE III Purified Purified Conditioned Isolated
Dimer Antibody Sol OP1 CHO Cell Media Pro Domain Subunits
"anti-pro" xx xx xx "anti- yy yy yy mature OP-1"
IX. Immunoassays
[0067] The ability to detect morphogens in solution and to
distinguish between soluble and mature dimeric morphogen forms
provides a valuable tool for diagnostic assays, allowing one to
monitor the level and type of morphogen free in the body, e.g., in
serum and other body fluids.
[0068] For example, OP-1 is an intimate participant in normal bone
growth and resorption. Thus, soluble OP-1 is expected to be
detected at higher concentrations in individuals experiencing high
bone turnover, such as children, and at substantially lower levels
in individuals with abnormally low rates of bone turnover, such as
patients with osteoporosis, osteosarcoma, Paget's disease and the
like. Monitoring the level of OP-1, or other bone targeted
morphogens such as BMP2 and BMP4, in serum thus provides a means
for evaluating the status of bone tissue in an individual, as well
as a means for monitoring the efficacy of a treatment to regenerate
damaged or lost bone tissue. Similarly, monitoring the level of
endogenous GDF-1, can provide diagnostic information on the health
of nerve tissue, particularly brain tissue. Moreover, following
this disclosure one can distinguish between the level of soluble
and mature forms in solution.
[0069] A currently preferred detection means for evaluating the
level of morphogen in a body fluid comprises an immunoassay
utilizing an antibody or other suitable binding protein capable of
reacting specifically with a morphogen and being detected as part
of a complex with the morphogen. Immunoassays may be performed
using standard techniques known in the art and antibodies raised
against a morphogen and specific for that morphogen. Antibodies
which recognize a morphogen protein form of interest may be
generated as described herein and these antibodies then used to
monitor endogenous levels of protein in a body fluid, such as
serum, whole blood or peritoneal fluid. To monitor endogenous
concentrations of soluble morphogen, the antibody chosen preferably
has binding specificity for the soluble form e.g., has specificity
for the pro domain. Such antibodies may be generated by using the
pro domain or a portion thereof as the antigen, essentially as
described herein. A suitable pro domain for use as an antigen may
be obtained by isolating the soluble complex and then separating
the noncovalently associated pro domain from the mature domain
using standard procedures, e.g., by passing the complex over an
HPLC column, as described above or by separation by gel
electrophoresis. Alternatively, the pro form of the protein in its
monomeric form may be used as the antigen and the candidate
antibodies screened by Western blot or other standard immunoassay
for those which recognize the pro domain of the soluble form of the
protein of interest, but not the mature form, also as described
above.
[0070] Monomeric pro forms can be obtained from cell lysates of CHO
produced cells, or from prokaryotic expression of a DNA encoding
the pro form, in for example, E. coli. The pro form, which has an
apparent molecular weight of about 50 kDa in mammalian cells, can
then be isolated by HPLC and/or by gel electrophoresis, as
described above.
[0071] In order to detect and/or quantitate the amount of
morphogenic protein present in a solution, an immunoassay may be
performed to detect the morphogen using a polyclonal or monoclonal
antibody specific for that protein. Here, soluble and mature forms
of the morphogen also may be distinguished by using antibodies that
discriminate between the two forms of the proteins as described
above. Currently preferred assays include ELISAS and
radioimmunoassays, including standard competitor assays useful for
quantitating the morphogen in a sample, where an unknown amount of
sample morphogen is allowed to react with anti-morphogen antibody
and this interaction is competed with a known amount of labeled
antigen. The level of bound or free labeled antigen at equilibrium
then is measured to quantitate the amount of unlabeled antigen in
solution, the amount of sample antigen being proportional to the
amount of free labeled antigen. Exemplary protocols for these
assays are provided below. However, as will be appreciated by those
skilled in the art, variations of these protocols, as well as other
immunoassays, are well known in the literature and within the skill
of the art. For example, in the ELISA protocol provided below,
soluble OP-1 is identified in a sample using biotinylated anti-pro
antiserum. Biotinylated antibodies can be visualized in a
colormetric assay or in a chemiluminescent assay, as described
below. Alternatively, the antibody can be radio-labeled with a
suitable molecule, such as .sup.125I. Still another protocol that
may be used is a solid phase immunoassay, preferably using an
affinity column with anti-morphogen antibody complexed to the
matrix surface and over which a serum sample may be passed. A
detailed description of useful immunoassays, including protocols
and general considerations is provided in, for example, Molecular
Cloning: A Laboratory Manual, Sambrook et al., eds. Cold Spring
Harbor Press, New York, 1989, particularly Section 18.
