Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W094/t0203 21 q 7598 PCT/US93/10520
OP-3-INDUCED MORPHOGENESIS
Field of the Invention
This invention relates generally to the field of
tissue morphogenesis and more particularly to a novel
protein that induces tissue morphogenesis in mammals.
sackqround of the Invention
Cell differentiation is the central characteristic
of morphogenesis which initiates in the embryo, and
continues to various degrees throughout the life of an
organism in adult tissue repair and regeneration
mechanisms. The degree of morphogenesis in adult
tissue varies among different tissues and is related,
among other things, to the degree of cell turnover in a
given tissue. On this basis, tissues can be divided
into three broad categories: (1) tissues with static
cell populations such as nerve and skeletal muscle
where there is no cell division and most of the cells
formed during early development persist throughout
adult life; (2) tissues containing conditionally
renewing populations such as liver where there is
generally little cell division but, in response to an
appropriate stimulus, cells can divide to produce
daughters of the same differentially defined type; and
(3) tissues with permanently renewing populations
including blood, testes and stratified squamous
WO94/10203 PCT/US93/10520
2 1~7 Sg~ - 2 -
epithelia which are characterized by rapid and
continuous cell turnover in the adult. Here, the
terminally differentiated cells have a relatively short
life span and are replaced through proliferation of a
distinct subpopulation of cells, known as stem or
progenitor cells.
The cellular and molecular events which govern the
stimulus for differentiation of these cells is an area
of intensive research. In the medical field, it is
anticipated that the discovery of factor(s) which
control cell differentiation and tissue morphogenesis
will advance significantly medicine's ability to repair
and regenerate diseased or damaged mammalian tissues
and organs. Particularly useful areas include
reconstructive surgery and in the treatment of tissue
degenerative diseases including arthritis, emphysema,
osteoporosis, cardiomyopathy, cirrhosis, and
degenerative nerve diseases.
A number of different factors have been isolated in
recent years which appear to play a role in cell
differentiation. Recently, various members of the
structurally related proteins of the transforming
growth factor (TGF)-~ superfamily of proteins have been
identified as true morphogens.
This "family" of proteins, sharing substantial
amino acid sequence homology within their
morphogenically active C-terminal domains, including a
conserved six or seven cysteine skeleton, are capable
W094/t0203 PCT/US93/10520
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of inducing tissue-specific morphogenesis in a variety
of organs and tissues, including bone, cartilage,
liver, dentin, periodontal ligament, cementum, nerve
tissue and the epithelial mucosa of the
gastrointestinal tract. The proteins apparently bind
to surface receptors or otherwise contact and interact
with progenitor cells, predisposing or stimulating the
cells to proliferate and differentiate in a
morphogenically permissive environment. The morphogens
are capable of inducing the developmental cascade of
cellular and molecular events that culminate in the
formation of new organ-specific tissue, including any
vascularization, connective tissue formation, and nerve
ennervation as required by the naturally occurring
tissue.
Among the proteins useful in tissue morphogenesis
are proteins originally identified as bone inductive
proteins, such as the OP-l, (also referred to in
related applications as "OPl"), OP-2 (also referred to
in related applications as "OP2"), and the CBMP2
proteins, as well as amino acid sequence-related
proteins such as BMP5, BMP6 and its murine homolog,
Vgr-l, DPP and 60A (from Drosophila), Vgl (from
Xenopus), and GDF-l (from mouse) see, for example, U.S.
Patent No. 5,011, 691 to Oppermann et al., Lee (1991!
PNAS 88: 4250-4254, and Wharton et al. (1991) PNAS 88:
9214-9218. These TGF-~ superfamily members comprise a
distinct subfamily of proteins different from other
members of the TGF-~ superfamily in that the family of
morphogenic proteins are able to induce the full
WO94/10203 PCT/US93/10520
. t . . ,
~. .,
cascade of events that result in tissue morphogenesis,
including stimulating cell proliferation and cell
differentiation of progenitor cells, and supporting the
growth and mainten~ce of differentiated cells. The
morphogenic proteins apparently can act as endocrine,
paracrine or autocrine factors. Specifically, the
endogenous morphogens may be synthesized by the cells
on which they act, by neighboring cells, or by cells of
a distant tissue, the secreted protein being
transported to the cells to be acted on. In addition,
the family of morphogenic proteins induce true tissue
morphogenesis, rather than inducing formation of
fibrotic (scar) tissue as, for example, TGF-~ does.
The morphogens are synthesized in the cell as a
precursor molecule approximately three times larger
than the mature protein that is processed to yield
mature disulfide-linked dimers comprising the
C-terminal domain of the precursor sequence. The
proteins are inactive when reduced e.g., in monomeric
form, but are active as oxidized homodimeric species
as well as when oxidized in combination with other
morphogens under conditions to produce heterodimers.
The proteins useful in tissue morphogenesis typically
require a suitable environment enabling cells to
migrate, proliferate and differentiate in a tissue-
specific m~nner into, e.g., cartilage-producing
chondroblasts, bone-producing osteoblasts, hemopoietic
cells, or liver cells, depending on the nature of the
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local environment. The proliferation and
differentiation of cells induced by the morphogenic
proteins requires a suitable local environment,
including a suitable substratum on which the cells can
anchor. The proliferating and differentiating cells
also require the presence of appropriate signals to
direct their tissue-specificity, such as cell surface
markers.
It is an object of this invention to provide a
novel purified morphogenic protein, ~'OP-3", including
the amino acid sequence defining it and nucleic acids
encoding it, including allelic, species, chimeric, and
other amino acid sequence variants thereof, whether
naturally occurring or biosynthetically constructed,
and methods for utilizing the protein to induce the
developmental cascade of tissue morphogenesis for a
variety of tissues in m~mm~l5. The morphogenic
properties of OP-3 include the ability to induce
proliferation and differentiation of progenitor cells,
and the ability to support and maintain the
differentiated phenotype through the progression of
events that results in the formation of adult tissue.
Another object is to provide methods for the expression
and isolation of morphogenically active species of OP-3
using recombinant DNA techniques. Yet another object
is to provide generic sequences defining useful
morphogens. Still another object is to provide tissue-
specific acellular matrices that may be used in
combination with OP-3, and methods for their
WO94/10203 PCT/US93/10520
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preparation. Other objects include utilizing OP-3 in a
variety of applications including methods for
increasing a progenitor cell population in a mammal;
methods for stimulating progenitor cells to
differentiate in vivo or in vitro and to maintain their
differentiated phenotype; methods for inducing tissue-
specific growth in vivo, and methods for the
replacement of diseased or damaged tissue in vivo.
These and other objects and features of the invention
will be apparent from`the description, drawings, and
claims which follow.
WO94/10203 PCT/US93/10520
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Summary of the Invention
A novel substantially pure genetic sequence
encoding a novel substantially pure protein referred to
herein as "OP-3" now has been discovered. This novel
protein is a member of the morphogenic protein family
previously described by Applicants (see, for example,
US92/01968 (WO92/15323), and US92/07432 (WO93/05751).
Accordingly, the invention provides methods for
utilizing OP-3 to induce the developmental cascade of
tissue morphogenesis in a mammal. Specifically,
methods are provided for utilizing OP-3 to induce the
proliferation of uncommitted progenitor cells, to
induce the differentiation of these stimulated
progenitor cells in a tissue-specific manner under
appropriate environmental conditions, and to support
the growth and maintenAnce of these differentiated
cells. The protein also may be used to stimulate the
"redifferentiation" of cells that have strayed from
their differentiated phenotypes. Accordingly, OP-3 can
be utilized to initiate and maintain the developmental
cascade of tissue morphogenesis in an appropriate,
morphogenically permissive environment.
As used herein, useful OP-3 morphogens include
proteins encoded by the DNA sequence provided in Seg.
ID No. 1 ("mOP-3") and allelic and species variants
thereof, as well as other naturally-occurring and
biosynthetic amino acid sequence variants, including
chimeric proteins, that are morphogenically active as
WO94/10203 PCT/US93/10520
2147~i9~ `
defined herein. "Morphogenically active fragment" is
understood to include all proteins and protein
fragments encoded by part or all of the sequence of
Seq. ID No. l and which have morphogenic activity as
defined herein. Specifically, as defined herein, a
morphogen is a dimeric protein comprising a pair of
polypeptide chains, wherein each polypeptide chain
comprises at least the C-terminal six cysteine skeleton
defined by residues 303 to 399 of Seq. ID No. l (or
residues 335-431 of OPl, Seq. ID no. 3), including
functionally equivalent arrangements of these cysteines
(e.g., amino acid insertions or deletions which alter
the linear arrangement of the cysteines in the sequence
but not their relationship in the folded structure),
such that, when the polypeptide chains are folded, the
dimeric protein species comprising the pair of
polypeptide chains has the appropriate three-
dimensional structure, including the appropriate intra-
or inter-chain disulfide bonds such that the protein is
capable of acting as a morphogen as defined herein.
Specifically, the morphogens generally are capable of
all of the following biological functions in a
morphogenically permissive environment: stimulating
proliferation of progenitor cells; stimulating the
differentiation of progenitor cells; stimulating the
proliferation of differentiated cells; and supporting
the growth and maintenAnce of differentiated cells.
In one aspect, the morphogens of this invention
comprise a morphogenically active dimeric species
comprising a pair of polypeptide chains, wherein at
WO94/10203 PCT/US93/10520
21 ~ 7~98
least one of the polypeptide ChA; nS comprises the amino
acid sequence defined by residues 303 to 399 of Seq. ID
No. 1 including allelic, species and other amino acid
sequence variants thereof. In preferred morphogens, at
least one polypeptide chain comprises the sequence
defined by residues 298-399, residues 261-399 or
residues 264-399 of Seq. ID No. 1. Alternatively, the
amino acid sequence of both polypeptide chains may be
defined by part or all of the amino acid sequence of
Seq. ID No. 1, including allelic, species and other
amino acid sequence variants thereof, including
naturally-occurring sequence or biosynthetically
constructed variants, and chimeric constructs as
described below. Where only one polypeptide chain is
defined by the amino acid sequence of part or all of
Seq. ID. No. 1, the other polypeptide chain preferably
comprises at least the sequence defining the C-terminal
six cysteine skeleton of any of the other known
morphogen family members, including OP-1, OP-2, CBMP2A,
CBMP2B, BMP3, BMP5, BMP6, Vgr-1, Vgl, 60A, DPP and GDF-
1, described, for example, in US92/07432 (WO93/05751),
including allelic, species and other amino acid
sequence variants thereof, including chimeric variants.
Other useful sequences include biosynthetic constructs,
such as are described in U.S. Pat. No. 5,011,691.
In still another aspect of the invention, generic
sequences are provided which accommodate the sequence
identity of useful morphogens and incorporate OP-3's
novel features.
W094/10203 2 1 4 7 5 9 8 PCT/US93/10520
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In another aspect of the invention, morphogens of
this invention comprise morphogenically active proteins
encoded by part or all of the genetic sequence listed
in Seq. ID No. l, including allelic, species and other
amino acid sequence variants thereof. In still another
aspect, the invention comprises morphogens encoded by
nucleic acids that hybridize to part or all of the pro
region of the OP-3 protein, bases 120 to 848 of Seq ID
No. 1, under stringent hybridization conditions. As
used herein, "stringent hybridization conditions" are
defined as hybridization in 40% formamide, 5 x SSPE,
5 x Denhardt's Solution, and 0.1% SDS at 37C
overnight, and washing in 0.1 x SSPE, 0.1% SDS at 50C.
In one aspect of the invention, morphogenically
active fragments of OP-3 are useful in the replacement
of diseased or damaged tissue in a mammal, including,
but not limited to, damaged lung tissue resulting from
emphysema; cirrhotic tissue, including cirrhotic kidney
or liver tissue; damaged heart or blood vessel tissue,
as may result from cardiomyopathies and/or
atherothrombotic or cardioembolic strokes; damaged
stomach and other mucosal tissues of the
gastrointestinal tract resulting from ulceric
perforations and/or their repair; damaged nerve tissue
as may result from physical injury, degenerative
diseases such as Alzheimer's disease, multiple
sclerosis, or strokes; damaged cartilage and bone
tissue as may result from metabolic bone diseases and
WO94/10203 PCT/US93/10520
- 21~7598
other bone remodeling disorders; damaged dentin,
periodontal and/or cementum tissue as may result from
disease or mechanical injury; and in the replacement of
damaged tissue as a result of inflammation and/or
chronic inflammatory disease.
As provided herein, morphogenically active
fragments of OP-3 are provided to a tissue-specific
locus in vivo, to induce the developmental cascade of
tissue morphogenesis at that site. Cells stimulated ex
vivo by contact with OP-3 also may be provided to the
tissue locus. In these cases the existing tissue
provides the necessary matrix requirements, providing a
suitable substratum or scaffold for the proliferating
and differentiating cells in a morphogenically
permissive environment, as well as providing the
necessary signals for directing the tissue-specificity
of the developing tissue. The proteins or stimulated
cells also may be combined with a formulated matrix and
implanted as a device at a locus in vivo. The
formulated matrix should be a biocompatible, preferably
biodegradable acellular matrix having the
characteristics described below. Where the necessary
signals for directing the tissue-specificity of the
developing tissue are not provided endogenously, the
matrix preferably also is tissue-specific.
In another aspect, the members of the morphogen
protein family also can control the body's cellular and
humoral inflammatory response to a foreign object or an
initial tissue injury. In many instances, the loss of
WO94/10203 PCT/US93/10520
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2ii47~i98
- 12 -
tissue function results from the tissue destructive
effects and the subsequent formation of scar tissue
associated with the body's immune/inflammatory response
to an initial or repeated injury to the tissue. The
degree of scar tissue formation generally depends on
the regenerative properties of the injured tissue, and
on the degree and type of tissue damage. Thus, in
another aspect, morphogenically active fragments of
OP-3 may be used to prevent or to substantially inhibit
the formation of scar tissue, including alleviating
immune response-mediated tissue damage, by providing
OP-3 or cells stimulated by exposure to OP-3 protein,
to a newly injured tissue locus. The OP-3 protein also
may be provided as a prophylactic, provided to a site
in anticipation of tissue injury, such as part of a
surgical or other clinical procedure likely to produce
tissue damage, and to induce an inflammatory/immune
response. In a particularly useful embodiment, OP-3
may be used as part of a transplant procedure, to
e~h~nce the tissue viability of the organ and/or tissue
to be transplanted. The morphogen may be provided to
the organ and/or tissue to be transplanted prior to
harvest, during its transport, and/or during
transplantation into the recipient host as described
below.
OP-3 also may be used to increase or regenerate a
mesenchymal progenitor or stem cell population in vitro
or in a m~mm~l~ For example, progenitor cells may be
isolated from an individual's bone marrow, stimulated
WO94/10203 21 ~ 7 PCT/US93/10520
1, ~., ~ ~ ".
- 13 -
ex vivo with morphogenic OP-3 for a time and at a
concentration sufficient to induce the cells to
proliferate, and returned to the bone marrow. Other
sources of progenitor cells that may be suitable
include biocompatible cells obtained from a cultured
cell line, stimulated in culture, and subsequently
provided to the body. Alternatively, OP-3 may be
provided by systemic (e.g., oral or parenteral)
administration, or it may be injected or otherwise
provided to a progenitor cell population in an
individual to induce its mitogenic activity in vivo.
For example, a morphogenically active fragment of OP-3
may be provided to the cells in vivo, e.g., by systemic
injection, to induce mitogenic activity. Similarly, a
particular population of hemopoietic stem cells may be
increased by exposure to OP-3, for example by perfusing
(plasmaphoresing) an individual's blood to extract the
cells of interest, stimulating these cells ex vivo, and
returning the stimulated cells to the blood.
It is anticipated that the ability to augment an
individual's progenitor cell population will enh~nce
existing methods for treating disorders resulting from
a loss or reduction of a renewable cell population
significantly. Two particularly significant
applications include the treatment of blood disorders
and diseases involving ;mr~ ired or lost immune
function.
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_
2147~98
The morphogens of this invention also can inhibit
proliferation of epithelial cell populations. The
ability to inhibit epithelial cell proliferation may be
exploited to reduce tissue damage associated with
psoriasis and dermatitis, and other inflammatory skin
diseases, as well as ulcerative diseases of the
gastrointestinal tract, such as, for example, in the
healing of ulcers, including gastric ulcers, and the
ulcerations induced in oral mucocitis and inflammatory
bowel disease. Morphogens may be used to particular
advantage as a cytoprotective agent in clinical
therapies likely to effect proliferating epithelial
populations, such as cancer radiotherapies and
chemotherapies that typically induce oral mucositis,
hair loss and/or skin disorders.
In another aspect of the invention, morphogenic
OP-3 may be used to support the growth and maintenance
of differentiated cells, inducing existing
differentiated cells to continue expressing their
phenotype. It is anticipated that this activity will
be particularly useful in the treatment of tissue
disorders where loss of function is caused by reduced
or lost metabolic function in which cells become
senescent or quiescent, such as may occur in aging
cells and/or may be manifested in osteoporosis and a
number of nerve degenerative diseases, including
Alzheimer's disease. Application of OP-3 directly to
the cells to be treated, or providing it systemically,
as by oral or parenteral administration, can stimulate
WO94/10203 2 PCT/US93/10520
~8
these cells to continue expressing their phenotype,
thereby significantly reversing the effects of the
dysfunction. In addition, a morphogenically active
fragment of OP-3 also may be used in gene therapy
protocols to stimulate the growth of quiescent cells,
thereby potentially e~h~ncing the ability of these
cells to incorporate exogenous DNA.
In yet another aspect of the invention, a
morphogenically active fragment of OP-3 also may be
used to induce "redifferentiation" of cells that have
strayed from their differentiation pathway, such as can
occur during tumorgenesis. It is anticipated that this
activity will be particularly useful in treatments to
reduce or substantially inhibit the growth of
neoplasms. The method also is anticipated to induce
the de- and/or re-differentiation of these cells. As
described supra, a morphogenically active OP-3 fragment
may be provided to the cells directly or systemically,
stimulating these cells to revert back to a morphology
and phenotype characteristic of untransformed cells.
In still another aspect of the invention, OP-3 may
be used to stimulate cell adhesion molecule (CAM)
expression levels in a cell. CAMs are molecules
defined as carrying out cell-cell interactions
necessary for tissue formation. CAMs are believed to
play a fundamental regulatory role in tissue
development, including tissue boundary formation,
WO94/10203 PCT/US93/10520
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98
embryonic induction and migration, and tissue
stabilization and regeneration. Altered CAM levels
have been implicated in a number of tissue disorders,
including congenital defects, neoplasias, and
degenerative diseases.
In particular, N-CAM expression is associated with
normal neuronal cell development and differentiation,
including retinal formation, synaptogenesis, and nerve-
muscle tissue adhesion. Inhibition of one or more ofthe N-CAM isoforms is known to prevent proper tissue
development. Altered N-CAM expression levels also are
associated with neoplasias, including neuroblastomas
(see infra), as well as with a number of neuropathies,
lS including normal pressure hydrocephalous and type II
schizophrenia. Application of the morphogen directly
to the cells to be treated, or providing the morphogen
to the mammal systemically, for example, parenterally,
or indirectly by oral A~rinistration, may be used to
induce cellular expression of one or more CAMs,
particularly N-CAMs and Ll.
CAMs also have been postulated as part of a
morphoregulatory pathway whose activity is induced by a
to date unidentified molecule (See, for example,
Edelman, G.M. (1986) Ann. Rev. Cell Biol., 2:81-116).
Without being limited to any given theory, the
morphogens described herein may act as inducers of this
pathway.
WO94/10203 PCT/US93/10520
l~8
The matrices utilized in the methods of the
invention may be derived from organ-specific tissue, or
they may be formulated synthetically. In one
embodiment of the invention, when OP-3 (or~a collection
of progenitor cells stimulated by OP-3) is provided at
a tissue-specific locus, e.g., by systemic
administration, implantation or injection at a tissue-
specific locus, the existing tissue at that locus,
whether diseased or damaged, has the capacity of acting
as a suitable matrix or scaffold for the
differentiation and proliferation of migrating
progenitor cells. Alternatively, a formulated matrix
may be provided externally together with the stimulated
progenitor cells or morphogenically active OP-3
fragment, as may be necessary when the extent of injury
sustained by the damaged tissue is large. The matrix
should be a biocompatible, suitably modified acellular
matrix having dimensions such that it allows the
differentiation and proliferation of migratory
progenitor cells, and is capable of providing a
morphogenically permissive environment. The matrix
also preferably allows cellular attachment and is
biodegradable. Where the necessary tissue-directing
signals can not be provided endogenously, the matrix
preferably also is tissue-specific.
Formulated matrices may be generated from
dehydrated organ-specific tissue prepared, for example,
- by treating the tissue with solvents to substantially
remove the intracellular, non-structural components
WO94/10203 ~i ` PCT/US93/10520
2 1 47 598 _ 18 -
from the tissue. Alternatively, the matrix may be
formulated synthetically using a biocompatible,
preferably in vivo biodegradable, structural molecule,
and may be formulated with suitable tissue-specific
cell attachment factors. The molecule may be a
naturally occurring one such as collagen, 1A~; ni n or
hyaluronic acid, or a synthetic polymer comprising, for
example, polylactic acid, polybutyric acid,
polyglycolic acid, and copolymers thereof. Currently
preferred structural polymers comprise tissue-specific
collagens. Currently preferred cell attachment factors
include glycosaminoglycans and proteoglycans. The
matrix further may be treated with an agent or agents
to increase the number of pores and micropits on its
surfaces, so as to enhAnce the influx, proliferation
and differentiation of migratory progenitor cells from
the body of the mammal.
The invention thus relates to compositions and
methods for the use of morphogenically active fragments
of OP-3, a novel species variant of the generic family
of morphogens disclosed in USSN 667,274 and USSN
752,764, as a tissue morphogen. Morphogenically active
OP-3 and protein fragments can be isolated from
naturally-occurring sources, or they may be constructed
Diosynthetically using conventional recombinant DNA
technology. Active OP-3 useful in the compositions and
methods of this invention may include forms having
varying glycosylation patterns, varying N-termini and
active truncated forms, e.g., produced by recombinant
WO94/10203 PCT/US93/10520
21~ 7~9~
-- 19 --
DNA techn;ques. Active OP-3 proteins also include
chimeric constructs as described below, comprising both
an OP-3 active domain and a non-OP-3 sequence as, for
example, the pro domain and/or the N-terrinAl region of
the mature protein. OP-3 protein can be expressed from
intact or truncated cDNA or from synthetic DNAs in
procaryotic or eucaryotic host cells, and purified,
cleaved, refolded, and dimerized to form
morphogenically active compositions. Useful host cells
include procaryotes, including E. coli, and eucaryotic
cells, including mammalian cells, such as CHO, COS,
melanoma or BSC cells, or the insect/baculovirus
system. Thus recombinant DNA techniques may be
utilized to produce large quantities of OP-3 capable of
inducing tissue-specific cell differentiation and
tissue morphogenesis in a variety of mammals, including
humans.
Brief Description of the Drawinqs
Figure 1 is a nucleotide sequence comparison of the
mouse cDNA sequence of OP-2 and OP-3. Exon boundaries
are indicated by bars beneath the sequence; diamonds
indicate nucleotide differences within exons 2 and 3;
and
Figure 2 is an immunoblot comparing mammalian cell
expression of an OPl/OP3 chimeric protein construct
(lanes 4-8) with that of authentic, recombinant OPl
(lane 1~.
WO94/10203 PCT/US93/10520
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Detailed Description
The invention provides a novel genetic sequence,
mOP-3, encoding a novel protein, OP-3, having
morphogenic properties. The genetic sequence
originally was identified in a mouse cDNA library, and
the invention provides methods for identifying and
isolating the gene from other species. As will be
appreciated by those skilled in the art, the methods
described herein also may be used to isolate the OP-3
gene from other libraries, including genomic libraries.
The invention also provides means for producing the
OP-3 genetic sequence and the encoded protein. The
invention further provides methods and compositions for
inducing the developmental cascade of tissue
morphogenesis in a mammal utilizing morphogenically
active fragments of OP-3. The methods and compositions
provided herein may be utilized in a range of
applications, including stimulating the proliferation
and/or differentiation of progenitor cells and inducing
the repair and regeneration of damaged tissue. The
morphogenic OP-3 species of the invention are novel
members of the family of morphogens disclosed in
US92/01968 (WO92/15323) and US92/07432 (WO93/05751).
As described herein, OP-3 may be isolated from natural
sources or constructed biosynthetically utilizing
conventional recombinant DNA technology or constructed
synthetically using st~n~rd chemical techniques.
Morphogenically active fragments of OP-3 are useful
for initiating and maintaining the tissue-specific
developmental cascade in a variety of tissues,
WO94/10203 PCT/US93/10520
21~7$98 ' ~ .,
including, but not limited to, bone, cartilage, dentin,
neural tissue, liver, periodontal ligament, cementum,
lung, heart, kidney and numerous tissues of the
gastrointestinal tract. When combined with naive
mesenchymal progenitor cells as disclosed herein, OP-3
can induce the proliferation and differentiation of
these progenitor cells. In the presence of appropriate
tissue-specific signals to direct the differentiation
of these cells, and a morphogenically permissive
environment, OP-3 is capable of reproducing the cascade
of cellular and molecular events that occur during
embryonic development to yield functional tissue. For
example, the protein can induce the de novo formation
of cartilage and endochondral bone, including inducing
the proliferation and differentiation of progenitor
cells into chondrocytes and osteoblasts, inducing
appropriate mineralization and bone remodeling,
inducing formation of an appropriate bone tissue
vascular supply and inducing formation of
differentiated bone marrow (see Example 7 below.)
Provided below is a detailed description of the
nucleic acid and amino acid sequences which describe
OP-3 proteins useful in the compositions and methods of
this invention, including a description of how to make
them, and methods and means for their therapeutic
~;n; stration. Also provided are numerous,
nonlimiting examples which (l) illustrate the
- suitability of these proteins as tissue morphogens and
therapeutic agents, and (2) provide assays with which
to test the morphogens encompassed by the invention in
WO94/10203 PCT/US93/10520
59~ ` st
different tissues. Also provided in Example 9 is a
method for screening compounds to identify morphogen
stimulating agents capable of stimulating endogenous
OP-3 expression and/or secretion. OP-3 stimulating
agents then may be used in any of the therapeutic
applications described herein in place of, or in
addition to, OP-3 protein administration.
I. Useful Morphogens
As defined herein a protein is morphogenic if it is
capable of inducing the developmental cascade of
cellular and molecular events that culminate in the
formation of new, organ-specific tissue and comprises
at least the conserved C-terminal six cysteine skeleton
or its functional equivalent (see supra).
Specifically, the morphogens generally are capable of
all of the following biological functions in a
morphogenically permissive environment: stimulating
proliferation of progenitor cells; stimulating the
differentiation of progenitor cells; stimulating the
proliferation of differentiated cells; and supporting
the growth and maintenAnce of differentiated cells.
Details of how the morphogen family of proteins
described herein first were identified, as well as a
description of how to make, use and test them for
morphogenic activity are disclosed, for example, in
international application US92/01968 (WO92/15323). As
disclosed therein, the morphogens may be purified from
naturally-sourced material or recombinantly produced
WO94/10203 PCT/US93/10520
21g7~8 ,,
from procaryotic or eucaryotic host cells, preferably
as described therein. Alternatively, novel morphogenic
sequences may be identified following the procedures
disclosed therein.
Particularly useful morphogens identified to date
include OP-1, OP-2, CBMP2A and CBMP2B (the
morphogenically active domains of proteins referred to
in the art as BMP2A and BMP2B, or BMP2 and BMP4,
respectively), BMP3, BMP5, BMP6, Vgr-1, GDF-1, Vgl, DPP
and 60A, including their allelic and species variants,
as well as other amino acid sequence variants,
including chimeric morphogens. Morphogenically active
biosynthetic constructs such as those disclosed in U.S.
Pat. No. 5,011,691, (e.g., COP-l, COP-3, COP-4, COP-5,
COP-7, and COP-16) also are envisioned to be useful.
The novel morphogen OP-3 and its genetic sequence
now have been identified. The OP-3 proteins useful in
the invention include any morphogenically active
fragment of the OP-3 amino acid sequence present in
Seq. ID No. 1, or allelic, species or other amino acid
sequence variants thereof. The morphogenically active
fragment of OP-3 also may include any morphogenically
active protein encoded by part or all of the nucleic
acid sequence presented in Seq. ID No. 1. The
morphogenic protein also may comprise a protein encoded
by part or all of a nucleic acid which hybridizes to at
least part of the nucleic acid sequence encoding the
"pro" region of the OP-3 protein, e.g., bases 120-848
of Seq. ID No. 1, under stringent conditions.
WO94/10203 PCT/US93/10520
2~4'l59~ - ~
- 24 -
The mOP-3 gene encodes a protein ("mOP-3") first
expressed as an immature translation product that is
399 amino acids in length. This precursor form,
referred to herein as the "prepro" form, (Seq. ID.
No. 1, amino acid residues 1-399) includes an
N-terminal signal peptide sequence, typically less than
about 20 residues, followed by a "pro" domain that is
cleaved to yield the mature sequence. The "pro" form of
the protein includes the pro domain and the mature
domain, and forms a soluble species that appears to be
the primary form secreted from cultured mammalian
cells. The signal peptide, anticipated to include
residues 1-17 for mOP3, 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
preferred form of morphogenically active OP-3 protein
comprises a processed sequence, including fragments
thereof, appropriately dimerized and disulfide bonded.
Where a soluble form of the protein is desired, the
protein preferably comprises both the mature domain, or
an active portion thereof, and part or all of the pro
domain.
By amino acid sequence homology with other, known
morphogens, the pro domain likely is cleaved at
residues 257-260 of Seq. ID No. 1, which represent the
canonical Arg-Xaa-Xaa-Arg cleavage site, to yield a
mature sequence 139 amino acids in length (Seq. ID
No. 1, residues 261-399). Alternatively, the pro
domain may be cleaved at residues 260-263 to yield a
WO94/10203 PCT/US93/10520
21 ~ 7598
shorter sequence 135 amino acids in length (Seq. ID
No. 1, amino acid residues 264-399). All morphogens,
including OP-l, OP-2 and the OP-3 proteins disclosed
herein, comprise at least a conserved six cysteine
skeleton in the amino acid sequence C-terminal domain
and, preferably, a conserved seven cysteine skeleton
(see, for example, US92/01968 (WO92/15323). The
conserved six cysteine skeleton in mOP-3 (Seq. ID No.
1) is defined by amino acid residues 303-399; the
conserved seven cysteine skeleton is defined by amino
acid residues 298-399. In addition to the conserved
six cysteine skeleton found in known morphogen family
members including OP-l, OP-2, CBMP2A, CBMP2B, BMP3,
BMP5, BMP6, Vgr-l, Vgl, 60A, DPP and GDF-l, described,
for example, in PCT/US92/07432 (W093/05751), the OP-3
proteins, like the OP-2 proteins, also has one
additional cysteine residue (residue 338 of Seq. ID
No. 1 ) in the conserved C-terminal domain.
The mature sequence of OP-3 shares significant
amino acid sequence homology with the morphogens
identified to date. Specifically, the seven cysteine
fragment shows greater than 79% amino acid identity
with the correspo~ing mOP-2 and hOP-2 sequences, and
greater than 66% identity with the corresponding OP-l
sequences. Like OP-2, OP-3 has an eighth cysteine
within the seven cysteine domain (e.g., at position 338
of Seq. ID No. 1). In addition, OP-3 is unique among
WO94/10203 ~ ~ ~ PCT/US93/10520
- 26 -
the morphogens identified to date in that the residue
at position 9 in the conserved seven cysteine domain
(e.g., residue 315 of Seg. ID No. 1) is a serine,
whereas other morphogens typically have a tryptophan at
this location (see Table I below, and Table II in
PCT/US92/07358 (W093/04692), for example.)
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 correspon~;ng 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 cAn~i~Ate sequence are identical to the
corresponding amino acid in the reference sequence.
WO94/10203 PCT/US93/10520
7~598
As used herein, all homologies and identities
calculated use OP-3 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.
Thus, useful OP-3 variants include, but are not
limited to, amino acid sequences derived from Seq. ID
No. 1 and wherein the cysteine at position 338 is
replaced with another amino acid, preferably a
tyrosine, histidine, isoleucine or serine and
conservative substitutions thereof, e.g., such as
defined by Dayoff et al., Atlas of Protein Sequence and
Structure; vol. 5, Suppl. 3, pp.345-362 (M.O. Dayoff,
ed., Nat'l BioMed. Research Fdn., Washington D.C.
1979.). Still other useful OP-3 variants include
proteins wherein the serine at position 315 is replaced
with another amino acid, preferably a tryptophan and
conservative substitutions thereof.
Generic Sequence 7 (Seq. ID No. 12) and Generic
Sequence 8 (Seq. ID No. 13) disclosed below,
accommodate the homologies shared among perferred
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
WO94/10203 PCT/US93/10520
?~ 5g~ -
- 28 -
Sequence Listing and Table I below) and/or in the art,
as well as in PCT publication US 92/07358, filed August
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.
Generic Sequence 7
Leu Xaa Xaa Xaa Phe
1 5
Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa Xaa
Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala
Xaa Tyr Cys Xaa Gly Xaa Cys Xaa
WO94/10203 PCT/US93/10520
2~7S98
Xaa Pro Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Asn His Ala Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys
Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Leu Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Val Xaa Leu Xaa
Xaa Xaa Xaa Xaa Met Xaa Val Xaa
Xaa Cys Xaa Cys 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 =
WO94/10203 PCT/US93/10520
?~4rl59~
- 30 -
(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, His,
Gln, Leu or Gly); Xaa at res.23 = (Tyr, Asn or Phe);
Xaa at res.26 = (Glu, His, 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, His, Ser or Ile); Xaa at res.37
= (Met, Phe, Gly or Leu); Xaa at res.38 = (Asn, Ser or
Lys); Xaa at res.39 = (Ala, Ser, Gly or Pro); Xaa at
res.40 = (Thr, Leu or Ser); Xaa at res.44 = (Ile, Val
or Thr); Xaa at res.45 = (Val, Leu, 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 = (His, 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.S9 = (Lys, Leu or Glu); Xaa at
res.60 = (Pro, Val or Ala); Xaa at res.63 = (Ala or
WO94/10203 ~7598 PCT/US93/10520
- 31 -
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).
As described above, Generic Sequence 8 (Seq. ID No.
13) includes all of Generic Sequence 7 and in addition
includes the following sequence at its N-terminus:
Cys Xaa Xaa Xaa Xaa
l 5
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
W094/10203 PCT/US93/10520
~ 2~
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 z
(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).
Table I, set forth below, compares the C-terminal
amino acid sequences defining the seven cysteine
skeleton of human OP-l, mouse OP-l, human OP-2, mouse
OP-2, and mouse OP-3 (mOP-3, Seq. ID No. l). In the
table, the sequences are aligned essentially following
the method of Needleman et al. (1970) J. Mol. Biol.,
48:443-453, calculated using the Align Program
(DNAstar, Inc.) In the table, three dots indicates
that the amino acid in that position is the same as the
amino acid in hOP-l. Three dashes indicate that no
amino acid is present in that position, and are
included for purposes of illustrating homologies. As
is apparent from the following amino acid sequence
comparisons, significant amino acid sequence homology
exists between mouse OP-3 and mouse and human OP-l and
OP-2.
TABLE I
hOP-l Cys Lys Lys His Glu Leu Tyr Val
mOP-l ... ... ... ... ... ... ... ...
hOP-2 ... Arg Arg ......... ... ... ... ...
mOP-2 ... Arg Arg ......... ... ... ... ...
mOP-3 ... Arg Arg ......... ... ... ... ...
l 5
W O 94/10203 ~ PCT/US93/10520
1~7p
~8
hOP-l Ser Phe Arg Asp Leu Gly Trp Gln Asp
mOP-l ... ... ... ... ... ... ... ... ...
hOP-2 ... ... Gln ....... ... ... ... Leu
mOP-2 ... ... ... ... ... ... ... Leu
mOP-3 ... ... ... ... ... ... ... Leu
hOP-l Trp Ile Ile Ala Pro Glu Gly Tyr Ala
mOP-l ... ... ... ... ... ... ... ... ...
hOP-2 ... Val ........ ... ... Gln ........ ... Ser
mOP-2 ... Val ........ ... ... Gln ........ ... Ser
mOP-3 Ser Val ............ ... ... Gln ........ ... Ser
20 25
hOP-l Ala Tyr Tyr Cys Glu Gly Glu Cys Ala
mOP-l ... ... ... ... ... ... ... ... ...
hOP-2 ... ... ... ... ... ... ... ... Ser
mOP-2 ... ... ... ... ... ... ... ... ...
mOP-3 ... ... ... ... Ala ....... ... ... Ile
30 35
hOP-lPhe Pro Leu Asn Ser Tyr Het Asn Ala
mOP-l ... ... ... ... ... ... ... ... ...
hOP-2 ... ... ... Asp ...... Cys ....... . ... ...
mOP-2 ... ... ... Asp ...... Cys ....... . ... ...
mOP-3 Tyr ........ ... ... ... Cys ........ ... Ser
hOP-l Thr Asn His Ala Ile Val Gln Thr Leu
mOP-l ... ... ... ... ... ... ... ... ...
hOP-2 ... ... ... ... ... Leu .............. Ser
mOP-2 ... ... ... ... ... Leu .............. Ser
mOP-3 ... ... ... ... Thr Het ............... Ala
45 50
hOP-l Val His Phe Ile Asn Pro Glu Thr Val
mOP-l ... ... ... ... ... ... Asp ........ .........
hOP-2 ... ... Leu Met Lys ........... Asn Ala
mOP-2 ... ... Leu Het Lys ........... Asp Val
mOP-3 ... ... Leu Het Lys ........... Asp Ile Ile
W 0 94/10203 P ~ /US93/10520
21~1598 ~ _
- 34 -
hOP-l Pro Lys Pro Cys Cys Ala Pro Thr Gln
mOP-l ... ... ... ... ... ... ... ... ...
hOP-2 ... ... Ala ........ ... ... ... ... Lys
mOP-2 ... ... Ala ........ ... ... ... ... Lys
mOP-3 ... ... Val ........ ... Val ........ ... Glu
hOP-l Leu Asn Ala Ile Ser Val Leu Tyr Phe
mOP-l ... ... ... ... ... ... ... ... ...
hOP-2 ... Ser ........ Thr ....... ... ... ... Tyr
mOP-2 ... Ser ........ Thr ....... ... ... ... Tyr
mOP-3 ... Ser ........ ... ... Leu ........ ... Tyr
hOP-l Asp Asp Ser Ser Asn Val Ile Leu Lys
mOP-l ... ... ... ... ... ... ... ... ...
hOP-2 ... Ser ........ Asn ....... ... ... ... Arg
mOP-2 ... Ser ........ Asn ....... ... ... ... Arg
mOP-3 ... Arg Asn Asn ........... ... ... ... Arg
hOP-l Lys Tyr Arg Asn Met Val Val Arg
mOP-l ... ... ... ... ... ... ... ...
hOP-2 ... His ........ ... ... ... ... Lys
mOP-2 ... His ........ ... ... ... ... Lys
mOP-3 Arg Glu ......... ... ... ... ... Gln
hOP-l Ala Cys Gly Cys His
mOP-l ... ... ... ... ...
hOP-2 ... ... ... ... ...
mOP-2 ... ... ... ... ...
mOP-3 ... ... ... ... ...
100
WO94/10203 PCT/US93/10520
II. Formulations and Methods for Administering OP-3
Protein as Therapeutic Agents
II.A OP-3 Protein Considerations
The morphogens 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 morphogen preferably comprises part
of an aqueous solution. The solution is
physiologically acceptable so that in addition to
delivery of the desired morphogen to the patient, the
solution does not otherwise adversely affect the
patient's electrolyte and volume balance. The aqueous
medium for the morphogen thus may comprise normal
physiologic saline (0.9% NaCl, 0.15M), pH 7-7.4. The
aqueous solution cont~i n; ng the morphogen can be made,
for example, by dissolving the protein in 50% ethanol,
or acetonitrile cont~ining 0.1% trifluoroacetic acid
(TFA) or 0.1% HCl, or equivalent solvents. One volume
of the resultant solution then is added, for example,
to ten volumes of phosphate buffered saline (PBS),
which further may include 0.l-0.2% human serum albumin
(HSA). The resultant solution preferably is vortexed
extensively.
WO94/10203 PCT/US93/10520
9a,.-;~ _
If desired, a given morphogen may be made more
soluble by association with a suitable molecule. For
example, association of the mature dimer with the pro
domain of the morphogen increases solubility of the
protein significantly. For example,the pro form of
OP-3 comprises a species that is soluble in
physiologically buffered solutions. In fact, the
endogenous protein is thought to be transported (e.g.,
secreted and circulated) to particular tissues in this
form. This soluble form of the protein may be obtained
from the culture medium of morphogen-secreting
mammalian cells. Alternatively, a soluble species may
be formulated by complexing the mature dimer (or an
active fragment thereof) with part or all of a pro
domain. Another molecule capable of Pnh~ncing
solubility and particularly useful for oral
administrations, is casein. For example, addition of
0.2% casein increases solubility of the mature active
form of OP-l by 80%. Other components found in milk
and/or various serum proteins also may be useful.
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 Reminqton'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.
WO94/10203 PCT/US93/10520
~ 7S90
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
morphogen in vivo.