[0072] For serum assays, the serum preferably first is partially
purified to remove some of the excess, contaminating serum
proteins, such as serum albumin. Preferably the serum is extracted
by precipitation in ammonium sulfate (e.g., 45%) such that the
complex is precipitated. Further purification can be achieved using
purification strategies that take advantage of the differential
solubility of soluble morphogen complex or mature morphogens
relative to that of the other proteins present in serum. Further
purification also can be achieved by chromatographic techniques
well known in the art.
[0073] Soluble OP-1 may be detected using a polyclonal antibody
specific for the OP-1 pro domain in an ELISA, as follows. 1
.mu.g/100 .mu.l of affinity-purified polyclonal rabbit IgG specific
for OP-1-pro is added to each well of a 96-well plate and incubated
at 37.degree. C. for an hour. The wells are washed four times with
0.167M sodium borate buffer with 0.15 M NaCl (BSB), pH 8.2,
containing 0.1% Tween 20. To minimize non-specific binding, the
wells are blocked by filling completely with 1% bovine serum
albumin (BSA) in BSB and incubating for 1 hour at 370.degree. C.
The wells are then washed four times with BSB containing 0.1% Tween
20. A 100 .mu.l aliquot of an appropriate dilution of each of the
test samples of cell culture supernatant or serum sample is added
to each well in triplicate and incubated at 37.degree. C. for 30
min. After incubation, 100 .mu.l biotinylated rabbit anti-pro serum
(stock solution is about 1 mg/ml and diluted 1:400 in BSB
containing 1% BSA before use) is added to each well and incubated
at 37.degree. C. for 30 min. The wells are then washed four times
with BSB containing 0.1% Tween 20. 100 .mu.l strepavidin-alkaline
(Southern Biotechnology Associates, Inc. Birmingham, Ala., diluted
1:2000 in BSB containing 0.1% Tween 20 before use) is added to each
well and incubated at 37.degree. C. for 30 min. The plates are
washed four times with 0.5M Tris buffered Saline (TBS), pH 7.2. 50
.mu.l substrate (ELISA Amplification System Kit, Life Technologies,
Inc., Bethesda, Md.) is added to each well incubated at room
temperature for 15 min. Then, 50 .mu.l amplifier (from the same
amplification system kit) is added and incubated for another 15 min
at room temperature. The reaction is stopped by the addition of 50
.mu.l 0.3 M sulphuric acid. The OD at 490 nm of the solution in
each well is recorded. To quantitate the level of soluble OP-1 in
the sample, a standard curve is performed in parallel with the test
samples. In the standard curve, known increasing amounts of
purified OP-1-pro is added. Alternatively, using, for example,
Lumi-phos 530 (Analytical Luminescence Laboratories) as the
substrate and detection at 300-650 nm in a standard luminometer,
complexes can be detected by chemiluminescence, which typically
provides a more sensitive assay than detection by means of a
visible color change.