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
con~;n;ng, 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.
Alternatively, the morphogens described herein may
be administered orally. Oral administration of
proteins as therapeutics generally is not practiced as
most proteins readily are degraded by digestive enzymes
and acids in the mammalian digestive system before they
can be absorbed into the bloodstream. However, the
morphogens described herein typically are acid-stable
and protease-resistant (see, for example, U.S. Pat. No.
4,968,590.) In addition, at least one morphogen, OP-l,
has been identified in bovine mammary gland extract,
colostrum and milk, as well as saliva. Moreover, the
OP-l purified from mammary gland extract is
morphogenically active. For example, this protein
WO94/10203 PCT/US93/10520
2~ 41~9~- _
- 38 -
induces endocho~ral bone formation in mammals when
implanted subcutaneously in association with a suitable
matrix material, using a stAn~Ard 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. These findings indicate that oral and
parenteral administration are viable means for
administering morphogens to an individual. Moreover,
while the mature forms of certain morphogens described
herein typically are sparingly soluble, the morphogen
form found in milk (and mammary gland extract and
colostrum) is readily soluble, probably by association
of the mature, morphogenically active form with the pro
domain of the intact sequence and/or by association
with one or more milk compo~ents. Accordingly, the
compounds provided herein also may be associated with
molecules capable of enhAncing their solubility in
vitro or in vivo, including, for example, part or all
of a morphogen pro domain, as described below, and
casein, as described above.
The compounds 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 OP-3 to bone tissue.
Alternatively, an antibody or other binding protein
that interacts specifically with a surface molecule on
W O 94/10203 PC~r/US93/10520
- 21~7598
- 39 -
the desired target tissue cells also may be used. Such
targeting molecules further may be covalently
associated to the morphogen, e.g., by chemical
crosslinking, or by using stAn~rd 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.
As described above, the morphogen family members
share significant sequence homology in the 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
sequence may be morphogen-specific. As described
above, it also is known that the various morphogens
identified to date are differentially expressed in the
different tissues. Accordingly, without being limited
to any given theory, it is likely that, under natural
conditions in the body, selected morphogens typically
act on a given tissue. Accordingly, part or all of
morphogen-specific sequences may serve as targeting
molecules for the morphogens described herein. For
example, the pro domains may interact specifically with
one or more molecules at the target tissue to direct
the morphogen associated with the pro domain to that
tissue. Thus, another useful targeting molecule for
targeting OP-3 to bone tissue, for example, may include
part or all of a morphogen-specific sequence, such as
WO 94/10203 ~59~ PCI~/US93/10520
- 40 -
part or all of a pro domain and/or the N-terminus of
the mature protein. Particularly useful are the
morphogen-specific sequences of OP-l, BMP2 or BMP4, all
of which proteins are found naturally associated with
bone tissue (see, for example, US Pat. No. 5,011,691).
Alternatively, the morphogen-specific sequences of
GDF-1 may be used to target morphogenic OP-3 to nerve
tissue, particularly brain tissue where GDF-1 appears
to be primarily expressed (see, for example, Lee,
(1991) PNAS, 88:4250-4254. As described above, pro
forms of the proteins may be obtAine~ from the culture
medium of morphogen-secreting mammalian cells.
Alternatively, a suitable species may be formulated by
complexing the mature dimer (or an active fragment
thereof~ with part or all of a pro domain. Chimeric
OP-3 proteins comprising, for example, non-OP-3 pro
domains and/or non-OP-3 N-termini, may be synthesized
using st~n~Ard recombinant DNA methodology and/or
automated chemical nucleic acid synthesis methodology
well described in the art and as disclosed below.
Finally, the OP-3 proteins 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 cofactoIs for stimulating bone
tissue growth in osteoporotic individuals, for example,
include but are not limited to, vitamin D3, calcitonin,
prostaglAn~;ns, parathyroid hormone, dexamethasone,
estrogen and IGF-I or IGF-II. Useful cofactors for
W O 94/10203 - P(~r/US93/10520
2l~7~98
- 41 -
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.
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 ~;n;stration, 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, (W092/10567). The
composition then may be painted, sprayed or otherwise
applied to the desired tissue surface.
The compositions can be formulated for parenteral
or oral ~r;ni stration to humans or other mammals in
therapeutically effective amounts, e.g., amounts which
provide appropriate concentrations of OP-3 to target
tissue for a time sufficient to induce morphogenesis,
including particular steps thereof, as described above.
W094/10203 PCT/US93/10520
147S9'8
- 42 -
Where OP-3 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
tissue or organ from the donor. OP-3 may be provided
to the donor host directly, as by injection of a
formulation comprising OP-3 into the tissue, or
indirectly, e.g., by oral or parenteral A~mi n; stration,
using any of the means described above.
Alternatively or, in addition, once removed from
the donor, the organ or living tissue may be placed in
a preservation solution contA; n; ng oP-3. In addition,
the recipient also preferably is provided with the
morphogen just prior to, or concommitant with,
transplantation. In all cases, OP-3 may be
A~m; n; stered 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.
Where OP-3 comprises part of a tissue or organ
preservation solution, any commercially available
preservation solution may be used to advantage. For
example, useful solutions known in the art include
Collins solution, Wisconsin solution, Belzer solution,
Eurocollins solution and lactated Ringer's solution.
Generally, an organ preservation solution usually
possesses one or more of the following properties: (a)
an osmotic pressure substantially equal to that of the
inside of a mammalian cell,(solutions typically are
WO94/l0203 21~7 PCT/US93/10520
- 43 -
hyperosmolar and have K+ and/or Mg++ ions present in an
amount sufficient to produce an osmotic pressure
slightly higher than the inside of a mammalian cell);
(b) the solution typically is capable of maintaining
substantially normal ATP levels in the cells; and (c)
the solution usually allows optimum maintPnAnce of
glucose metabolism in the cells. Organ preservation
solutions also may contain anticoagulants, energy
sources such as glucose, fructose and other sugars,
metabolites, heavy metal chelators, glycerol and other
materials of high viscosity to e~hAnce survival at low
temperatures, free oxygen radical inhibiting and/or
scavenging agents and a pH indicator. A detailed
description of preservation solutions and useful
components may be found, for example, in US Patent No.
5,002,965.
OP-3 is envisioned to be useful in enhancing
viability of any organ or living tissue to be
transplanted. The morphogens may be used to particular
advantage in lung, heart, liver, kidney or pancreas
transplants, as well as in the transplantation and/or
grafting of bone marrow, skin, gastrointestinal mucosa,
and other living tissues.
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 A~in;stered, the chemical characteristics (e.g.,
WO94/10203 PCT/US93/10520
2-147~98
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
0.00l to 10% w/v compound for parenteral
A~;n; stration. Typical dose ranges are from about l0
ng/kg to about l g/kg of body weight per day; a
preferred dose range is from about 0.l ~g/kg to
l00 mg/kg of body weight. No obvious morphogen-induced
pathological lesions are induced when mature morphogen
(e.g., OP-l, 20 ~g) is AA~;n;stered daily to normal
growing rats for 21 consecutive days. Moreover, l0 ~g
systemic injections of morphogen (e.g., OP-l) injected
daily for l0 days into normal newborn mice does not
produce any gross abnormalities.
II.B Matrix Preparation
A morphogenically active fragment of OP-3 may be
implanted surgically, dispersed in a biocompatible,
preferably in vivo biodegradable matrix appropriately
modified to provide a structure or scaffold in which
the OP-3 may be dispersed and which allows the
differentiation and proliferation of migrating
WO94/10203 PCT/US93/10520
~t~ 8
- 45 -
progenitor cells. The matrix also may provide signals
capable of directing the tissue specificity of the
differentiating cells, as well as providing a
morphogenically permissive environment, being
essentially free of growth inhibiting signals.
The formulated matrix may be shaped as desired in
anticipation of surgery or may be shaped by the
physician or technician during surgery. Thus, the
material may be used in topical, subcutaneous,
intraperitoneal, or intramuscular implants to repair
tissue or to induce its growth de novo. The matrix
preferably is biodegradable in vivo, being slowly
absorbed by the body and replaced by new tissue growth,
in the shape or very nearly in the shape of the
implant. The ~atrix also may be particulate in nature.
Details of how to make and how to use the matrices
useful in this invention are disclosed below.
II.B(i) Tissue-Derived Matrices
Suitable biocompatible, in vivo biodegradable
acellular matrices may be prepared from naturally-
occurring tissue. The tissue is treated with suitableagents to substantially extract the cellular,
nonstructural components of the tissue. The agents
also should be capable of extracting any morphogenesis
inhibiting components associated with the tissue. The
resulting material is a porous, acellular matrix,
substantially depleted in nonstructurally-associated
components.
WO94/10203 PCT/US93/10520
~,~ 4~9~
- 46 -
The matrix also may be further treated with agents
that modify the matrix, increasing the number of pores
and micropits on its surfaces. Those skilled in the
art will know how to determine which agents are best
suited to the extraction of nonstructural components
for different tissues. For example, soft tissues such
as liver and lung may be thin-sectioned and exposed to
a nonpolar solvent such as, for example, 100~ ethanol,
to destroy the cellular structure of the tissue and
extract nonstructural components. The material then
may be dried and pulverized to yield nonadherent porous
particles or it may be maintained as a gel-like
solution. Structural tissues such as cartilage and
dentin where collagen is a primary proteinaceous
component may be demineralized and extracted with
guanidinium hydrochloride, essentially following the
method of Sampath et al. (1983) PNAS 80:6591-6595. For
example, pulverized and demineralized dentin is
extracted with five volumes of 4M guanidinium-HCl, 50mM
Tris-HCl, pH 7.0 for 16 hours at 4C. The suspension
then is filtered. The insoluble material that remains
is collected and used to fabricate the matrix. The
material is mostly collagenous in matter. It is devoid
of morphogenic activity. The matrix particles may
further be treated with a collagen fibril-modifying
agent that extracts potentially unwanted components
from the matrix, and alters the surface structure of
the matrix material. Useful agents include acids,
organic solvents or heated aqueous media. A detailed
description of these matrix treatments are disclosed,
for example, in U.S. Patent No. 4,975,526 and PCT
publication US90/00912, published September 7, 1990
(W090/10018).
WO94/10203 PCT/US93/10520
2~7~8~.
- 47 -
The currently most preferred agent is a heated
aqueous fibril-modifying medium such as water, to
increase the matrix particle surface area and porosity.
The currently most preferred aqueous medium is an
acidic aqueous medium having a pH of less than about
4.5, e.g., within the range of about pH 2 - pH 4 which
may help to "swell~ the collagen before heating. 0.1
acetic acid, which has a pH of about 3, currently is
most preferred. O.1 M acetic acid also may be used.
Various amounts of delipidated, demineralized
guanidine-extracted bone collagen are heated in the
aqueous medium (lg matrix/30ml aqueous medium) under
constant stirring in a water jacketed glass flask, and
maintained at a given temperature for a predetermined
period of time. Preferred treatment times are about
one hour, although exposure times of between about 0.5
to two hours appear acceptable. The temperature
employed is held constant at a temperature within the
range of about 37C to 65C. The currently preferred
heat treatment temperature is within the range of about
45C to 60C.
After the heat treatment, the matrix is filtered,
washed, lyophilized and used for implant. Where an
acidic aqueous medium is used, the matrix also is
preferably neutralized prior to washing and
lyophilization. A currently preferred neutralization
buffer is a 200mM sodium phosphate buffer, pH 7Ø To
neutralize the matrix, the matrix preferably first is
. ,
WO94/10203 PCT/US93/10520
~j98
- 48 -
allowed to cool following thermal treatment, the acidic
aqueous medium (e.g., 0.1% acetic acid) then is removed
and replaced with the neutralization buffer and the
matrix agitated for about 30 minutes. The
neutralization buffer then may be removed and the
matrix washed and lyophilized.
Other useful fibril-modifying treatments include
acid treatments (e.g., trifluoroacetic acid and
hydrogen fluoride) and solvent treatments such as
dichloromethane, acetonitrile, isopropanol and
chloroform, as well as particular acid/solvent
combinations.
After contact with the fibril-modifying agent, the
treated matrix may be washed to remove any extracted
components, following a form of the procedure set forth
below:
1. Suspend matrix preparation in TBS (Tris-
buffered saline) lg/200 ml and stir at 4C for 2 hrs;
or in 6 M urea, 50 mM Tris-HCl, 500 mM NaCl, pH 7.0
(UTBS) or water and stir at room temperature (RT) for
30 minutes (sufficient time to neutralize the pH);
2. Centrifuge and repeat wash step; and
3. Centrifuge; discard supernatant; water wash
residue; and then lyophilize.
WO94/10203 I q 7598 PCT/US93/10520
- 49 -
Alternatively, suitable matrix materials may be
obtA; nP~ commercially. For example, an extracellular
matrix extract such as MatrigelTM, (Collaborative
Research, Inc., Bedford) derived from mouse sarcoma
cells, may be used to advantage.
II.B(ii) Synthetic Matrices
In addition to the naturally-derived tissue-
specific matrices described above, useful tissue-
specific matrices may be formulated synthetically.
These porous biocompatible, in vivo biodegradable
synthetic matrices are disclosed in PCT publication
US91/03603, published December 12, 1991 (WO91/18558).
Briefly, the matrix comprises a porous crosslinked
structural polymer of biocompatible, biodegradable
collagen and appropriate, tissue-specific
glycosaminoglycans as tissue-specific cell attachment
factors. Collagen derived from a number of sources may
be suitable for use in these synthetic matrices,
including insoluble collagen, acid-soluble collagen,
collagen soluble in neutral or basic aqueous solutions,
as well as those collagens which are commercially
available.
Glycosaminoglycans (GAGs) or mucopolysaccharides
are hexosamine-containinq polysaccharides of ~n i ~1
origin that have a tissue specific distribution, and
therefore may be used to help determine the tissue
WO94/10203 PCT/US93/10520
9Q~ _
- 50 -
specificity of the morphogen-stimulated differentiating
cells. Reaction with the GAGs also provides collagen
with another valuable property, i.e., inability to
provoke an immune reaction (foreign body reaction) from
an animal host.
Chemically, GAGs are made up of residues of
hexosamines glycosidically bound and alternating in a
more-or-less regular manner with either hexouronic acid
or hexose moieties (see~ e.g., Dodgson et al. in
Carbohydrate Metabolism and its Disorders (Dickens et
al., eds.) Vol. 1, Academic Press (1968)). Useful GAGs
include hyaluronic acid, heparin, heparin sulfate,
chondroitin 6-sulfate, chondroitin 4-sulfate, dermatan
sulfate, and keratin sulfate. Other GAGs are suitable
for forming the matrix described herein, and those
skilled in the art will either know or be able to
ascertain other suitable GAGs using no more than
routine experimentation. For a more detailed
description of mucopolysaccharides, see Aspinall,
Polysaccharides, Pergamon Press, Oxford (1970). For
example, as disclosed in U.S. Application Serial
No. 529,852, chondroitin-6-sulfate can be used where
endochondral bone formation is desired. Heparin
sulfate, on the other hand, may be used to formulate
synthetic matrices for use in lung tissue repair.
Collagen can be reacted with a GAG in aqueous
acidic solutions, preferably in diluted acetic acid
solutions. By adding the GAG dropwise into the aqueous
WO94/10203 PCT/US93/10520
`~ 1 ~ 7~98
collagen dispersion, coprecipitates of tangled collagen
fibrils coated with GAG results. This tangled mass of
fibers then can be homogenized to form a homogeneous
dispersion of fine fibers and then filtered and dried.
Insolubility of the collagen-GAG products can be
raised to the desired degree by covalently cross-
linking these materials, which also serves to raise the
resistance to resorption of these materials. In
general, any covalent cross-linking method suitable for
cross-linking collagen also is suitable for cross-
linking these composite materials, although
crosslinking by a dehydrothermal process is preferred.
When dry, the crosslinked particles are essentially
spherical, with diameters of about 500 ym. Scanning
electron miscroscopy shows pores of about 20 ym on the
surface and 40 ym on the interior. The interior is
made up of both fibrous and sheet-like structures,
providing surfaces for cell attachment. The voids
interconnect, providing access to the cells throughout
the interior of the particle. The material appears to
be roughly 99.5% void volume, making the material very
efficient in terms of the potential cell mass that can
be grown per gram of microcarrier.
Another useful synthetic matrix is one formulated
from biocompatible, in vivo biodegradable synthetic
polymers, such as those composed of glycolic acid,
lactic acid and/or butyric acid, including copolymers
WO94/10203 PCT/US93/10520
,,; .~ . .
?.'~ 41 S9~
- 52 -
and derivatives thereof. These polymers are well
described in the art and are available commercially.
For example, polymers composed of polyactic acid (e.g.,
MW lO0 kDa), 80% polylactide/20~ glycoside or poly
3-hydroxybutyric acid (e.g., MW 30 kDa) all may be
purchased from PolySciences, Inc. The polymer
compositions generally are obtained in particulate
form. In addition, one can alter the morphology of the
polymer compositions, for example to increase porosity,
using any of a number of particular solvent treatments
known in the art. Where the morphogen is adsorbed to
the matrix surface, the steps preferably are performed
under conditions which avoid hydrolysis of the polymers
(e.g., non-aqueous conditions such as in an ethanol-
trifluoro-acetic acid solution).
The OP-3 proteins described herein can be combined
and dispersed in a suitable matrix using any of the
methods described below:
l. Ethanol Precipitation
Matrix is added to the morphogen dissolved in
guanidine-HCl. Samples are vortexed and incubated at a
low temperature. Samples are then further vortexed.
Cold absolute ethanol is added to the mixture which is
then stirred and incubated. After centrifugation
(microfuge, high speed) the supernatant is discarded.
The matrix is washed with cold concentrated ethanol in
water and then lyophilized.
WO94/10203 PCT/US93/10520
- 21 ~ 7598
.
- 53 -
2. Acetonitrile Trifluoroacetic Acid Lyophilization
In this procedure, a morphogenically active
fragment of OP-3 in an acetonitrile trifluroacetic acid
(ACN/TFA) solution is added to the carrier material.
Samples are vigorously vortexed many times and then
lyophilized.
3. Buffered Saline Lyophilization
A preparation of a morphogenically active fragment
of OP-3 in physiological saline also may be vortexed
with the matrix and lyophilized to produce
morphogenically active material.
Tissue morphogenesis requires a morphogenically
permissive environment. Clearly, in fully-functioning
healthy tissue that is not composed of a permanently
renewing cell population, there must exist signals to
prevent continued tissue growth. Thus, it is
postulated that there exists a control mechanism, such
as a feedback control mechanism, which regulates the
control of cell growth and differentiation. In fact,
it is known that both TGF-~, and MIS are capable of
inhibiting cell growth when present at appropriate
concentrations. In addition, using the bone model
system it can be shown that osteogenic devices
comprising a bone-derived carrier (matrix) that has
been demineralized and guanidine-extracted to
substantially remove the noncollagenous proteins does
WO94/10203 PCT/US93/10520
~L~4~''g~ _
allow endochondral bone formation when implanted in
association with an osteoinductive morphogen. If,
however, the bone-derived carrier is not demineralized
but rather is washed only in low salt, for example,
induction of endochondral bone formation is inhibited,
suggesting the presence of one or more inhibiting
factors within the carrier.
III. Examples
Example 1. Recombinant Production of OP-3
OP-3 proteins useful in the methods and
compositions of this invention may be purified from
natural sources or produced using st~nd~rd recombinant
methodology. General considerations for the
recombinant production of OP3 morphogens are described
below.
A. Identification of Novel mOP-3 Sequences
A genetic sequence encoding the morphogenic OP-3
protein was identified using a 0.3 kb EcoRI-BamHl OP-2
fragment from a mouse OP-2 cDNA as a hybridization
probe, specific to the mid-pro region of OP-2
(corresponding to amino acid residues 125 to 225 of the
pre-pro protein) essentially as described in
USSN 667,274. The 3 2 P-labeled probe was prepared using
the random he~ cleotide priming method, and the
WO94/10203 PCT/US93/lOS20
21q75
hybridizations were performed using the following
conditions: 40% formamide, 5 X SSPE, 5 X Denhardt's
Solution, 0.1~ SDS, at 37C overnight, and washing in
0.1 X SSPE, 0.1% SDS at 50C. Approximately 1 X 106
phages from a mouse cDNA (carried in lambda zapII)
library made from the teratocarcinoma cell line PCC4
(Stratagene, Inc., La Jolla, CA, cat # 936301) were
screened. This screening yielded four individual
clones which were purified over three rounds of
screening. The plasmid DNA containing the cDNAs was
obtained using the lambda zapII excision process
following manufacturer's directions. Three of the four
clones were shown by DNA sequencing to encode OP-3.
The DNA sequence, referred to herein as mOP-3 and
described in Seq. ID No. 1, was identified by this
procedure.
The isolated mOP-3 DNA sequence, in accordance with
other known morphogens, encodes a protein comprising a
"pro" region (defined essentially by residues 20-260 or
20-263 of Seq. ID No. 1) and a mature region (defined
essentially by residues 261-399 or 264-399 of Seq. ID
No. 1), including a functional domain comprising the
conserved cysteine skeleton.
Like OP-2, OP-3 is marked by an eighth cysteine
within the seven cysteine domain (e.g., at position 338
of Seq. ID No.l). The extra cysteine likely helps
stabilize the folded structure, possibly by providing
inter-molecular disulfide bonding. The extra cysteine
WO94/10203 PCT/US93/10520
- . _
j9~
- 56 -
also allows for heterodimer formation between OP-3 and
another morphogen comprising the "eighth" cysteine,
like OP-2 for example, or a modified OP-l, wherein an
extra cysteine has been inserted at the appropriate
location. The extra cysteine also may allow tetramer
formation. The extra cysteine does not inhibit
synthesis or reduce the stability of the translated
sequence significantly as expressed proteins comprising
the extra cysteine are readily detected by SDS gel
electrophoresis. A primary glycosylation site occurs
just C terminal to the extra cysteine in both OP-2 and
OP-3, which may provide a protective effect.
The cDNA sequences for both human and mouse OP-2
are provided in Seq. ID Nos. 7 and 9, and the genomic
sequence for human OP-2 is provided in Seq. ID No. 11,
wherein the exons defining the coding region of these
proteins are indicated. The exon boundaries also are
indicated in Fig. 1, described below. The human OP-2
locus was isolated from a genomic library (Clontech,
EMBL-3 #HL1067J) on three overlapping phage clones,
using stAn~Ard cloning procedures. The OP-2 coding
information was spread over 27 kb and, like OP-l,
contains 7 exons. A comparison of exon-intron
boundaries in the 7 cysteine domain showed matching
locations with those of OP-l. The first OP-2 exon
contains 334 bp of coding sequence (111 amino acids),
including the signal peptide, and is followed by the
largest intron (14.6 kb). The second exon (190 bp,
64 amino acids) is separated by a short intron (0.4 kb)
W094/10203 PCT/US93/10520
- 21~7598
- 57 -
from exon 3 (149 bp, 49 amino acids). It follows a
large third intron of 9.5 kb. The fourth exon (195 bp,
65 amino acids) encodes the maturation site
("OP-2-Ala") and is followed by a 0.8 kb intron. The
7 cysteine domains resides on exons 5 to 7: exon 5
(80 bp, 27 amino acids) encodes the first cysteine of
mature OP-2 and is followed by intron 5 (0.5 kb in
length), exon 6 (111 bp, 37 amino acids) is separated
by a 2.5 kb intron from the seventh, last exon with
147 bp (49 amino acids) of coding sequence. As stated
above, the exon-intron boundaries are conserved between
human OP-l and OP-2, two different members of the
morphogen family of proteins. By analogy, the exon-
intron boundaries between human and mouse OP-2, two
species variants of a morphogen, are anticipated to be
conserved as well.
Figure 1 shows the alignment of the murine OP-2 and
murine OP-3 coding regions of the cDNA. The exon
boundaries are indicated by bars beneath the sequence.
Both sequences have the same number of nucleotides.
The nucleotide sequence is about 80% conserved in the
N-terminal and C-terminal regions. In the figure,
nucleotide identity between the sequences is indicated
by stippling. In addition, the central region of the
sequence is highly conserved and this conserved region
falls into the boundaries of exon 2 and 3. There are
only three nucleotide differences in this region,
indicated in the figure by diamonds.
WO94/10203 PCT/US93/10520
- 58 -
The high degree of conservation in the nucleotide
sequences indicates that OP-2 and OP-3 likely share the
nucleotide sequence of exon 2 and 3. The different
proteins may result from alternatively spliced
transcripts, or they may arise from independent genes
which share part of their coding sequence. Intron 1,
which lies upstream of exon 2 in OP-2 (see Seq. ID
No.ll) is large (14.6kb) and could include the star~ of
the OP-3 gene and/or its first exon sequence.
Certainly, as has been found for other mammalian genes,
one or more of the introns of these morphogens may
include sequences having a transcription regulatory
function.
Using the screening procedure described herein and
in USSN 752,764, and the labelled OP-2 fragment, or
preferably a labelled OP-3 fragment, OP-3 genetic
sequences from other species and other libraries may be
isolated. Alternatively, or in addition, a probe to
the N-terminal region of the mature protein, or the 3'
noncoding region flanking and immediately following the
stop codon, also may be used to screen for other OP-3
species variants. These sequences vary substantially
among the morphogens and represent morphogen-specific
sequences. Mammalian cell expression of OP-3 readily
can be achieved using COS (simian kidney ATCC, CRL-
1650) or CH0 (Chinese hamster ovary) cells (e.g., CHO-
DXBII, from Lawrence Chasin, Columbia University, NY).
An exemplary protocol for mammalian cell expression is
provided below. Other useful eukaryotic cell systems
include the insect/baculovirus system or the mammalian
complement system.
wos4/1n203 ~7S9~ PCT/US93/10520
- 59 -
B. Expression of Novel OP-3 Sequences
To express the OP-3 protein, the OP-3 DNA is
subcloned into an insertion site of a suitable,
commercially available pUC-type vector (e.g., pUC-l9,
ATCC #37254, Rockville, MD), along with a suitable
promoter/e~h~ncer sequences and 3' termination
sequences. Currently preferred promoter/e~hAncer
sequences are the CMV-MIE promoter (human
cytomegalovirus major intermediate-early promoter,
preferably the intron-free or "short" form of the
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
preferably contains a selectable marker, most
preferably an amplifiable marker such as DHFR, e.g.,
under SV40 early promoter control (ATCC #37148).
Transfection, cell culturing, gene amplification and
protein expression conditions are 5~An~Ard 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),
stably transfected high expression cell lines obtained
by subcloning and evaluated by stAndArd Northern blot.
Southern blots also are used to assess the state of
integrated OP-3 sequences and the extent of their copy
number amplification.
WO94/10203 PCT/US93/10520
900
- 60 -
Chimeric OP-3 morphogens, e.g., comprising an OP-3
active domain and, for example, part or all of a pro
domain from another, different morphogen may be
constructed using st~n~Ard recombinant DNA technology
and/or an automated DNA synthesizer to construct the
desired sequence. Useful chimeras include those
wherein the non-OP-3 sequence is joined to the OP-3
sequence encoding the mature OP-3 protein, and the non-
OP-3 sequence encodes part or all of the sequence
between the signal peptide processing site and the
"Arg-Xaa-Xaa-Arg" processing sequence from at least one
morphogen. Alternatively, the non-OP-3 sequence may be
joined to an OP-3 sequence encoding, for example, the 6
or 7 cysteine skeletons, wherein the non-OP-3 sequence
includes the sequence encoding the N-terminus of the
mature protein. As will be appreciated by persons
skilled in the art, the non-OP-3 sequences may be
composed of sequences from one or morphogens and/or may
comprise novel biosynthetic sequences.
Mammalian expression of a biosynthetic gene
construct encoding a chimeric OPl-OP3 polypeptide chain
is demonstrated in the immunoblot presented in Fig. 2.
A vector carrying the construct under CMV promoter
control was transfected into CHO cells (CHO-DXBll)
using st~n~rd procedures and as described herein.
A chimeric gene was constructed by replacing the
conserved seven cysteine domain of OP-l with that of
OP-3. The resulting chimeric gene contains the entire
pre-pro-domain of human OP-1 and the region of mature
WO94/10203 PCT/US93/10520
-
~lg~7s~
OP-l between the maturation site and the first cysteine
of the conserved C-terminal seven cysteine domain,
fused to the conserved seven cysteine domain of mouse
OP-3, but with two arginine residues in place of the
native lysine residues found in OP-3 at the start of
the seven cysteine domain.
The gene fusion was accomplished by splicing the
SacI site of OP-3 (near the first cysteine of the seven
cysteine domain) with a newly created SacI site in
OP-l, created at the matching residues by silent
mutagenesis. The SacI site encodes the Glu-Leu
dipeptide in the sequence Cys-Arg-Arg-His-Glu-Leu of
OP-l and Cys-Lys-Lys-His-Glu-Leu of OP-3, respectively.
The chimeric gene was placed downstream of the CMV
(Cytomegalovirus) MIE "short~ (intron-free) promoter
and upstream of the SV40 transcriptional terminator in
a pUC vector. This plasmid was cotransfected with DNA
encoding the DHFR marker and viral trans-activating
elements (e.g., VAl, ElA) into a CHO dhfr(-) host and
subjected to Methotrexate selection and one round of
amplification at l mM Methotrexate including
subcloning. l0 ~l of "spent" culture supernatant
(3 days old) was analysed by "Western blot"
(immllnohlot), as follows.
The l0 ~l harvested medium was briefly heated with
concentrated SDS sample buffer, containing ~-mercapto
ethanol (5~) and directly analysed by electrophoresis
on a 15~ SDS- polyacrylamide gel (in the buffer system
WO94/10203 PCT/US93/10520
'~
~ - 62 -
of Laemmli) along with a set of prest~;ne~ molecular
weight st~n~Ards (Bio-rad, Richmond, CA). Proteins
were transferred from the gel to Immobilon membrane by
the "Western blot" procedure. The chimeric OP-l/OP-3
protein was detected by reaction with rabbit serum
raised against a synthetic peptide representing the
first 17 amino acids of mature OP-l, starting with
serine-threonine-glycine-serine-. Authentic
recombinant OP-l, expressed in CHO cells was included
for comparison. In the figure sample lanes were as
follows: lane l: OP-l; lanes 4, 5, 6, 7, and 8:
chimeric OP-l/OP-3; lanes 9 and l0: prestained
molecular weight st~n~rds. The apparent mobility of
the recombinant proteins, at approximately 20 kDa on
this gel, is due to glycosylation of the OP-l and OP-3
proteins which may also be the cause of the multiple
species observed.
The expressed protein then can be purified as
follows. For a typical 2L preparation of transfected
mammalian cells conditioned in 0.5% FCS, for example,
the total protein is typically about 700 mg. The
amount of OP-3 in the media, estimated by Western blot,
is between about 0.1-5.0 mg. OP-3 media then is
diluted in a low salt, physiologically buffered 6M urea
solution, and loaded onto an S-Sepharose column, which
acts as a strong cation exchanger. OP-3 binds to the
column in low salt, and serum proteins are removed.
The column subsequently is developed with an NaCl
gradient, e.g., O.lM NaCl-1.0M NaCl, in 6M urea, 20mM
HEPES, pH 7Ø Most contaminants are removed at the
start of the gradient, and OP-3 is eluted primarily at
a higher salt concentration.
W094/10203 21~ 7~ PC~/US93/l0s20
- 63 -
The sample then is loaded onto a phenyl-Sepharose
column (hydrophobic interaction chromatography). OP-3
binds phenyl-Sepharose in the presence of high
concentrations of a weak chaotropic salt (e.g., lM
(NH4)2S04 in a physiologically buffered 6M urea
solution). Once OP-3 is bound, the column is developed
with a decreasing ammonium sulfate gradient, e.g.,
0.6M-O.OM (NH4)2S04 gradient in a physiologically
buffered, 6M urea solution. Again, most contaminants
are removed at the start of the gradient, and OP-3
elutes primarily at low or no ammonium sulfate
concentrations.
The OP-3 eluted from the phenyl-Sepharose column
then is dialyzed against water, and prepared for
loading onto a reverse phase chromatography column
(e.g., C-18 HPLC), for example, by dialyzing against
30% acetonitrile, 0.1% TFA.
An alternative chromatography protocol is to
perform the S-Sepharose chromatography in the absence
of 6 M urea. The bound proteins then are eluted with
salt step elutions (e.g., O.1-0.6M NaCl). Remaining
OP-3 then can be eluted in the presence of 6M urea.
The 6M urea elution also may be used in place of the
non-urea elution to achieve maximum recovery in one
step. In addition, OP-3 may be eluted from the phenyl-
Sepharose column in 38% ethanol-0.01% TFA, thereby
eliminating the need to dialyze the eluent before
applying it to the C-18 column. Finally, multiple C-18
columns may be used (e.g., three), to further enhance
purification and concentration of the protein.
WO94/10203 PCT/US93/10520
0~
OP-3 also will bind hydroxyapatite efficiently,
typically in the absence of 6 M urea and at low
phosphate concentrations (less than 5 mM phosphate).
Bound OP-3 can be removed from the column with an
elution gradient of about .00l-0.5M step elution of
phosphate in a physiologically buffered solution.
Additionally, urea (6M) may be added during the elution
step.
Other related chromatography methods also may be
useful in purifying OP-3 from eucaryotic cell culture
systems. For example, heparin-Sepharose may be used in
combination with the S-Sepharose column.
Alternatively, immobilized metal-ion affinity
chromatography (IMAC) (e.g., CU2 or Zn ) and a
physiologically buffered phosphate solution may be used
to advantage.
C. Soluble OP3 Complexes
A currently preferred form of the OP-3 morphogen
useful in therapeutic formulations, having improved
solubility in aqueous solutions and consisting
essentially of amino acids, is a dimeric morphogenic
protein comprising at least the l00 amino acid peptide
sequence having the pattern of seven or more cysteine
residues characteristic of the morphogen family
complexed with a peptide comprising part or all of a
pro region of a member of the morphogen family, or an
WO94/10203 PCT/US93/10520
~lg~s9~
allelic, species or other sequence variant thereof.
Preferably, the dimeric morphogenic protein is
complexed with two peptides. AlSo, the dimeric
morphogenic protein preferably is noncovalently
complexed with the pro region peptide or peptides. The
pro region peptides also preferably comprise at least
the N-terminal eighteen amino acids that define the
OP-3 morphogen pro region (e.g., residues 18-35 of Seq.
ID No. 1). In a most preferred embodiment, peptides
defining substantially the full length pro region are
used.
Other soluble forms of morphogens include dimers of
the uncleaved pro forms of these proteins, 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
peptide.
As described above, useful pro domains include the
full length pro regions, as well as various truncated
forms hereof, particularly truncated forms cleaved at
proteolytic Arg-Xaa-Xaa-Arg cleavage sites. In OP-3,
possible pro sequences cleaved at Arg-Xaa-Xaa-Arg sites
include sequences defined by residues 18-260 of Seq. ID
No. 1 (anticipated full length form); or by residues
- 18-263. Accordingly, currently preferred pro sequences
are those encoding the full length form of the pro
region for OP-3 or another, known morphogen. Other pro
WO94/10203 PCT/US93/10520
. ,.; : _
~4~9 66 -
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.
As will be appreciated by those having ordinary
skill in the art, useful sequences encoding the pro
region may be obtained from genetic sequences encoding
known morphogens. Alternatively, chimeric pro regions
can be constructed from the sequences of one or more
known morphogens. Still another option is to create a
synthetic sequence variant of one or more known pro
region sequences.
In another preferred aspect, useful pro region
peptides include polypeptide chains comprising an amino
acid sequence encoded by a nucleic acid that hybridizes
under stringent conditions with a DNA or RNA sequence
encoding at least the N-terminal eighteen amino acids
of the pro region sequence for OP-3 e.g., nucleotides
120-173 of Seq. ID No. 1.
In yet another preferred aspect, useful pro region
peptides include polypeptide chains comprising an amino
acid sequence encoded by a nucleic acid that hybridizes
under stringent conditions with a DNA or RNA sequence
encoding at least the N-terminal eighteen amino acids
of the pro region sequence for OPl or OP2, e.g.,
nucleotides 136-192 and 152-211 of Seq. ID No. 3 and 7,
respectively.
WO94/10203 PCT/US93/10520
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- 67 -
C.l. Isolation of Soluble morphoqen complex from
conditioned media or body fluid
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.
Soluble OP-3 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 exchanqe 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 s~n~rd
procedures and, for example, using antibody specific
WO94/10203 PCT/US93/10520
214~5'98
- 68 -
for a the OP-3 pro domain (complexed, for example, to a
protein A-conjugated Sepharose column.) Protocols for
developing imr~1noAffinity columns are well described in
the art, (see, for example, Guide to Protein
Purification, M. Deutscher, ed., Academic Press, San
Diego, l990, particularly sections VII and XI.)
In this experiment OP-l was expressed in mammalian
CHO (chinese hamster ovary) cells as described in the
art (see, for example, international application
US90/05903 (WO9l/05802).) The CHO cell conditioned
media cont~;n;ng 0.5% FBS was initially purified using
Immobilized Metal-Ion Affinity Chromatography (IMAC).
The soluble OP-l 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-l
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-l is next
applied to an S-Sepharose cation-exchange column
equilibrated in 20 mM NaPO4 (pH 7.0) with 50 mM NaCl.
This S-Sepharose step serves to further purify and
concentrate the soluble OP-l 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
peritone~l fluid.
WO94/10203 PCT/US93/10520
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- 69 -
IMAC was performed using Chelating-Sepharose
(Pharmacia) that had been charged with three column
volumes of 0.2 M ZnSO4. 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 cont~in~ting proteins were
eluted with 35 mM imidazole (pH 7.0) in equilibration
buffer. The soluble OP-l complex then is eluted with
50 mM imidazole (pH 8.0) in 20 mM HEPES and 500 mM
NaCl.
The 50 mM imidazole eluate contA;n;ng the soluble
OP-l complex was diluted with nine volumes of 20 mM
NaP04 ( pH 7.0) and applied to an S-Sepharose
(Pharmacia) column equilibrated in 20 mM NaPO4 (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
NaPO4 (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 X 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 lO mL fractions. The apparent
molecular of the soluble OP-l was determined by
comparison to protein molecular weight stAnAArds
WO94/10203 PCT/US93/10520
;
4~1r~gS
- 70 -
(alcohol dehydrogenase (ADH, 150 kDa), bovine serum
albumin (~SA, 68 kDa), carbonic anhydrase (CA, 30 kDa)
and cytochrome C (cyt C, 12.5 kDa). The purity of the
S-200 column fractions was determined by separation on
st~n~Ard 15% polyacrylamide SDS gels stained with
coomassie blue. The identity of the mature OP-l and
the pro-domain was determined by N-terminal sequence
analysis after separation of the mature OP-l from the
pro-domain using st~n~Ard reverse phase C18 HPLC.
The soluble OP-l complex elutes with an apparent
molecular weight of 110 kDa. This agrees well with the
predicted composition of the soluble OP-l complex with
one mature OP-l 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.
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 st~nd~rd 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
st~n~rd 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 36kD, 39kDa proteins
confirmed as mature morphogen and isolated, cleaved pro
domain, respectively. N-terminal sequencing of the
WO94/10203 PCT/US93/10520
2l~7s9s
- 71 -
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. 16) and a
truncated form, (beginning at residue 48 of Seq. ID No.
16.) 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. 16, all of which are active as demonstrated by the
stAnd~rd bone induction assay.
C.2. In Vitro Soluble Morphogen Complex Formation
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
WO94/10203 PCT/US93/10520
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- 72 -
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.
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.
C3. Stability of Soluble Morphogen Complexes
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 18-35 of Seq. ID NO. 1 for
OP-3), and preferably is the full length pro region.
Residues 18-35 show sequence homology to the N-terminal
portion of other morphogens and are believed to have
particular utility in enh~ncing complex stability for
all morphogens. Other useful means for enh~ncing the
stability of soluble morphogen complexes include three
classes of additives. 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).
WO94/10203 PCT/US93/10520
~75
Useful concentrations of 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.
Example 2. Mitogenic Effect of OP-3
2.1 Mitogenic Effect of Morphogen on Rat and
Human Osteoblasts
The following example can be used to demonstrate
the ability of OP-3 to induce proliferation of
osteoblasts in vitro using the following assay. In
this and all examples involving osteoblast cultures,
rat osteoblast-enriched primary cultures preferably are
used. Although these cultures are heterogeneous in
that the individual cells are at different stages of
differentiation, the culture is believed to more
accurately reflect the metabolism and function of
osteoblasts _ vivo than osteoblast cultures obtained
from established cell lines. Unless otherwise
indicated, all chemicals referenced are stAnd~rd,
commercially available reagents, readily available from
a number of sources, including Sigma Chemical, Co., St.
Louis; Calbiochem, Corp., San Diego and Aldrich
Chemical Co., Milwaukee.
Rat osteoblast-enriched primary cultures are
prepared by sequential collagenase digestion of newborn
suture-free rat calvaria (e.g., from 1-2 day-old
animals, Long-Evans strain, Charles River Laboratories,
Wilmington, MA), following stAn~rd procedures, such as
are described, for example, in Wong et al., (1975) PNAS
WO94/10203 PCT/US93/10520
72:3167-3171. Rat osteoblast single cell suspensions
then are plated onto a multi-well plate (e.g., a
24 well plate) at a concentration of 50,000 osteoblasts
per well in alpha MEM (modified Eagle's medium, Gibco,
Inc., Long Island) containing 10% FBS (fetal bovine
serum), L-glutamine and penicillin/streptomycin. The
cells are incubated for 24 hours at 37C, at which time
the growth medium is replaced with alpha MEM contA;n;ng
1% FBS and the cells incubated for an additional
24 hours so that cells are in serum-deprived growth
medium at the time of the experiment.