[0074] Morphogen (soluble or mature form) may be detected in a
standard plated-based radioimmunoassay as follows. Empirically
determined limiting levels of anti-morphogen antibody (e.g.,
anti-OP-1, typically 50-80 ng/well) are bound to wells of a PVC
plate e.g., in 50 .mu.l PBS phosphate buffered saline. After
sufficient incubation to allow binding at room temperature,
typically one hour, the plate is washed in a PBS/Tween 20 solution,
("washing buffer"), and 200 .mu.l of block (3% BSA, 0.1.mu. lysine
in 1.times.BSB) is added to each well and allowed to incubate for 1
hour, after which the wells are washed again in washing buffer. 40
.mu.l of a sample composed of serially diluted plasma (preferably
partially purified as described above) or morphogen standard (e.g.,
OP-1) is added to wells in triplicate. Samples preferably are
diluted in PTTH (15 mM KH.sub.2PO.sub.4, 8 mM Na.sub.2PO.sub.4, 27
mM KCl, 137 mM NaCl, 0.05% Tween 20, 1 mg/ml-HSA, 0.05% NaN.sub.3,
pH 7.2). 10 .mu.l of labelled competitor antigen, preferably
100,000-500,000 cpm/sample is added (e.g., .sup.125I OP-1,
radiolabelled using standard procedures), and plates are incubated
overnight at 4.degree. C. Plates then are washed in washing buffer,
and allowed to dry. Wells are cut apart and bound labelled OP-1
counted in a standard gamma counter. The quantities of bound
labelled antigen (e.g., .sup.125I OP-1) measured in the presence
and absence of sample then are compared, the difference being
proportional to the amount of sample antigen (morphogen) present in
the sample fluid.
[0075] As a corollary assay method, immunoassays may be developed
to detect endogenous anti-morphogen antibodies, and to distinguish
between such antibodies to soluble or mature forms. Endogenous
anti-morphogen antibodies have been detected in serum, and their
level is known to increase, for example, upon implanting of an
osteogenic device in a mammal. Without being limited to a
particular theory, these antibodies may play a role in modulating
morphogen activity by modulating the level of available protein in
serum. Assays that monitor the level of endogenous antibodies in
blood or their body fluids thus can be used in diagnostic assays to
evaluate the status of a tissue, as well as to provide a means for
monitoring the efficacy of a therapy for tissue regeneration.
[0076] The currently preferred means for detecting endogenous
anti-morphogen antibodies is by means of a standard Western blot.
See, for example, Molecular Cloning: A Laboratory Manual Sambrook
et al., eds., Cold Spring Harbor Press, New York, 1989,
particularly pages 18.60-18.75, incorporated herein by reference,
for a detailed description of these assays. Purified mature or
soluble morphogen is electrophoresed on an SDS polyacrylamide gel
under oxidized or reduced conditions designed to separate the
proteins in solution, and the proteins then transferred to a
polyvinylidene difluoride microporus membrane (0.45 .mu.m pore
sizes) using standard buffers and procedures. The filter then is
incubated with the serum being tested (at various dilutions).
Antibodies bound to either the pro domain or the mature morphogen
domain are detected by means of an anti-human antibody protein,
e.g., goat anti-human Ig. Titers of the antimorphogen antibodies
can be determined by further dilution of the serum until no signal
is detected.
X. Formulations and Methods for Administering Soluble Morphogens as
Therapeutic Agents
[0077] The soluble morphogens of this invention are particularly
useful as therapeutic agents to regenerate diseased or damaged
tissue in a mammal, particularly a human.
[0078] The soluble morphogen complexes may be used to particular
advantage in regeneration of damaged or diseased lung, heart,
liver, kidney, nerve or pancreas tissue, as well as in the
transplantation and/or grafting of these tissues and bone marrow,
skin, gastrointestinal mucosa, and other living tissues.
[0079] The soluble morphogen complexes described herein may be
provided to an individual by any suitable means, preferably
directly or systemically, e.g., parenterally or orally. Where the
morphogen is to be provided directly (e.g., locally, as by
injection, to a desired tissue site), or parenterally, such as by
intravenous, subcutaneous, intramuscular, intraorbital, ophthalmic,
intraventricular, intracranial, intracapsular, intraspinal,
intracisternal, intraperitoneal, buccal, rectal, vaginal,
intranasal or by aerosol administration, the soluble morphogen
complex preferably comprises part of an aqueous solution. The
solution is physiologically acceptable so that in addition to
delivery of the desired morphogen to the patient, the solution does
not otherwise adversely affect the patient's electrolyte and volume
balance. The aqueous medium for the soluble morphogen thus may
comprise normal physiologic saline (0.9% NaCl, 0.15M), pH
7-7.4.