The cultured cells are divided into three groups:
(1) wells which receive, for example, 0.1, 1.0, 10.0,
40 and 80.0 ng of OP-3; (2) wells which receive 0.1,
1.0, 10.0 and 40 ng of a local-acting growth factor
(e.g., TGF-~); and (3) the control group, which receive
no growth factors. The cells then are incubated for an
additional 18 hours after which the wells are pulsed
with 2~Ci/well of 3 H-thymidine and incubated for six
more hours. The excess label then is washed off with a
cold solution of 0.15 M NaCl and then 250 ~1 of 10%
tricholoracetic acid is added to each well and the
wells incubated at room temperature for 30 minutes.
The cells then are washed three times with cold
distilled water, and lysed by the addition of 250 ~1 of
1% sodium dodecyl sulfate (SDS) for a period of
30 minutes at 37C. The resulting cell lysates are
harvested using stAnA~rd means well known in the art,
and the incorporation of 3 H-thymidine into cellular DNA
determined by liquid scintillation as an indication of
mitogenic activity of the cells. In the experiment,
OP-3 is anticipated to stimulate 3 H-thymidine
incorporation into DNA, and thus promote osteoblast
WO94/10203 PCT/US93/10520
`- 21~7S98
- 75 -
cell proliferation. By contrast, the effect of TGF-
~is transient and biphasic. At high concentrations,
TGF-~ has no significant effect on osteoblast cell
proliferation.
The in vitro effect of OP-3 on osteoblast
proliferation also may be evaluated using human primary
osteoblasts (obtained from bone tissue of a normal
adult patient and prepared as described above) and on
human osteosarcoma-derived cell lines. In all cases
OP-3 is anticipated to induce cell proliferation in
accordance with the morphogen's ability to induce
endochondral bone formation (see Example 7, below).
2.2 Proqenitor Cell Stimulation
The following example demonstrates the ability of
OP-3 to stimulate the proliferation of mesenchymal
progenitor cells. Useful naive stem cells include
pluripotential stem cells, which may be isolated from
bone marrow or umbilical cord blood using conventional
methodologies, (see, for example, Faradji et al.,
(1988) Vox Sanq., 55 (3):133-138 or Broxmeyer et al.,
(1989) PNAS 86:3828-3832), as well as naive stem cells
obtained from blood. Alternatively, embryonic cells
(e.g., from a cultured mesodermal cell line) may be
useful.
Another method for obt~i~ing progenitor cells and
for determining the ability of OP-3 fragments to
stimulate cell proliferation is to capture progenitor
cells from an in vivo source. For example, a
biocompatible matrix material able to allow the influx
of migratory progenitor cells may be implanted at an in
WO94/10203 PCT/US93/10520
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vivo site long enough to allow the influx of migratory
progenitor cells. For example, a bone-derived,
guanidine-extracted matrix, formulated as disclosed for
example in Sampath et al. ((1983) PNAS 80:6591-6595),
or U.S. Patent No. 4,975,526, may be implanted into a
rat at a subcutaneous site, essentially following the
method of Sampath et al. After three days the implant
is removed, and the progenitor cells associated with
the matrix dispersed and cultured.
Progenitor cells, however obtained, then are
incubated in vitro with OP-3 under st~nd~Ard cell
culture conditions well described in the art and
described hereinabove. In the absence of external
stimuli, the progenitor cells do not, or only
m;n;~-lly, proliferate on their own in culture.
However, progenitor cells cultured in the presence of a
morphogenically active fragment of OP-3 are anticipated
to proliferate. Cell growth can be determined visually
or spectrophotometrically using stAn~Ard methods well
known in the art.
Example 3. Morphoqen-Induced Cell Differentiation
3.1 Embryonic Mesenchyme Differentiation
Morphogenically active fragments of OP-3 can be
utilized to induce cell differentiation. The ability
of OP-3 to induce cell differentiation can be
demonstrated by culturing early mesenchymal cells in
the presence of OP-3 and then studying the histology of
WO94/10203 PCT/US93/10520
1~7s98 "
- 77 -
the cultured cells by staining with tol~ ;ne blue
using st~nA~rd cell culturing and cell sta;~;~g
methodologies well described in the art. For example,
it is known that rat mesenchymal cells destined to
become mandibular bone, when separated from the
overlying epithelial cells at stage 11 and cultured in
vitro under st~nd~rd tissue culture conditions, e.g.,
in a chemically defined, serum-free medium, containing
for example, 67% DMEM (Dulbecco's modified Eagle's
medium), 22% F-12 medium, lOmM Hepes pH 7, 2mM
glutamine, 50 ~g/ml transferrin, 25 ~g/ml insulin,
trace elements, 2mg/ml bovine serum albumin coupled to
oleic acid, with HAT (0.1 mM hypoxanthine, lO~M
aminopterin, 12 ~M thymidine, will not continue to
differentiate. However, if these same cells are left
in contact with the overlying endoderm for an
additional day, at which time they become stage
12 cells, they will continue to differentiate on their
own in vitro to form chondrocytes. Further
differentiation into osteoblasts and, ultimately,
mandibular bone, requires an appropriate local
environment, e.g., a vascularized environment.
Stage 11 mesenchymal cells, cultured in vitro in
the presence of OP-3, e.g., 10-100 ng/ml, are
anticipated to continue to differentiate in vitro to
form chondrocytes just as they continue to
differentiate _ vitro if they are cultured with the
cell products harvested from the overlying endodermal
cells. This experiment may be performed with different
mesenchymal cells to demonstrate the cell
differentiation capability of OP-3 in different
tissues.
WO94/10203 PCT/US93/10520
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~41~9~ 78 -
As another example of morphogen-induced cell
differentiation, the ability of OP-3 to induce
osteoblast differentiation may be demonstrated in vitro
using primary osteoblast cultures, or osteoblast-like
cells lines, and assaying for a variety of bone cell
markers that are specific markers for the
differentiated osteoblast phenotype, e.g., alkaline
phosphatase activity, parathyroid hormone-mediated
cyclic AMP (cAMP) production, osteocalcin synthesis,
and ~nh~nced mineralization rates.
3.2 Alkaline Phosphatase Induction of
Osteoblasts by OP-3
The cultured cells in serum-free medium are
incubated with, a range of OP-3 concentrations, for
example, 0.l, l.0, l0.0, 40.0 or 80.0 ng OP-3/ml
medium; or with a similar range of TGF-~
concentrations. 72 hours after the incubation period
the cell layer is extracted with 0.5 ml of 1% Triton
X-l00. The resultant cell extract then, is
centrifuged, and l00 ~l of the extract is added to
90 ~l of paranitrosophenylphospate (PNPP)/glycerine
mixture and incubated for 30 minutes in a 37C water
bath and the reaction stopped with l00 ~l NaOH. The
samples then are run through a plate reader (e.g.,
Dynatech MR700 plate reader, and absorbance measured at
400 nm, using p-nitrophenol as a st~n~rd) to determine
the presence and amount of alkaline phosphate activity.
Protein concentrations are determined by the Biorad
method. Alkaline phosphatase activity is calculated in
units/~g protein, where l unit=l nmol p-nitrophenol
liberated/30 minutes at 37~C.
WO94/10203 2 PCT/US93/10520
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- 79 -
OP-3 alone stimulates the production of alkaline
phosphatase in osteoblasts, and thus promotes the
growth and expression of the osteoblast differentiated
phenotype.
The long term effect of OP-3 morphogen on the
production of alkaline phosphatase by rat osteoblasts
also may be demonstrated as follows.
Rat osteoblasts are prepared and cultured in multi-
well plates as described above. In this example six
sets of 24 well plates are plated with 50,000 rat
osteoblasts per well. The wells in each plate,
prepared as described above, then are divided into
three groups: (l) those which receive, for example,
l ng of OP-3 per ml of medium; (2) those which receive
40 ng of OP-3 per ml of medium; and (3) those which
received 80 ng of OP-3 per ml of medium. Each plate
then is incubated for different lengths of time:
0 hours (control time), 24 hours, 48 hours, 96 hours,
120 hours and 144 hours. After each incubation period,
the cell layer is extracted with 0.5 ml of 1% Triton X-
l00. The resultant cell extract is centrifuged, and
alkaline phosphatase activity determined as for
Example 3.l, using paranitroso-phenylphosphate (PNPP).
OP-3 stimulates the production of alkaline phosphatase
in osteoblasts in dose-~epen~e~t manner so that
increasing doses of OP-3 further increase the level of
alkaline phosphatase production, and moreover, the
OP-3-stimulated elevated levels of alkaline phosphatase
in the treated osteoblasts is anticipated to last for
an extended period of time.
WO94/10203 PCT/US93/10520
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- 80 -
3.3 OP-3 Protein Induction of PTH-Mediated cAMP.
$he effect of a OP-3 on parathyroid hormone-
mediated cANP production in rat osteoblasts in vitro
may be demonstrated as follows.
Rat osteoblasts are prepared and cultured in a
multiwell plate as described above. The cultured cells
then are divided into three groups: (1) wells which
receive, for example, 1.0, 10.0 and 40.0 nq OP-3/ml
medium); (2) wells which receive for example, TGF-~, at
similar concentration ranges; and (3) a control group
which receives no growth factors. The plate is then
incubated for another 72 hours. At the end of the
72 hours the cells are treated with medium containing
0.5% bovine serum albumin (BSA) and lmM 3-isobutyl-1-
methylxanthine for 20 minutes followed by the addition
into half of the wells of human recombinant parathyroid
hormone (hPTH, Sigma, St. Louis) at a concentration of
200 ng/ml for 10 minutes. The cell layer then is
extracted from each well with 0.5 ml of 1% Triton
X-100. The cAMP levels then are determined using a
radioimmunoassay kit (e.g., Amersham, Arlington
Heights, Illinois). OP-3 alone stimulates an increase
in the PTH-mediated cAMP response, and thus promotes
the growth and expression of the osteoblast
differentiated phenotype.
WO94/10203 PCT/US93/10520
21~7S~
3.4 OP-3 Protein Induction of Osteocalcin
Production
Osteocalcin is a bone-specific protein synthesized
by osteoblasts which plays an integral role in the rate
of bone mineralization in vivo. Circulating levels of
osteocalcin in serum are used as a marker for
osteoblast activity and bone formation in vivo.
Induction of osteocalcin synthesis in osteoblast-
enriched cultures can be used to demonstrateOP-3 morphogenic efficacy in vitro.
Rat osteoblasts are prepared and cultured in a
multi-well plate as above. In this experiment the
medium is supplemented with 10%FBS, and on day 2, cells
are fed with fresh medium supplemented with fresh l0 mM
~-glycerophosphate (Sigma, Inc.). Beginning on day 5
and twice weekly thereafter, cells are fed with a
complete mineralization medium contAining all of the
above components plus fresh L(+)-ascorbate, at a final
concentration of 50~g/ml medium. OP-3 then is added to
the wells directly, e.g., in 50% acetonitrile (or 50%
ethanol) containing 0.1% trifluoroacetic acid (TFA), at
no more than 5~1 morphogen/ml medium. Control wells
receive solvent vehicle only. The cells then are
re-fed and the conditioned medium sample diluted l:l in
stAn~Ard radioimmunoassay buffer contAining stAn~Ard
protease inhibitors and stored at -20 C until assayed
for osteocalcin. Osteocalcin synthesis is measured by
5~nAArd radioimmunoassay using a commercially
available osteocalcin-specific antibody.
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Mineralization is determined on long term cultures
(13 day) using a modified von Kossa st~;~ing technique
on fixed cell layers: cells are fixed in fresh 4%
paraformaldehyde at 23 C for lO min, following rinsing
cold 0.9% NaCl. Fixed cells then are stained for
endogenous alkaline phosphatase at pH 9.5 for lO min,
using a commercially available kit (Sigma, Inc.)
Purple st~ine~ cells then are dehydrated with methanol
and air dried. after 30 min incubation in 3% AgNO3 in
the dark, H2O-rinsed samples are exposed for 30 sec to
254 nm W light to develop the black silver-stained
phosphate nodules. Individual mineralized foci (at
least 20 ~m in size) are counted under a dissecting
microscope and expressed as nodules/culture.
OP-3 stimulates osteocalcin synthesis in osteoblast
cultures. The increased osteocalcin synthesis in
response to OP-3 is dose dependent and shows a
significant increase over the basal level after 13 days
of incubation. The enhanced osteocalcin synthesis also
can be confirmed by detecting the elevated osteocalcin
mRNA message (20-fold increase) using a rat
osteocalcin-specific probe. In addition, the increase
in osteoclacin synthesis correlates with increased
mineralization in long term osteoblast cultures as
determined by the appearance of mineral nodules. OP-3
increases the initial mineralization rate significantly
compared to untreated cultures.
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3.5 Morphogen-Induced CAM Expression
The morphogens described herein induce CAM
expression, particularly N-CAM expression, as part of
their induction of morphogenesis (see copending
USSN 922,813). CAMs are morphoregulatory molecules
identified in all tissues as an essential step in
tissue development. N-CAMs, which comprise at least 3
isoforms (N-CAM-180, N-CAM-140 and N-CAM-120, where
~180~ 140~ and "120~ indicate the apparent molecular
weights of the isoforms as measured by SDS
polyacrylamide gel electrophoresis) are expressed at
least transiently in developing tissues, and
permanently in nerve tissue. Both the N-CAM-180 and N-
CAM-140 isoforms are expressed in both developing and
adult tissue. The N-CAM-120 isoform is found only in
adult tissue. Another neural CAM is Ll.
The ability of OP-3 to stimulate CAM expression can
be demonstrated using the following protocol, using
NG108-15 cells. NG108-15 is a transformed hybrid cell
line (neuroblastoma x glioma, America Type Tissue
Culture (ATCC), Rockville, MD), exhibiting a morphology
characteristic of transformed embryonic neurons. As
described in Example 4, below, untreated NG108-15 cells
exhibit a fibroblastic, or minimally differentiated,
morphology and express only the 180 and 140 isoforms of
N-CAM normally associated with a developing cell.
Following morphogen treatment these cells exhibit a
morphology characteristic of adult neurons and express
~nh~nced levels of all three N-CAM isoforms.
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In this example, NG108-15 cells are cultured for
4 days in the presence of increasing concentrations of
OP-3 using stAn~Ard culturing procedures, and stAn~Ard
Western blots performed on whole cell extracts. N-CAM
isoforms are detected with an antibody which
crossreacts with all three isoforms, mAb H28.123,
obtained from Sigma Chemical Co., St. Louis, the
different isoforms being distinguishable by their
different mobilities on an electrophoresis gel.
Control NG108-15 cells (untreated) express both the 140
kDa and the 180 kDa isoforms, but not the 120 kDa, as
determined by western blot analyses using up to 100 ~g
of protein. Treatment of NG108-15 cells with OP-3
results in a dose-depPn~ent increase in the expression
of the 180 kDa and 140 kDa isoforms, as well as the
induction of the 120 kDa isoform induced. In addition,
OP-3-induced CAM expression correlates with cell
aggregation, as determined by histology.
Example 4. OP-3 Protein-Induced Redifferentiation of
Transformed Phenotype
The OP-3 morphogens described herein also can
induce redifferentiation of transformed cells to a
morphology characteristic of untransformed cells. The
examples provided below detail morphogen-induced
redifferentiation of a transformed human cell line of
neuronal origin (NG108-15); as well as mouse
neuroblastoma cells (NlE-115), and human embryo
carcinoma cells, to a morphology characteristic of
untransformed cells.
W094/10203 PCT/US93/10520
As described above, NG108-15 is a transformed
hybrid cell line produced by fusing neuroblastoma x
glioma cells (obtained from ATTC, Rockville, MD), and
exhibiting a morphology characteristic of transformed
embryonic neurons, e.g., having a fibroblastic
morphology. Specifically, the cells have polygonal
cell bodies, short, spike-like processes and make few
contacts with neighboring cells (see copending
USSN 922,813). Incubation of NG108-15 cells, cultured
in a chemically defined, serum-free medium, with 0.1 to
300 ng/ml of morphogen (e.g; OP-3) for four hours is
anticipated to induce an orderly, dose-dependent change
in cell morphology.
In the example, NG108-15 cells are subcultured on
poly-L-lysine coated 6 well plates. Each well contains
40-50,000 cells in 2.5 ml of chemically defined medium.
On the third day, 2.5 ~1 of morphogen (e.g., OP-3) in
60% ethanol cont~;ning 0.025% trifluoroacetic is added
to each well. Morphogenic OP-3 of varying
concentrations are tested (typically, concentration
ranges of 0-300 ng/ml are tested). The media is
changed daily with new aliquots of morphogen. OP-3 is
anticipated to induce a dose-depen~ent
redifferentiation of the transformed cells, including a
rounding of the soma, an increase in phase brightness,
extension of the short neurite processes, and other
significant changes in the cellular ultrastructure.
After several days treated cells should begin to form
epithelioid sheets that then become highly packed,
multi-layered aggregates, as determined visually by
microscopic examination.
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Moreover, morphogen-induced redifferentiation
occurs without any associated changes in DNA synthesis,
cell division, or cell viability, making it unlikely
that the morphologic changes are secondary to cell
differentiation or a toxic effect of the morphogen. In
addition, the morphogen-induced redifferentiation does
not inhibit cell division, as determined by
3 H-thymidine uptake, unlike other molecules which have
been shown to stimulate differentiation of transformed
cells, such as butyrate, DMSO, retanoic acid or
Forskolin in analogous experiments. Thus, OP-3
maintains cell stability and viability after inducing
redifferentiation.
The OP-3 morphogens described herein accordingly
provide useful therapeutic agents for the treatment of
neoplasias and neoplastic lesions of the nervous
system, particularly in the treatment of
neuroblastomas, including retinoblastomas, and gliomas.
As yet another, related example, the ability of
OP-3 to induce the "redifferentiation" of transformed
human cells may be demonstrated using the following
assay. Specifically, the effect of OP-3 on human EC
cells (embryo carcinoma cells, e.g., NTERA-Z CL.Dl,
ATCC, Rockville, MD) may be determined. In the absence
of an external stimulant, these cells can be maintained
as undifferentiated stem cells, and can be induced to
grow in serum free media (SFM). In the absence of
treatment with a morphogen, the cells proliferate
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rampantly and are anchorage-inderen~ent. In the
presence of morphogen, EC cells grow as flattened
cells, becoming anchorage dependent and forming
aggregates. In addition, growth rate is reduced
approximately 10 fold. Ultimately, the cells are
induced to differentiate. In the example, varying
concentrations of OP-3 (e.g., 0-300 ng/ml) are added
daily to cultured cells (e.g., 40-50,000 cells in
2.5 ml chemically defined medium), and the effects of
treatment determined by visual e~A~;nAtion. OP-3 is
anticipated to stimulate redifferentiation of these
cells to a morphology characteristic of untransformed
embryo cells.
Example 5. MaintPnAnce of Phenotype
Morphogenically active fragments of OP-3 also may
be used to maintain a cell's differentiated phenotype.
This application is particularly useful for inducing
the continued expression of phenotype in senescent or
quiescent cells.
5.1 In Vitro Model for Phenotypic Mainte~Ance
The phenotypic maintPnAnce capability of morphogens
is determined readily. A number of differentiated
cells become senescent or quiescent after multiple
passages in vitro under stAn~Ard tissue culture
conditions well described in the art (e.g., Culture of
Animal Cells: A ~An~Al of Basic Techniques, C.R.
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Freshney, ed., Wiley, 1987). However, if these cells
are cultivated in vitro in association with a morphogen
such as OP-3, cells are stimulated to maintain
expression of their phenotype through multiple
passages. For example, the alkaline phosphatase
activity of cultured osteoblasts, such as cultured
osteosarcoma cells and calvaria cells, is significantly
reduced after multiple passages in vitro. However, if
the cells are cultivated in the presence of OP-3,
alkaline phosphatase activity should be maintained over
extended periods of time. Similarly, phenotypic
expression of myocytes also is maintained in the
presence of a morphogen. In the experiment,
osteoblasts are cultured as described in Example 2.
The cells are divided into groups, incubated with
varying concentrations of OP-3 (e.g., 0-300 ng/ml) and
passaged multiple times (e.g., 3-5 times) using
stAn~Ard methodology. Passaged cells then are tested
for alkaline phosphatase activity, as described in
Example 3 as an indication of differentiated cell
metabolic function. Osteoblasts cultured in the
absence of OP-3 should have reduced alkaline
phosphatase activity, as compared to OP-3-treated
cells.