[0080] Soluble morphogens of this invention are readily purified
from cultured cell media into a physiological buffer, as described
above. In addition, and as described above, if desired, the soluble
complexes may be formulated with one or more additional additives,
including basic amino acids (e.g., L-arginine, lysine, betaine);
non-ionic detergents (e.g. Tween-80 or NonIdet-120) and carrier
proteins (e.g., serum albumin and casein).
[0081] Useful solutions for oral or parenteral administration may
be prepared by any of the methods well known in the pharmaceutical
art, described, for example, in Remington's Pharmaceutical
Sciences, (Gennaro, A., ed.), Mack Pub., 1990. Formulations may
include, for example, polyalkylene glycols such as polyethylene
glycol, oils of vegetable origin, hydrogenated naphthalenes, and
the like. Formulations for direct administration, in particular,
may include glycerol and other compositions of high viscosity.
Biocompatible, preferably bioresorbable polymers, including, for
example, hyaluronic acid, collagen, tricalcium phosphate,
polybutyrate, polylactide, polyglycolide and lactide/glycolide
copolymers, may be useful excipients to control the release of the
soluble morphogen in vivo.
[0082] Other potentially useful parenteral delivery systems for
these morphogens include ethylene-vinyl acetate copolymer
particles, osmotic pumps, implantable infusion systems, and
liposomes. Formulations for inhalation administration may contain
as excipients, for example, lactose, or may be aqueous solutions
containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate and deoxycholate, or oily solutions for administration
in the form of nasal drops, or as a gel to be applied
intranasally.
[0083] The soluble morphogens described herein also may be
administered orally. Oral administration of proteins as
therapeutics generally is not practiced as most proteins readily
are degraded by digestive enzymes and acids in the mammalian
digestive system before they can be absorbed into the bloodstream.
However, the mature domains of the morphogens described herein
typically are acid-stable and protease-resistant (see, for example,
U.S. Pat. No. 4,968,590.) In addition, at least one morphogen,
OP-1, has been identified, in mammary gland extract, colostrum and
milk, as well as saliva. Moreover, the OP-1 purified from mammary
gland extract is morphogenically active. For example, this protein
induces-endochondral bone formation in mammals when implanted
subcutaneously in association with a suitable matrix material,
using a standard in vivo bone assay, such as is disclosed in U.S.
Pat. No. 4,968,590. In addition, endogenous morphogen also is
detected in human serum (see above). Finally, comparative
experiments with soluble and mature morphogens in a number of
experiments defining morphogenic activity indicate that the
non-covalent association of the pro domain with the dimeric species
does not interfere with morphogenic activity. These findings
indicate that oral and parenteral administration are viable means
for administering morphogens to an individual, and that soluble
morphogens have utility in systemic administration protocols.
[0084] The soluble complexes provided herein also may be associated
with molecules capable of targeting the morphogen to a desired
tissue. For example, tetracycline and diphosphonates
(bisphosphonates) are known to bind to bone mineral, particularly
at zones of bone remodeling, when they are provided systemically in
a mammal. Accordingly, these molecules may be included as useful
agents for targeting soluble morphogens to bone tissue.
Alternatively, an antibody or other binding protein that interacts
specifically with a surface molecule on the desired target tissue
cells also may be used. Such targeting molecules further may be
covalently associated to the morphogen complex, e.g., by chemical
crosslinking, or by using standard genetic engineering means to
create, for example, an acid labile bond such as an Asp-Pro
linkage. Useful targeting molecules may be designed, for example,
using the single chain binding site technology disclosed, for
example, in U.S. Pat. No. 5,091,513.
[0085] Finally, the soluble morphogen complexes provided herein may
be administered alone or in combination with other molecules known
to have a beneficial effect on tissue morphogenesis, including
molecules capable of tissue repair and regeneration and/or
inhibiting inflammation. Examples of useful cofactors for
stimulating bone tissue growth in osteoporotic individuals, for
example, include but are not limited to, vitamin D.sub.3,
calcitonin, prostaglandins, parathyroid hormone, dexamethasone,
estrogen and IGF-I or IGF-II. Useful cofactors for nerve tissue
repair and regeneration may include nerve growth factors. Other
useful cofactors include symptom-alleviating cofactors, including
antiseptics, antibiotics, antiviral and antifungal agents and
analgesics and anesthetics.
[0086] The compounds provided herein can be formulated into
pharmaceutical compositions by admixture with pharmaceutically
acceptable nontoxic excipients and carriers. As noted above, such
compositions may be prepared for parenteral administration,
particularly in the form of liquid solutions or suspensions; for
oral administration, particularly in the form of tablets or
capsules; or intranasally, particularly in the form of powders,
nasal drops or aerosols. Where adhesion to a tissue surface is
desired the composition may include the morphogen dispersed in a
fibrinogen-thrombin composition or other bioadhesive such as is
disclosed, for example in PCT US91/09275, the disclosure of which
is incorporated herein by reference. The composition then may be
painted, sprayed or otherwise applied to the desired tissue
surface.
[0087] The compositions can be formulated for parenteral or oral
administration to humans or other mammals in therapeutically
effective amounts, e.g., amounts which provide appropriate
concentrations of the morphogen to target tissue for a time
sufficient to induce morphogenesis, including particular steps
thereof, as described above.
[0088] Where the soluble morphogen complex is to be used as part of
a transplant procedure, the morphogen may be provided to the living
tissue or organ to be transplanted prior to removal of the tissue
or organ from the donor. The morphogen may be provided to the donor
host directly, as by injection of a formulation comprising the
soluble complex into the tissue, or indirectly, e.g., by oral or
parenteral administration, using any of the means described
above.
[0089] Alternatively or, in addition, once removed from the donor,
the organ or living tissue may be placed in a preservation solution
containing the morphogen. In addition, the recipient also
preferably is provided with the morphogen just prior to, or
concomitant with, transplantation. In all cases, the soluble
complex may be administered directly to the tissue at risk, as by
injection to the tissue, or it may be provided systemically, either
by oral or parenteral administration, using any of the methods and
formulations described herein and/or known in the art.
[0090] Where the morphogen comprises part of a tissue or organ
preservation solution, any commercially available preservation
solution may be used to advantage. A useful preservation solution
is described in U.S. Ser. No. 07/938,337, filed Aug. 28, 1992, and
in PCT/US92/07358, both incorporated herein by reference.
[0091] As will be appreciated by those skilled in the art, the
concentration of the compounds described in a therapeutic
composition will vary depending upon a number of factors, including
the dosage of the drug to be administered, the chemical
characteristics (e.g., hydrophobicity) of the compounds employed,
and the route of administration. The preferred dosage of drug to be
administered also is likely to depend on such variables as the type
and extent of tissue loss or defect, the overall health status of
the particular patient, the relative biological efficacy of the
compound selected, the formulation of the compound, the presence
and types of excipients in the formulation, and the route of
administration. In general terms, the compounds of this invention
may be provided in an aqueous physiological buffer solution
containing about 0.001 to 10% w/v compound for parenteral
administration. Typical dose ranges are from about 10 ng/kg to
about 1 g/kg of body weight per day; a preferred dose range is from
about 0.1 .mu.g/kg to 100 mg/kg of body weight. No obvious
morphogen-induced pathological lesions are induced when mature
morphogen (e.g., OP-1, 20 .mu.g) is administered daily to normal
growing rats for 21 consecutive days. Moreover, 10 .mu.g systemic
injections of morphogen (e.g., OP-1) injected daily for 10 days
into normal newborn mice does not produce any gross
abnormalities.
[0092] Where morphogens are administered systemically, in the
methods of the present invention, preferably a large volume loading
dose is used at the start of the treatment. The treatment then is
continued with a maintenance dose. Further administration then can
be determined by monitoring at intervals the levels of the
morphogen in the blood.
OTHER EMBODIMENTS
[0093] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The present embodiments are therefore to be considered in
all respects as illustrative and not restrictive, the scope of the
invention being indicated by the appended claims rather than by the
foregoing description, and all changes which come within the
meaning and range of equivalency of the claims are therefore
intended to be embraced therein.
Sequence CWU 1
1
* * * * *