5.2 In Vivo Model for Phenotypic Maint~nA~ce
Phenotypic main~e~Ance capability also may be
demonstrated in vivo, using a rat model for
osteoporosis, as disclosed in international application
PCT/US92/07432 (WO93/05751). As described therein,
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Long Evans female rats (Charles River Laboratories,
Wilmington, MA) are Sham-operated (control animals) or
ovariectomized using st~ rd surgical techn; ques, to
produce an osteoporotic condition resulting from
decreased estrogen production. Shortly following
surgery, e.g., 200 days after ovariectomy, rats are
systemically provided with phosphate buffered saline
(PBS) or morphogen, (e.g., OP-3, 1-100 ~g) for 21 days
(e.g., by daily tail vein injection.) The rats then
are sacrificed and serum alkaline phosphatase levels,
serum calcium levels, and serum osteocalcin levels are
determined, using stAn~Ard methodologies as described
therein and above. Elevated levels of osteocalcin and
alkaline phosphatase should be observed in the rats
treated with an effective amount of OP-3. Moreover,
histomorphometric analysis on the tibial diasypheal
bone is anticipated to show improved bone mass in
OP-3-treated animals as compared with untreated,
ovariectomized rats. In fact, the bone mass of OP-3-
animals is anticipated to be comparable to (e.g.,approaches) that of the sham-operated (e.g.,
nonovarectomized) rats.
Example 6. Proliferation of Proqenitor Cell Populations
Progenitor cells may be stimulated to proliferate
_ vivo or ex vivo. The cells may be stimulated in
vivo by injecting or otherwise providing a sterile
preparation cont~i n i ng the morphogenically active
fragment of OP-3 into the individual. For example, the
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hemopoietic pluripotential stem cell population of an
individual may be stimulated to proliferate by
injecting or otherwise providing an appropriate
concentration of OP-3 to the individual's bone marrow.
Progenitor cells may be stimulated ex vivo by
contacting progenitor cells of the population to be
~nhAnced with a morphogenically active fragment of OP-3
under sterile conditions at a concentration and for a
time sufficient to stimulate proliferation of the
cells. Suitable concentrations and stimulation times
may be determined empirically, essentially following
the procedure described in Example 2, above. A
morphogen concentration of between about 0.1-100 ng/ml
and a stimulation period of from about 10 minutes to
about 72 hours, or, more generally, about 24 hours,
typically should be sufficient to stimulate a cell
population of about 104 to 106 cells. The stimulated
cells then are provided to the individual as, for
example, by injecting the cells to an appropriate in
vivo locus. Suitable biocompatible progenitor cells
may be obtained by any of the methods known in the art
or described here;nAhove.
Example 7. Reqeneration of Damaged or Diseased Tissue
OP-3 may be used to repair diseased or damaged
mammalian tissue. The tissue to be repaired preferably
is assessed first, and excess necrotic or interfering
scar tissue removed as needed, e.g., by ablation or by
surgical, chemical, or other methods known in the
medical arts.
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OP-3 then may be provided directly to the tissue
locus as part of a sterile, biocompatible composition,
either by surgical implantation or injection. The
morphogen also may be provided systemically, as by oral
or parenteral administration. Alternatively, a
sterile, biocompatible composition cont~;ning
progenitor cells stimulated by a morphogenically active
fragment of OP-3 may be provided to the tissue locus.
The existing tissue at the locus, whether diseased or
damaged, provides the appropriate matrix to allow the
proliferation and tissue-specific differentiation of
progenitor cells. In addition, a damaged or diseased
tissue locus, particularly one that has been further
assaulted by surgical means, provides a morphogenically
permissive environment. Systemic provision of OP-3
should be sufficient for certain applications (e.g., in
the treatment of osteoporosis and other disorders of
the bone remodeling cycle, as an example).
In some circumstances, particularly where tissue
damage is extensive, the tissue may not be capable of
providing a sufficient matrix for cell influx and
proliferation. In these instances, it may be necessary
to provide OP-3 or progenitor cells stimulated by OP-3
to the tissue locus in association with a suitable,
biocompatible, formulated matrix, prepared by any of
the means described below. The matrix preferably is in
vivo biodegradable. The matrix also may be
-- tissue-specific and/or may comprise porous particles
having dimensions within the range of 70-850~m, most
preferably 150-420~m.
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OP-3 also may be used to prevent or substantially
inhibit immune/inflammatory response-mediated tissue
damage and scar tissue formation following an injury.
OP-3 is provided to a newly injured tissue locus, to
induce tissue morphogenesis at the locus, preventing
the aggregation of migrating fibroblasts into non-
differentiated connective tissue. OP-3 preferably is
provided as a sterile pharmaceutical preparation
injected into the tissue locus within five hours of the
injury. Where an immùne/inflammatory response is
unavoidably or deliberately induced, as part of, for
example, a surgical or other aggressive clinical
therapy, OP-3 preferably is provided prophylactically
to the patient, prior to, or concomitant with, the
therapy.
Below are several examples, describing protocols
for demonstrating OP-3-induced tissue morphogenesis in
bone, liver, nerve, dentin, cementum and periodontal
tissue.
7.1 OP-3-Induced Bone Morphoqenesis
A particularly useful mammalian tissue model system
for demonstrating and evaluating the morphogenic
activity of a protein is the endochondral bone tissue
morphogenesis model known in the art and described, for
example, in U.S. Pat. No. 4,968,590. The ability to
induce endochondral bone formation includes the ability
to induce the proliferation of progenitor cells into
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21 ~ 759~
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chon~roblasts and osteoblasts, the ability to induce
cartilage matrix formation, cartilage calcification,
and bone remodeling, and the ability to induce
formation of-an appropriate vascular supply and
hematopoeitic bone marrow differentiation.
The local environment in which the morphogenic
material is placed is important for tissue
morphogenesis. As used herein, "local environment" is
understood to include the tissue structural matrix and
the environment surrounding the tissue. For example,
in addition to needing an appropriate anchoring
substratum for their proliferation, the cells
stimulated by morphogens need signals to direct the
tissue-specificity of their differentiation. These
signals vary for the different tissues and may include
cell surface markers. In addition, vascularization of
new tissue requires a local environment which supports
vascularization.
The following sets forth various procedures for
evaluating the in vivo morphogenic utility of OP-3 and
OP-3-containing compositions. The compositions may be
injected or surgically implanted in a mammal, following
any of a number of procedures well known in the art.
For example, surgical implant bioassays may be
performed essentially following the procedure of
Sampath et al. (1983) PNAS 80:6591-6595 and U.S. Pat
No. 4,968,590.
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Histological sectioning and staining is preferred
to determine the extent of morphogenesis in vivo,
particularly in tissue repair procedures. Excised
implants are fixed in Bouins Solution, embedded in
paraffin, and cut into 6-8 ~m sections. Staining with
toluidine blue or hemotoxylin/eosin demonstrates
clearly the ultimate development of the new tissue.
Twelve day implants are usually sufficient to determine
whether the implants contain newly induced tissue.
Successful implants exhibit a controlled
progression through the stages of induced tissue
development allowing one to identify and follow the
tissue-specific events that occur. For example, in
endocho~ral bone formation the stages include:
(l) leukocytes on day one; (2) mesenchymal cell
migration and proliferation on days two and three;
(3) chondrocyte appearance on days five and six;
(4) cartilage matrix formation on day seven;
(5) cartilage calcification on day eight; (6) vascular
invasion, appearance of osteoblasts, and formation of
new bone on days nine and ten; (7) appearance of
osteoclastic cells, and the commencement of bone
remodeling and dissolution of the implanted matrix on
days twelve to eighteen; and (8) hematopoietic bone
marrow differentiation in the resulting ossicles on day
twenty-one.
In addition to histological evaluation, biological
markers may be used as markers for tissue
morphogenesis. Useful markers include tissue-specific
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enzymes whose activities may be assayed (e.g.,
spectrophotometrically) after homogenization of the
implant. These assays may be useful for quantitation
and for rapidly obtaining an estimate of tissue
formation after the implants are removed from the
animal. For example, alkaline phosphatase activity may
be used as a marker for osteogenesis.
Incorporation of systemically provided OP-3 may be
followed using tagged fragments (e.g., radioactively
labelled) and determining their localization in the new
tissue, and/or by monitoring their disappearance from
the circulatory system using a stAn~Ard labeling
protocol and pulse-chase procedure. OP-3 also may be
provided with a tissue-specific molecular tag, whose
uptake may be monitored and correlated with the
concentration of OP-3 provided. As an example, ovary
removal in female rats results in reduced bone alkaline
phosphatase activity, and renders the rats predisposed
to osteoporosis (as described in Example 5). If the
female rats now are provided with OP-3, a reduction in
the systemic concentration of calcium should be seen,
which correlates with the presence of the provided OP-3
and which is anticipated to correspond with increased
alkaline phosphatase activity.
7.2 Morphogen-Induced Liver Reqeneration
As another example, a method for inducing
morphogenesis of substantially injured liver tissue
following a partial hepatectomy utilizing OP-3 is
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2147598 96 -
presented. Variations on this general protocol may be
used to test morphogen activity of OP-3 in other
different tissues. The general method involves
excising an-essentially nonregenerating portion of a
tissue and providing OP-3, preferably as a soluble
pharmaceutical preparation to the excised tissue locus,
closing the wound, and e~r;n;ng the site at a future
date. Like bone, liver has a potential to regenerate
upon injury during post-fetal life.
OP-3, e.g., l mg/ml, in a biocompatible solution,
for example, (e.g., a purified recombinant mature form
of OP-3, is solubilized in 50% ethanol, or compatible
solvent, cont~;n;ng 0.1% trifluoroacetic acid, or
compatible acid. Alternatively, the mature protein may
be solubilized by association with a pro domain. The
injectable OP-3 solution is prepared, e.g., by diluting
one volume of OP-3 solvent-acid stock solution with
9 volumes of 0.2% rat serum albumin in sterile PBS
(phosphate-buffered saline).
In the experiment, growing rats or aged rats (e.g.,
Long Evans, Charles River Laboratories, Wilmington) are
anesthetized by using ketamine. Two of the liver lobes
(left and ri~ht) are cut out (approximately l/3 of the
lobe) and the OP-3 is injected locally at multiple
sites along the cut ends. The amount of OP-3 injected
may be, e.g., lO0 ~g in lO00 ~l of PBS/RSA (phosphate
buffered saline/rat serum albumin) injection buffer.
Placebo samples are injection buffer only. In
experimental essays, five rats in each group preferably
are used. The wound is closed and the rats are allowed
to eat normal food and drink tap water.
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After 12 days, the rats are sacrificed and liver
regeneration is observed visually, to evaluate the
effects of the OP-3 on liver regeneration most
effectively. The OP-3 fragment-injected group is
anticipated to show, e.g., complete liver tissue
regeneration with no sign remaining of any cut in the
liver. sy contrast, the control group into which only
Pss is injected, show only mini~-l regeneration with
the incision remaining in the sample. Previous
experiments with other morphogens (e.g., OP-l) show
these morphogens alone induce liver tissue
regeneration.
7.3 Morphoqen-Induced Dentin, Cementum and
Periodontal Liqament Regeneration
As still another example, the ability of OP-3 to
induce dentinogenesis also may be demonstrated. To
date, the unpredictable response of dental pulp tissue
to injury is a basic clinical problem in dentistry.
Cynomolgus monkeys are chosen as primate models as
monkeys are presumed to be more indicative of human
dental biology than models based on lower non-primate
mammals.
Using stAnAArd dental surgical procedures, small
areas (e.g., 2mm) of dental pulps are surgically
exposed by removing the enamel and dentin immediately
- above the pulp (by drilling) of sample teeth,
performing a partial amputation of the coronal pulp
- tissue, inducing hemostasis, application of the pulp
treatment, and sealing and filling the cavity by
5~An~Ard procedures.
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Pulp treatments used may include: a
morphogenically active fragment of OP-3 dispersed in a
carrier matrix; carrier matrix alone, and no treatment.
Twelve teeth per animal (four for each treatment) are
prepared, and two animals are used. At four weeks,
teeth are extracted and processed histologically for
analysis of dentin formation, and/or ground to analyze
dentin mineralization. The effect of OP-3 on
osteodentin reparation may be observed visually by
comparing control samples treatment (PBS) with OP-3.
OP-3 plus a carrier matrix induces formation of
reparative or osteodentin bridges on surgically exposed
healthy dental pulps. sy contrast, pulps treated with
carrier matrix alone, do not form reparative dentin.
Similarly, implanting demineralized teeth and OP-3
into surgically prepared canine tooth sockets is
anticipated to stimulate new periodontal tissue
formation, including new cementum and periodontal
ligament, as well as new alveolar bone and dentin
tissue, as described for OP-l in international
application PCT/US92/08742, filed 9/15/93. By
contrast, untreated teeth or teeth treated with carrier
vehicle alone do not induce periodontal tissue growth.
7.4 Morphoqen-Induced Nerve Tissue Repair
As yet another example, the induction of
regenerative effects on central nervous system (CNS)
repair, by a morphogenically active fragment of OP-3,
may be demonstrated using a rat brain stab model. In
the experiment, male Long Evans rats are anesthetized
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_ 99 _
and the head area prepared for surgery. The calvariae
is exposed using st~nA~rd surgical procedures and a
hole drilled toward the center of each lobe using a
0.035K wire, just piercing the calvariae. 25~1
solutions cont~in;ng either morphogen (e.g., OP-3,
25~g) or PBS then is provided to each of the holes by
Hamilton syringe. Solutions are delivered to a depth
approximately 3 mm below the surface, into the
underlying cortex, corpus callosum and hippocampus.
The skin then is sutured and the animal allowed to
recover.
Three days post surgery, rats are sacrificed by
decapitation and their brains processed for sectioning.
Scar tissue formation is evaluated by immunofluoresence
st~;ning for glial fibrillary acidic protein, a marker
protein for glial scarring, to qualitatively determine
the degree of scar formation. Sections also are probed
with OP-3-specific antibody to determine the presence
of the protein. Reduced levels of glial fibrillary
acidic protein are anticipated to be observed in the
tissue sections of animals treated with OP-3,
evidencing the ability of the morphogen to inhibit
glial scar formation, thereby stimulating nerve
regeneration.
The ability of OP-3 to stimulate peripheral nervous
system axonal growth over extended distances may be
demonstrated using the following model. Neurons of the
peripheral nervous system can sprout new processes on
their own following injury, but without guidance these
sproutings typically fail to connect appropriately and
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die. Where the break is extensive, e.g., greater than
5 or 10 mm, regeneration is poor or nonexistent.
Previous experiments with other morphogens, e.g., OP-l,
show that morphogens stimulate peripheral nervous
system axonal growth over extended distances, allowing
repair and regeneration of damaged peripheral neural
pathways.
In this example OP-3 stimulation of nerve
regeneration is demonstrated using the rat sciatic
nerve model. The rat sciatic nerve can regenerate
spontaneously across a 5 mm gap, and occasionally
across a 10 mm gap, provided that the severed ends are
inserted in a saline-filled nerve guidance ch~nnel. In
this experiment, nerve regeneration across at least a
12mm gap is tested.
Adult female Sprague-Dawley rats (Charles River
Laboratories, Inc.) weighing 230-250 g are anesthetized
with intraperitoneal injections of sodium pentobarbital
(35 mg/kg body weight). A skin incision is made
parallel and just posterior to the femur. The
avascular intermuscular plane between vastus lateralis
and hamstring muscles are entered and followed to the
loose fibroareolar tissue surrounding the sciatic
nerve. The loose tissue is divided longitll~;n~lly
thereby freeing the sciatic nerve over its full extent
without devascularizing any portion. Under a surgical
microscope the sciatic nerves are transected with
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-- 101 --
microscissors at mid-thigh and grafted with a OP-3 gel
graft that separates the nerve stumps by 12 mm. The
graft region is encased in a silicone tube 20 mm in
length with a 1.5 mm inner diameter, the interior of
which is filled with the morphogen solution.
Specifically, the central 12 mm of the tube consists of
an OP-3 gel prepared by mixing 1 to 5 ~g of
substantially pure recombinantly produced OP-3 protein
with approximately 100 ~l of MATRIGELTM (from
Collaborative Research, Inc., Bedford, MA), an
extracellular matrix extract derived from mouse sarcoma
tissue, and containing solubilized tissue basement
membrane, including lA~in;n, type IV collagen, heparin
sulfate, proteoglycan and entactin, in phosphate-
buffered saline. The morphogen-filled tube then is
implanted directly into the defect site, allowing 4 mm
on each end to insert the nerve stumps. Each stump is
abutted against the morphogen gel and is secured in the
silicone tube by three stitches of commercially
available surgical 10-0 nylon through the epineurium,
the fascicle protective sheath.
In addition to OP-3 gel grafts, control grafts of
empty silicone tubes, silicone tubes filled with gel
only and "reverse" autografts, wherein 12 mm transected
segments of the animal~s sciatic nerve are rotated 180
prior to suturing, preferably also are grafted. All
experiments preferably are performed in quadruplicate.
All wounds preferably are closed by wound clips that
are removed after 10 days. Rats can be grafted on both
WO94/10203 PCT/US93/10520
2147S98
- 102 -
legs. At 3 weeks the animals are sacrificed, and the
grafted segments removed and frozen on dry ice
immediately. Frozen sections then are cut throughout
the graft site, and e~A~ined for axonal regeneration by
immunofluorescent staining using anti-neurofilament
antibodies labeled with flurocein (obtained, for
example, from Sigma Chemical Co., St. Louis).
Regeneration of the sciatic nerve is anticipated to
occur across the entire 12 mm distance in all graft
sites wherein the gap is filled with the OP-3 gel. By
contrast, empty silicone tubes, gel alone and reverse
autografts do not show nerve regeneration.
Example 8. Identification of Morphoqen-Expressinq
Tissue
Deter~ining the tissue distribution of morphogens
may be used to identify different morphogens expressed
in a given tissue, as well as to identify new, related
morphogens. Tissue distribution also may be used to
identify useful morphogen-producing-tissue for use in
screening and identifying cAn~i~Ate morphogen-
stimulating agents. The morphogens (or their mRNA
transcripts) readily are identified in different
tissues using stAnAArd methodologies and minor
modifications thereof in tissues where expression may
be low. For example, protein distribution may be
determined using stAn~Ard Western blot analysis or
immunofluorescent techniques, and antibodies specific
WO94/10203 PCT/US93/10520
2147598
.
~ 103 ~
to the morphogen or morphogens of interest. Similarly,
the distribution of morphogen transcripts may be
determined using st~nA~rd Northern hybridization
protocols and transcript-specific probes.
Any probe capable of hybridizing specifically to a
transcript, and distinguishing the transcript of
interest from other, related transcripts may be used.
secause the morphogens described herein share such high
sequence homology in their active, C-terminal Ao~-;ns,
the tissue distribution of a specific morphogen
transcript may best be determined using a probe
specific for the pro region of the immature protein
and/or the N-terminal region of the mature protein.
Another useful sequence is the 3' non-coding region
flanking and immediately following the stop codon.
These portions of the sequence vary substantially among
the morphogens of this invention, and accordingly, are
specific for each protein. For example, a particularly
useful OP-3-specific probe sequence is one derived from
a portion of the 3' untranslated sequence, e.g.,
nucleotides 1310-1674 of Seq. ID No. 1, which shares
little or no homology with other morphogen sequences,
including OP-2. The chosen fragment then is labelled
using st~nA~rd means well known and described in the
art.
Using these morphogen-specific probes, which may be
synthetically engineered or obtained from cloned
sequences, morphogen transcripts can be identified in
mammalian tissue, using st~nA~rd methodologies well
WO94/10203 PCT/US93/10520
21~7~98
- 104 -
known to those having ordinary skill in the art. A
detailed description of a suitable hybridization
protocol is described in Ozkaynak, et al., (1991)
Biochem. Biophys. Res. Commn. 179:116-123, and
Ozkaynak, et al. (1992) J. Biol. Chemistry
267:25220-25227. Briefly, total RNA is prepared from
various tissues (e.g., murine embryo and developing and
adult liver, kidney, testis, heart, brain, thymus,
stomach) by a st~n~Ard methodology such as by the
method of Chomczyaski et al. ((1987) Anal. Biochem
162:156-159) and described below. Poly (A)+ RNA is
prepared by using oligo (dT)-cellulose chromatography
(e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.).
Poly (A)+ RNA (generally 15 ~g) from each tissue is
fractionated on a 1% agarose/formaldehyde gel and
transferred onto a Nytran membrane (Schleicher &
Schuell). Following the transfer, the membrane is
baked at 80C and the RNA is cross-linked under W
light (generally 30 seconds at 1 mW/cm2). Prior to
hybridization, the appropriate probe is denatured by
heating. The hybridization is carried out in a lucite
cylinder rotating in a roller bottle apparatus at
approximately 1 rev/min for approximately 15 hours at
37C using a hybridization mix of 40% formamide,
5 x Denhardts, 5 x SSPE, and 0.1% SDS. Following
hybridization, the non-specific counts are washed off
the filters in 0.1 x SSPE, 0.1% SDS at 50C.
An OP-3-specific 0.5 kb probe was made from a StuI-
BglII fragment of OP-3 cDNA. The fragment contains the
3' untranslated sequence from nucleotides 1310-1674,
plus an additional 140 bases. The fragment was
WO94/10203 PCT/US93/10520
21~7~g8
- 105 -
labelled using stAn~Ard techni~ues and the
hybridization performed as described. To date, OP-3,
like OP-2, appears to be expressed primarily in early
embryonic tissue. Specifically, Northern blots of
murine embryos show abundant OP-3 expression in 8-day
embryos, demonstrated by a strong band at 2.9 kb and a
weaker band at 2.3 kb.
Example 9. Screening Assay for Candidate Compounds
which Alter Endoqenous Morphogen Levels
Candidate compound(s) which may be ~drin;stered to
affect the level of endogenous OP-3 morphogen may be
found using the following screening assay, in which the
level of OP-3 production by a cell type which produces
measurable levels of the morphogen is deter~;~e~ with
and without incubating the cell in culture with the
compound, in order to assess the effects of the
compound on the cell. This can be accomplished by
detection of the morphogen either at the protein or RNA
level. A detailed description also may be found in
international application PCT/US92/07359, (WO93/05172).
9.1 Growth of Cells in Culture
Cell cultures of kidney, adrenals, urinary bladder,
brain, or other organs, may be prepared as described
widely in the literature. For example, kidneys may be
explanted from neonatal or new born or young or adult
rodents (mouse or rat) and used in organ culture as
whole or sliced (1-4 mm) tissues. Primary tissue
WO94/10203 PCT/US93/10520
2147S98
- 106 -
cultures and established cell lines, also derived from
kidney, adrenals, urinary, bladder, brain, mammary, or
other tissues may be established in multiwell plates (6
well or 24 well) according to conventional cell culture
5 techn; ques, and are cultured in the absence or presence
of serum for a period of time (1-7 days). Cells may be
cultured, for example, in Dulbecco's Modified Eagle
medium (Gibco, Long Island, NY) containing serum (e.g.,
fetal calf serum at 1%-10%, Gibco) or in serum-deprived
medium, as desired, or in defined medium (e.g.,
containing insulin, transferrin, glucose, albumin, or
other growth factors).
Samples for testing the level of morphogen
production includes culture supernatants or cell
lysates, collected periodically and evaluated for
morphogen production by immunoblot analysis (Sambrook
et al., eds., 1989, Molecular Cloning, Cold Spring
Harbor Press, Cold Spring Harbor, NY), or a portion of
the cell culture itself, collected periodically and
used to prepare polyA+ RNA for RNA analysis. To
monitor de novo morphogen synthesis, some cultures are
labeled according to conventional procedures with an
3 5 S-methionine/3 5 S-cysteine mixture for 6-24 hours and
then evaluated for morphogenic protein synthesis by
conventional immunoprecipitation methods.
9.2 Determination of Level of Morphogenic Protein
In order to quantitate the production of a
morphogenic protein, e.g., OP-3, by a cell type, an
immunoassay may be performed to detect the morphogen
WO94/10203 PCT/US93/10520
21~759~
- 107 -
using a polyclonal or monoclonal antibody specific for
that protein. For example, OP-3 may be detected using
a polyclonal antibody specific for OP-3 in an ELISA, as
follows.
1 ~g/100 ~1 of affinity-purified polyclonal rabbit
IgG specific for OP-3 is added to each well of a
96-well plate and incubated at 37C for an hour. The
wells are washed four times with 0.167M sodium borate
buffer with 0.15 M NàCl (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 37C.
The wells are then washed four times with BSB
contA;n;ng 0.1% Tween 20. A 100 ~1 aliquot of an
appropriate dilution of each of the test samples of
cell culture supernatant is added to each well in
triplicate and incubated at 37C for 30 min. After
incubation, 100 ~1 biotinylated rabbit anti-OP-3 serum
(stock solution is about 1 mg/ml and diluted 1:400 in
BSB contA;n;ng 1~ BSA before use) is added to each well
and incubated at 37C for 30 min. The wells are then
washed four times with BSB contAin;ng 0.1~ Tween 20.
100 ~1 strepavidin-alkaline (Southern Biotechnology
Associates, Inc. Birmingham, Alabama, diluted 1:2000 in
BSB containing 0.1% Tween 20 before use) is added to
each well and incubated at 37C for 30 min. The plates
are washed four times with 0.5M Tris buffered Saline
(TBS), pH 7.2. 50~1 substrate (ELISA Amplification
System Kit, Life Technologies, Inc., Bethesda, MD) is
added to each well incubated at room temperature for 15
WO94/10203 PCT/US93/10520
2147S98
- 108 -
min. Then, 50 ~1 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 ~1 0.3 M sulphuric acid.
The OD at 490 nm of the solution in each well is
recorded. To quantitate OP-3 in culture media, an OP-3
stAn~rd curve is performed in parallel with the test
samples.
Polyclonal antibody may be prepared as follows.
Each rabbit is given a primary immunization of 100
ug/500 ~1 recombinantly-produced OP-3 protein or
protein fragment in 0.1~ SDS mixed with 500 ~1
Complete Freund's Adjuvant. The antigen is injected
subcutaneously at multiple sites on the back and flanks
of the animal. The rabbit is boosted after a month in
the same manner using incomplete Freund's Adjuvant.
Test bleeds are taken from the ear vein seven days
later. Two additional boosts and test bleeds are
performed at monthly intervals until antibody against
OP-3 is detected in the serum using an ELISA assay.
Then, the rabbit is boosted monthly with 100 ~g of
antigen and bled (15 ml per bleed) at days seven and
ten after boosting.
Monoclonal antibody specific for a given morphogen
may be prepared as follows. A mouse is given two
injections of OP-3 protein or a protein fragment
specific for OP-3. The protein preferably is
recombinantly produced. The first injection contains
100~g of OP-3 in complete Freund's adjuvant and is
given subcutaneously. The second injection contains 50
~g of OP-3 in incomplete adjuvant and is given
intraperitoneally. The mouse then receives a total of
WO94/10203 PCT/US93/10520
21 ~ 7598
-- 109 --
230 ~g of OP-3 in four intraperi~one~l injections at
various times over an eight month period. One week
prior to fusion, the mouse is boosted intraperitoneally
with OP-3 (e.g., 100 ~g) and may be additionally
boosted with an OP-3-specific peptide (e.g.,
corresponding to the N-terminus of the mature protein)
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
OP-3-specific antibodies using OP-3 as antigen. The
cell fusion and monoclonal screening steps readily are
performed according to stAn~rd procedures well
described in st~n~Ard texts widely available in the
art.
Other Embodiments
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 inte~e~ to be embraced
therein.
WO 94/10203 PCT/US93/10520
Sg8 - 110-
S~QU~NCE LISTING
(1) G~NFRAT INFORMATION:
(i) APPLICANT
(A) NAME CREATIVE BIOHOLECULES, INC.
(B) STREET 45 SOUTH STREET
( C ) CITY: HOP~ 1O1I
(D) STATE MA
(E) COUN-nY: USA
(F) POSTAL CODE (ZIP): 01748
(G) TELEPHONE: 1-508-435-9001
(H) TELEFA~ 1-508-435-0454
(I) TELE~
(ii) TITLE OF 1NV~11ON OP3-INDUCED MORPHOGENESIS
(iii) NUNBER OF SEQUENCES 13
(iV) CORRF~PONDENCE ADDRESS
(A) ADDRESSEE CREATIVE BIOMOLECULES, INC.
(B) STREET 45 SOUTH STREET
( C) CITY: HOP~1N1ON
(D) STATE MA
(E) COUN1~Y: USA
(F) ZIP: 01748
(V) CONPUTER READABLE FORM:
(A) MEDIUM m E F1OPPY disk
(B) COhrU.~: IBM PC COmPatib1e
(C) OPERATING SYSTEM: PC_DOS/MS-DOS
(D) SOFTWARE PatentIn Re1eaSe #1.0, VerSiOn #1.25
(vi) CURRENT APPLICATION DATA
(A) APPLICATION NUMBER
(B) FILING DATE
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUHBER US 07/667,274
(B) FILING DATE 11-~AR-1991
(Vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER US 07/752,764
(B) FILING DATE 30-AUG-1991
(Vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER: US 07/753,059
(B) FILING DATE 30-AUG-1991
W O 94/10203 PCr/US93/10520
- 21 ~ 7598
111
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/752,857
(B) FILING DATE: 30-AUG-1991
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NI~RF.R: US 07/923,780
(B) FILING DATE: 31-JUL-1992
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMRF.R: US 07/922,813
(B) FILING DATE: 31-JUL-1992
(viii) AllORN~:Y/AGENT INFORMATION:
(A) NAME: PITCHER ESQ, EDMUND R
(B) REGISTRATION NUMBER: 27,829
(C) REFERENCE/DOCKET NUNBER: CRP-076PC
(iX) TF~RF~coM~uNlcATIoN INFORMATION:
(A) TELEPHONE: (508)435-9001
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1674 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
30(ii) HOLECULE m E: protein
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 69... 1268
(D) OTHER INFORMATION: /note= "mOP3-PP"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
GGATCCGCGG CGCTGTCCCA TC~ lC~l CGAGGCGTCG CTGGATGCGA GTCCGCTAAA 60
CGTCCGAG ATG GCT GCG CGT CCG GGA CTC CTA TGG CTA CTG GGC CTG GCT 110
Met Ala Ala Arg Pro Gly Leu Leu Trp Leu Leu Gly Leu Ala
45 1 5 10
CTG TGC GTG TTG GGC GGC GGT CAC CTC TCG CAT CCC CCG CAC GTC m 158
Leu Cys Val Leu Gly Gly Gly His Leu Ser His Pro Pro His Val Phe
15 20 25 30
W O 94/10203 PCT/US93/10520
a~ ~7S9~ `
~
- 112 -
CCC CAG CGT CGA CTA GGA GTA CGC GAG CCC CGC GAC ATG CAG CGC GAG 206
Pro Gln Arg Arg Leu Gly Val Arg Glu Pro Arg Asp Met Gln Arg Glu
35 40 45
5 ATT CGG GAG GTG CTG GGG CTA GCC GGG CGG CCC CGA TCC CGA GCA CCG 254
Ile Arg Glu Val Leu Gly Leu Ala Gly Arg Pro Arg Ser Arg Ala Pro
50 55 60
GTC GGG GCT GCC CAG CAG CCA GCG TCT GCG CCC CTC m ATG TTG GAC 302
10 Val Gly Ala Ala Gln Gln Pro Ala Ser Ala Pro Leu Phe Met Leu Asp
65 70 75
CTG TAC CGT GCC ATG ACG GAT GAC AGT GGC GGT GGG ACC CCG CAG CCT 350
Leu Tyr Arg Ala Met Thr Asp Asp Ser Gly Gly Gly Thr Pro Gln Pro
80 85 90
CAC TTG GAC CGT GCT GAC CTG ATT ATG AGC m GTC M C ATA GTG G M 398
His Leu Asp Arg Ala Asp Leu Ile Met Ser Phe Val Asn Ile Val Glu
95 100 105 110
CGC GAC CGT ACC CTG GGC TAC CAG GAG CCA CAC TGG M G G M TTC CAC 446
Arg Asp Arg Thr Leu Gly Tyr Gln Glu Pro His Trp Lys Glu Phe His
115 120 125
25 TTT GAC CTA ACC CAG ATC CCT GCT GGG GAG GCT GTC ACA GCT GCT GAG 494
Phe Asp Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Thr Ala Ala Glu
130 135 140
TTC CGG ATC TAC MA GAA CCC AGT ACC CAC CCG CTC M C ACA ACC CTC 542
30 Phe Arg Ile Tyr Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu
145 150 155
CAC ATC AGC ATG TTC GAA GTG GTC CM GAG CAC TCC M C AGG GAG TCT 590
His Ile Ser Net Phe Glu Val Val Gln Glu His Ser Asn Arg Glu Ser
160 165 170
GAC TTG TTC m TTG GAT CTT CAG ACG CTC CGA TCT GGG GAC GAG GGC 638
Asp Leu Phe Phe Leu Asp Leu Gln Thr Leu Arg Ser Gly Asp Glu Gly
175 180 185 190
TGG CTG GTG CTG GAC ATC ACA GCA GCC AGT GAC CGA TGG CTG CTG M C 686
Trp Leu Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn
195 200 205
45 CAT CAC M G GAC CTA GGA CTC CGC CTC TAT GTG GAA ACC GAG GAT GGG 734
His His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp Gly
210 215 220
WO 94/10203 PCr/US93/10520
2147598
-- 113 --
CAC AGC ATA GAT CCT GGC CTA GCT GGT CTG CTT GGA CGA CM GCA CCA 782
His Ser Ile Asp Pro Gly Leu Ala Gly Leu Leu Gly Arg Gln Ala Pro
- 225 230 235
5 CGC TCC AGA CAG CCT TTC ATG GTT GGT TTC TTC AGG GCC MC CAG AGT 830
- Arg Ser Arg Gln Pro Phe Met Val Gly Phe Phe Arg Ala Asn Gln Ser
240 245 250
CCT GTG CGG GCC CCT CGA ACA GCA AGA CCA CTG MG MG MG CAG CTA 878
10 Pro Val Arg Ala Pro Arg Thr Ala Arg Pro Leu Lys Lys Lys Gln Leu
255 260 265 270
MT CM ATC MC CAG CTG CCG CAC TCC MC MA CAC CTA GGA ATC CTT 926
Asn Gln Ile Asn Gln Leu Pro His Ser Asn Lys His Leu Gly Ile Leu
275 280 285
GAT GAT GGC CAC GGT TCT CAC GGC AGA GM GTT TGC CGC AGG CAT GAG 974
Asp Asp Gly His Gly Ser His Gly Arg Glu Val Cys Arg Arg His Glu
290 295 300
CTC TAT GTC AGC TTC CGT GAC CTT GGC TGG CTG GAC TCT GTC ATT GCC 1022
Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Ser Val Ile Ala
305 310 315
CCC CAG GGC TAC TCC GCC TAT TAC TGT GCT GGG GAG TGC ATC TAC CCA 1070
Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys Ala Gly Glu Cys Ile Tyr Pro
320 325 330
CTG AAC TCC TGT ATG MC TCC ACC AAC CAC GCC ACT ATG CAG GCC CTG 1118
Leu Asn Ser Cys Met Asn Ser Thr Asn His Ala Thr Met Gln Ala Leu
335 340 345 350
GTA CAT CTG ATG MG CCA GAT ATC ATC CCC MG GTG TGC TGT GTG CCT 1166
Val His Leu Net Lys Pro Asp Ile Ile Pro Lys Val Cys Cys Val Pro
355 360 365
ACT GAG CTG AGT GCC ATT TCT CTG CTC TAC TAT GAT AGA MC MT MT 1214
Thr Glu Leu Ser Ala Ile Ser Leu Leu Tyr Tyr Asp Arg Asn Asn Asn
370 375 380
GTC ATC CTG CGC AGG GAG CGC MC ATG GTA GTC CAG GCC TGT GGC TGC 1262
Val Ile Leu Arg Arg Glu Arg Asn Net Val Val Gln Ala Cys Gly Cys
385 390 395
CAC TGA~lCC~,-lG CCCMCAGCC TGCTGCCATC CCATCTATCT AGTCAGGCCT1315
His
400
CTCTTCCMG GCAGGMMCC AACAAAGAGG GAAGGCAGTG CmCMCTC CATGTCCACA 1375
W O 94/10203 PCT/US93/10520
214~S98 `i
- 114 -
TTCACAGTCT lGGCCCl~lC l~ lllll GCCAAGGCTG Ar~AAGATGGT CCTAGTTATA 1435
ACC~lG~lGA CCTCAGTAGC CCGATCTCTC Al~-lCCCCAA ACTCCCCAAT GCAGCCAGGG 1495
GCATCTATGT CC m GGGAT TGGGCACAGA AGTCCAA m ACC M CTTAT TCATGAGTCA 1555
CTACTGGCCC AGCCTGGACT TGAACCTGGA ACACAGGGTA GAGCTCAGGC TCTTCAGTAT 1615
CCATCAGAAG A m AGGTGT GTGCArAr-AT GACCACACTC CCCCTAGCAC TCCATAGCC 1674
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 399 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ala Ala Arg Pro Gly Leu Leu Trp Leu Leu Gly Leu Ala Leu Cys
1 5 10 15
Val Leu Gly Gly Gly His Leu Ser His Pro Pro His Val Phe Pro Gln
20 25 30
Arg Arg Leu Gly Val Arg Glu Pro Arg Asp Met Gln Arg Glu Ile Arg
35 40 45
Glu Val Leu Gly Leu Ala Gly Arg Pro Arg Ser Arg Ala Pro Val Gly
Ala Ala Gln Gln Pro Ala Ser Ala Pro Leu Phe Net Leu Asp Leu Tyr
65 70 75 80
Arg Ala Met Thr Asp Asp Ser Gly Gly Gly Thr Pro Gln Pro His Leu
85 90 95
Asp Arg Ala Asp Leu Ile Net Ser Phe Val Asn Ile Val Glu Arg Asp
100 105 110
Arg Thr Leu Gly Tyr Gln Glu Pro His Trp Lys Glu Phe His Phe Asp
115 120 125
Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg
130 135 140
WO 94/10203 PCI/US93/10520
-- 2I ~ 7S9:8` --
-- 115 --
Ile Tyr Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu His Ile
145 150 155 160
Ser Met Phe Glu Val Val Gln Glu His Ser Asn Arg Glu Ser Asp Leu
165 170 175
Phe Phe Leu Asp Leu Gln Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu
180 185 190
10 Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His
195 200 205
Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp Gly His Ser
210 215 220
Ile Asp Pro Gly Leu Ala Gly Leu Leu Gly Arg Gln Ala Pro Arg Ser
225 230 235 240
Arg Gln Pro Phe Met Val Gly Phe Phe Arg Ala Asn Gln Ser Pro Val
245 250 255
Arg Ala Pro Arg Thr Ala Arg Pro Leu Lys Lys Lys Gln Leu Asn Gln
260 265 270
25 Ile Asn Gln Leu Pro His Ser Asn Lys His Leu Gly Ile Leu Asp Asp
275 280 285
Gly His Gly Ser His Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr
290 295 300
Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Ser Val Ile Ala Pro Gln
305 310 315 320
Gly Tyr Ser Ala Tyr Tyr Cys Ala Gly Glu Cys Ile Tyr Pro Leu Asn
325 330 335
Ser Cys llet Asn Ser Thr Asn His Ala Thr Het Gln Ala Leu Val His
340 345 350
40 Leu Met Lys Pro Asp Ile Ile Pro Lys Val Cys Cys Val Pro Thr Glu
355 360 365
Leu Ser Ala Ile Ser Leu Leu Tyr Tyr Asp Arg Asn Asn Asn Val Ile
370 375 380
Leu Arg Arg Glu Arg Asn Met Val Val Gln Ala Cys Gly Cys His
385 390 395
W 0 94/10203 P ~ /US93/10520
.:
2147~9~ `
- 116 -
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQu~ CHARACTERISTICS:
(A) LENGTH: 1822 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) oRrANTsH: HOMO SAPIENS
(F) TISSUE l~rE: HIPPOCAMPUS
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 49..1341
(C) IDENTIFICATION METHOD: experimental
(D) OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN"
/product= nhOP1-PP"
/note= "hOPl cDNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
30 GGTGCGGGCC CGGAGCCCGG AGCCCGGGTA GCGCGTAGAG CCGGCGCG ATG CAC GTG 57
Met His Val
CGC TCA CTG CGA GCT GCG GCG CCG CAC AGC TTC GTG GCG CTC TGG GCA 105
Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala
CCC CTG TTC CTG CTG CGC TCC GCC CTG GCC GAC TTC AGC CTG GAC AAC 153
Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn
4020 25 30 35
GAG GTG CAC TCG AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG 201
Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg
40 45 50
CGG GAG ATG CAG CGC GAG ATC CTC TCC ATT TTG GGC TTG CCC CAC CGC 249
Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg
WO 94/10203 PCI/US93/10520
2147~98
-- 117 --
CCG CGC CCG CAC CTC CAG GGC MG CAC MC TCG GCA CCC ATG TTC ATG 297
Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Het Phe net
70 75 80
5 CTG GAC CTG TAC AAC GCC ATG GCG GTG GAG GAG GGC GGC GGG CCC GGC 345
Leu Asp Leu Tyr Asn Ala net Ala Val Glu Glu Gly Gly Gly Pro Gly
85 90 95
GGC CAG GGC TTC TCC TAC CCC TAC AAG GCC GTC TTC AGT ACC CAG GGC 393
10 Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly
100 105 110 115
CCC CCT CTG GCC AGC CTG CM GAT AGC CAT TTC CTC ACC GAC GCC GAC 441
Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp
120 125 130
ATG GTC ATG AGC TTC GTC MC CTC GTG GAA CAT GAC MG GM TTC TTC 489
Het Val net Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe
135 140 145
CAC CCA CGC TAC CAC CAT CGA GAG TTC CGG m GAT CTT TCC MG ATC 537
His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile
150 155 160
25 CCA GAA GGG GAA GCT GTC ACG GCA GCC GAA TTC CGG ATC TAC MG GAC 585
Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp
165 170 175
TAC ATC CGG GM CGC TTC GAC MT GAG ACG TTC CGG ATC AGC GTT TAT 633
30 Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile Ser Val Tyr
180 185 190 195
CAG GTG CTC CAG GAG CAC TTG GGC AGG GAA TCG GAT CTC TTC CTG CTC 681
Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu Phe Leu Leu
200 205 210
GAC AGC CGT ACC CTC TGG GCC TCG GAG GAG GGC TGG CTG GTG m GAC 729
Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp
215 220 225
ATC ACA GCC ACC AGC AAC CAC TGG GTG GTC MT CCG CGG CAC AAC CTG 777
Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu
230 235 240
45 GGC CTG CAG CTC TCG GTG GAG ACG CTG GAT GGG CAG AGC ATC MC CCC 825
Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile Asn Pro
245 250 255
WO 94/10203 PCI/US93/10520
2147598
-- 118 --
MG TTG GCG GGC CTG ATT GGG CGG CAC GGG CCC CAG MC AAG CAG CCC 873
Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys Gln Pro
260 265 270 275
5 TTC ATG GTG GCT TTC TTC MG GCC ACG GAG GTC CAC TTC CGC AGC ATC 921
Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser Ile
280 285 290
CGG TCC ACG GGG AGC MM CAG CGC AGC CAG MC CGC TCC MG ACG CCC 969
10 Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro
295 300 305
MG MC CAG GM GCC CTG CGG ATG GCC AAC GTG GCA GAG MC AGC AGC 1017
Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu Asn Ser Ser
310 315 320
AGC GAC CAG AGG CAG GCC TGT AAG MG CAC GAG CTG TAT GTC AGC TTC 1065
Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe
325 330 335
CGA GAC CTG GGC TGG CAG GAC TGG ATC ATC GCG CCT GM GGC TAC GCC 1113
Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala
340 345 350 355
25 GCC TAC TAC TGT GAG GGG GAG TGT GCC TTC CCT CTG MC TCC TAC ATG 1161
Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met
360 365 370
MC GCC ACC AAC CAC GCC ATC GTG CAG ACG CTG GTC CAC TTC ATC MC 1209
30 Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn
375 380 385
CCG GAA ACG GTG CCC MG CCC TGC TGT GCG CCC ACG CAG CTC MT GCC 1257
Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala
390 395 400
ATC TCC GTC CTC TAC TTC GAT GAC AGC TCC MC GTC ATC CTG MG MM 1305
Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile Leu Lys Lys
405 410 415
TAC AGA MC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAGCTCCTCC 1351
Tyr Arg Asn 2~et Val Val Arg Ala Cys Gly Cys His
420 425 430
GAGMTTCAG ACC(,-lllæG GCCMGmT TCTGGATCCT CCATTGCTCG CCTTGGCCAG 1411
GAACCAGCAG ACCMCTGCC ~ lGAGA C~, luCCC~,lC CCTATCCCCA ACTTTMMGG 1471
WO 94/10203 PCr/US93/10520
21475y~
- 119 -
TGTGAGAGTA TTAGr,AAAr~A TGAGCAGCAT AlGG~ llG ATCAGTTTTT CAGTGGCAGC 1531
ATCCMTGM CMGATCCTA CMGCTGTGC AGGCAAMCC TAGcAr~AAA AAAAAAr,AAc 1591
5 GcATAAAr~AA MATGGCCGG GCCAGGTCAT TGGCTGGGM GTCTCAGCCA TGCACGGACT 1651
CGmCCAGA GGTMTTATG AGCGCCTACC AGCCAGGCCA CCCAGCCGTG GGAGGMGGG 1711
GGCGTGGCM GGGGlGGGCA CA~ C TGTGCGMMG GMMTTGAC CCGGMGTTC 1771
CTGTAATAAA TGTCACAATA MACGMTGA ATr.AAAAAAA AAAAAAAAAA A 1822
(2) INFORHATION FOR SEQ ID NO: 4:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 431 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
25 Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala
5 10 15
Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser
20 25 30
Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser
35 40 45
Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu
50 55 60
Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro
0 Met Phe Met Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly
Gly Pro Gly Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser
100 105 110
Thr Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr
115 120 125
W O 94/10203 ~ ~ . PCT/US93/10520
'
-
2147Sg~ 120 -
Asp Ala Asp Het Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys
130 135 140
Glu Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu
145 150 155 160
Ser Lys Ile Pro Glu Gly Glu Ala Val Thr Ala Ala Glu Phe Arg Ile
165 170 175
0 Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile
180 185 190
Ser Val Tyr Gln Val Leu Gln Glu His Leu Gly Arg Glu Ser Asp Leu
195 200 205
Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu
210 215 220
Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg
225 230 235 240
His Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser
245 250 255
5 Ile Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn
260 265 270
Lys Gln Pro Phe Net Val Ala Phe Phe Lys Ala Thr Glu Val His Phe
275 280 285
Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser
290 295 300
Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val Ala Glu
305 310 315 320
Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr
325 330 335
0 Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu
340 345 350
Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn
355 360 365
Ser Tyr ~et Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His
370 375 380
Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln
385 390 395 400
W O 94/10203 ~ 7 S 9 8 PCT/US93/10520
- 121 - ~
Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Ile
405 410 415
Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His
420 425 430
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1873 base pairs
(B) m E: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HURTn~F
(F) TISSUE TYPE: ENBRYO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 104..1393
(D) OTHER INFORNATION: /function= "OSTEOGENIC PROTEIN"
/product= "MOP1-PP"
/note= nHOP1 (cDNA)~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
35 CTGCAGCAAG TGACClCGGG TCGTGGACCG CTGCCCTGCC CCClCCGCTG CCACCTGGGG 60
CGGCGCGGGC CCGGTGCCCC GGATCGCGCG TAGAGCCGGC GCG ATG CAC GTG CGC 115
Net His Val Arg
TCG CTG CGC GCT GCG GCG CCA CAC AGC TTC GTG GCG CTC TGG GCG CCT 163
Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala Leu Trp Ala Pro
5 10 15 20
45 CTG TTC TTG CTG CGC TCC GCC CTG GCC GAT TTC AGC CTG GAC AAC GAG 211
Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser Leu Asp Asn Glu
WO 94/10203 PCI'/US93/10520
2~4~98 122-
GTG CAC TCC AGC TTC ATC CAC CGG CGC CTC CGC AGC CAG GAG CGG CGG 259
Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg Arg
40 45 50
5 GAG ATG CAG CGG GAG ATC CTG TCC ATC TTA GGG TTG CCC CAT CGC CCG 307
Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu Pro His Arg Pro
55 60 65
CGC CCG CAC CTC CAG GGA MG CAT MT TCG GCG CCC ATG TTC ATG TTG 355
10 Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Het Phe Het Leu
70 75 . 80
GAC CTG TAC MC GCC ATG GCG GTG GAG GAG AGC GGG CCG GAC GGA CAG 403
Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Ser Gly Pro Asp Gly Gln
15 85 90 95 100
GGC TTC TCC TAC CCC TAC MG GCC GTC TTC AGT ACC CAG GGC CCC CCT 451
Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly Pro Pro
105 110 115
TTA GCC AGC CTG CAG GAC AGC CAT TTC CTC ACT GAC GCC GAC ATG GTC 499
Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp Ala Asp Het Val
120 125 130
25 ATG AGC TTC GTC MC CTA GTG GM CAT GAC MA GM TTC TTC CAC CCT 547
Het Ser Phe Val Asn Leu Val Glu His Asp Lys Glu Phe Phe His Pro
135 140 145
CGA TAC CAC CAT CGG GAG TTC CGG m GAT CTT TCC MG ATC CCC GAG 595
30 Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser Lys Ile Pro Glu
150 155 160
GGC GM CGG GTG ACC GCA GCC GAA TTC AGG ATC TAT MG GAC TAC ATC 643
Gly Glu Arg Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Asp Tyr Ile
35 165 170 175 180
CGG GAG CGA TTT GAC MC GAG ACC TTC CAG ATC ACA GTC TAT CAG GTG 691
Arg Glu Arg Phe Asp Asn Glu Thr Phe Gln Ile Thr Val Tyr Gln Val
185 190 195
CTC CAG GAG CAC TCA GGC AGG GAG TCG GAC CTC TTC TTG CTG GAC AGC 739
Leu Gln Glu His Ser Gly Arg Glu Ser Asp Leu Phe Leu Leu Asp Ser
200 205 210
45 CGC ACC ATC TGG GCT TCT GAG GAG GGC TGG TTG GTG TTT GAT ATC ACA 787
Arg Thr Ile Trp Ala Ser Glu Glu Gly Trp Leu Val Phe Asp Ile Thr
215 220 225
WO 94/10203 PCl/US93/10520
- 21~7~98
-- 123 --
GCC ACC AGC MC CAC TGG GTG GTC MC CCT CGG CAC MC CTG GGC TTA 835
Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His Asn Leu Gly Leu
230 235 240
5 CAG CTC TCT GTG GAG ACC CTG GAT GGG CAG AGC ATC MC CCC MG TTG 883
Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile Asn Pro Lys Leu
245 250 255 260
GCA GGC CTG ATT GGA CGG CAT GGA CCC CAG MC AAG CM CCC TTC ATG 931
10 Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys Gln Pro Phe Net
265 270 275
GTG GCC TTC TTC MG GCC ACG GM GTC CAT CTC CGT AGT ATC CGG TCC 979
Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg Ser Ile Arg Ser
280 285 290
ACG GGG GGC MG CAG CGC AGC CAG MT CGC TCC MG ACG CCA MG MC 1027
Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys Thr Pro Lys Asn
295 300 305
CM GAG GCC CTG AGG ATG GCC AGT GTG GCA GM MC AGC AGC AGT GAC 1075
Gln Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn Ser Ser Ser Asp
310 315 320
25 CAG AGG CAG GCC TGC MG MM CAT GAG CTG TAC GTC AGC TTC CGA GAC 1123
Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val Ser Phe Arg Asp
325 330 335 340
CTT GGC TGG CAG GAC TGG ATC ATT GCA CCT GM GGC TAT GCT GCC TAC 1171
30 Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly Tyr Ala Ala Tyr
345 350 355
TAC TGT GAG GGA GAG TGC GCC TTC CCT CTG MC TCC TAC ATG MC GCC 1219
Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser Tyr Met Asn Ala
360 365 370
ACC MC CAC GCC ATC GTC CAG ACA CTG GTT CAC TTC ATC MC CCA GAC 1267
Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe Ile Asn Pro Asp
375 380 385
ACA GTA CCC MG CCC TGC TGT GCG CCC ACC CAG CTC MC GCC ATC TCT 1315
Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu Asn Ala Ile Ser
390 395 400
45 GTC CTC TAC TTC GAC GAC AGC TCT MT GTC GAC CTG MG MG TAC AGA 1363
Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu Lys Lys Tyr Arg
405 410 415 420
W O 94/10203 PCT/US93/10520
21~175g8
- 124 -
AAC ATG GTG GTC CGG GCC TGT GGC TGC CAC TAG~-1111CC TGAGACCCTG 1413
Asn Net Val Val Arg Ala Cys Gly Cys His
425 430
ACC m GCGG GGCCACACCT TTCCAAATCT TCGATGTCTC ACCATCTAAG TCTCTCACTG 1473
CCCACCTTGG crAGrAGAAc AGACCAACCT CTCCTGAGCC llCCClCACC TCCCAACCGG 1533
AAGCATGTAA GG~llCCAGA AACCTGAGCG TGCAGCAGCT GATGAGCGCC ~ CCll~l 1593
GGCACGTGAC GrAcAAGATc CTACCAGCTA CCACAGCAAA CGCCT M GAG CAGGAAAAAT 1653
GTCTGCCAGG AAAGTGTCCA GTGTCCACAT GGCCCCTGGC GCTCTGAGTC TTTGAGGAGT 1713
AATCGCAAGC ClCGllCAGC TGCAGCAGAA GrAAr~GGcTT AGCCAGGGTG GGCGCTGGCG 1773
~ llGA AGGGAAACCA AGCAGAAGCC ACTGTAATGA TATGTCACAA TAAAACCCAT 1833
GAATGAAAAA AAAAAAAAAA AAAAAAAAAA AAAAr~AATTc 1873
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 430 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala
1 5 10 15
Leu Trp Ala Pro Leu Phe Leu Leu Arg Ser Ala Leu Ala Asp Phe Ser
20 25 30
Leu Asp Asn Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser
35 40 45
Gln Glu Arg Arg Glu Met Gln Arg Glu Ile Leu Ser Ile Leu Gly Leu
Pro His Arg Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro
WO 94/10203 PCI`/US93/10520
~14 759~
- 125 -
net Phe Met Leu Asp Leu Tyr Asn Ala Het Ala Val Glu Glu Ser Gly
85 90 95
Pro Asp Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr
100 105 110
Gln Gly Pro Pro Leu Ala Ser Leu Gln Asp Ser His Phe Leu Thr Asp
115 120 125
10 Ala Asp Met Val Met Ser Phe Val Asn Leu Val Glu His Asp Lys Glu
130 135 140
Phe Phe His Pro Arg Tyr His His Arg Glu Phe Arg Phe Asp Leu Ser
145 150 155 160
Lys Ile Pro Glu Gly Glu Arg Val Thr Ala Ala Glu Phe Arg Ile Tyr
165 170 175
Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Gln Ile Thr
180 185 190
Val Tyr Gln Val Leu Gln Glu His Ser Gly Arg Glu Ser Asp Leu Phe
195 200 205
25 Leu Leu Asp Ser Arg Thr Ile Trp Ala Ser Glu Glu Gly Trp Leu Val
210 215 220
Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg His
225 230 235 240
Asn Leu Gly Leu Gln Leu Ser Val Glu Thr Leu Asp Gly Gln Ser Ile
245 250 255
Asn Pro Lys Leu Ala Gly Leu Ile Gly Arg His Gly Pro Gln Asn Lys
260 265 270
Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Leu Arg
275 280 285
40 Ser Ile Arg Ser Thr Gly Gly Lys Gln Arg Ser Gln Asn Arg Ser Lys
290 295 300
Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Ser Val Ala Glu Asn
305 310 315 320
Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu Leu Tyr Val
325 330 335
W O 94/10203 PCT/US93/lOS20
21~7S98
- 126 -
Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu Gly
340 345 350
Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn Ser
355 360 365
Tyr Net Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu Val His Phe
370 375 380
Ile Asn Pro Asp Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln Leu
-385 390 395 400
Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn Val Asp Leu
405 . 410 415
Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys His
420 425 430
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1723 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(F) TISSUE TYPE: HIPPOCAMPUS
(ix) FEATURE:
(A) NANE/KEY: CDS
(B) LOCATION: 490.. 1696
(D) OTHER INFORMATION: /function= ~OSTEOGENIC PROTEIN"
/product= nhOP2-PP"
/note= "hOP2 (cDNA)"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GGCGCCGGCA GAGCAGGAGT GGCTGGAGGA GC~ llG GAGCAGGAGG TGGCACGGCA 60
GGGCTGGAGG G~lCC~lATG AGTGGCGGAG ACGGCCCAGG AGGCGCTGGA GCAACAGCTC 120
CCACACCGCA CC M GCGGTG GCTGCAGGAG CTCGCCCATC GCCCCTGCGC TGCTCGGACC 180
W O 94/10203 PCT/US93/10S20
21 ~ 75~
- 127 -
GCGGCCACAG CCGGACTGGC GGGTACGGCG GCGACAGAGG CATTGGCCGA GAGTCCCAGT 240
-CCGCAGAGTA GCCCCGGCCT CGAGGCGGTG GCGlCCCG~l C~l~lCC~lC CAGGAGCCAG 300
5 GACAGGTGTC GCGCGGCGGG GCTCCAGGGA CCGCGCCTGA GGCCGGCTGC CCGCCCGlCC 360
CGCCCCGCCC CGCCGCCCGC CGCCCGCCGA GCCCAGCCTC ~llGCC61CG GGGCGTCCCC 420
A6GCC~lGGG TCGGCCGCGG AGCCGATGCG CGCCCG~lGA GCGCCCCAGC TGAGCGCCCC 480
CGGCCTGCC ATG ACC GCG CTC CCC GGC CCG CTC TGG CTC CTG GGC CTG 528
Met Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu
1 5 10
15 GCG CTA TGC GCG CTG GGC GGG GGC GGC CCC GGC CTG CGA CCC CCG CCC 576
Ala Leu Cys Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro
15 20 25
GGC TGT CCC CAG CGA CGT CTG GGC GCG CGC GAG CGC CGG GAC GTG CAG 624
20 Gly Cys Pro Gln Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gln
30 35 40 45
CGC GAG ATC CTG GCG GTG CTC GGG CTG CCT GGG C6G CCC CGG CCC CGC 672
Arg Glu Ile Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg
S0 SS 60
GCG CCA CCC GCC GCC TCC CGG CTG CCC GCG TCC GCG CCG CTC TTC ATG 720
Ala Pro Pro Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Het
65 70 75
CTG GAC CTG TAC CAC GCC ATG GCC GGC GAC GAC GAC GAG GAC GGC GCG 768
Leu Asp Leu Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala
80 85 90
35 CCC GCG GAG CGG CGC CTG GGC CGC GCC GAC CTG GTC ATG AGC TTC GTT 816
Pro Ala Glu Arg Arg Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val
95 100 105
AAC ATG GTG GAG CGA GAC CGT GCC CTG GGC CAC CAG GAG CCC CAT TGG 864
40 Asn Het Val Glu Arg Asp Arg Ala Leu Gly His Gln Glu Pro His Trp
110 115 120 125
AAG GAG TTC CGC m GAC CTG ACC CAG ATC CCG GCT GGG GAG GCG GTC 912
Lys Glu Phe Arg Phe Asp Leu Thr Gln Ile Pro Ala Gly Glu Ala Val
130 135 . 140
ACA GCT GCG GAG TTC CGG ATT TAC AAG GTG CCC AGC ATC CAC CTG CTC 960
Thr Ala Ala Glu Phe Arg Ile Tyr Lys Val Pro Ser Ile His Leu Leu
145 150 155
WO 94/10203 PCI/US93/10520
2~475~8 _
-- 128 --
MC AGG ACC CTC CAC GTC AGC ATG TTC CAG GTG GTC CAG GAG CAG TCC 1008
Asn Arg Thr Leu His Val Ser Net Phe Gln Val Val Gln Glu Gln Ser
160 165 170
5 MC AGG GAG TCT GAC TTG TTC m TTG GAT CTT CAG ACG CTC CGA GCT 1056
Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gln Thr Leu Arg Ala
175 180 185
GGA GAC GAG GGC TGG CTG GTG CTG GAT GTC ACA GCA GCC AGT GAC TGC 1104
10 Gly Asp Glu Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys
190 195 200 205
TGG TTG CTG MG CGT CAC MG GAC CTG GGA CTC CGC CTC TAT GTG GAG 1152
Trp Leu Leu Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu
210 215 220
ACT GAG GAC GGG CAC AGC GTG GAT CCT GGC CTG GCC GGC CTG CTG GGT 1200
Thr Glu Asp Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly
225 230 235
CM CGG GCC CCA CGC TCC CM CAG CCT TTC GTG GTC ACT TTC TTC AGG 1248
Gln Arg Ala Pro Arg Ser Gln Gln Pro Phe Val Val Thr Phe Phe Arg
240 245 250
25 GCC AGT CCG AGT CCC ATC CGC ACC CCT CGG GCA GTG AGG CCA CTG AGG 1296
Ala Ser Pro Ser Pro Ile Arg Thr Pro Arg Ala Val Arg Pro Leu Arg
255 260 265
AGG AGG CAG CCG MG MM AGC MC GAG CTG CCG CAG GCC AAC CGA CTC 1344
30 Arg Arg Gln Pro Lys Lys Ser Asn Glu Leu Pro Gln Ala Asn Arg Leu
270 275 280 285
CCA GGG ATC m GAT GAC GTC CAC GGC TCC CAC GGC CGG CAG GTC TGC 1392
Pro Gly Ile Phe Asp Asp Val His Gly Ser His Gly Arg Gln Val Cys
290 295 300
CGT CGG CAC GAG CTC TAC GTC AGC TTC CAG GAC CTC GGC TGG CTG GAC 1440
Arg Arg His Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Leu Asp
305 310 315
TGG GTC ATC GCT CCC CM GGC TAC TCG GCC TAT TAC TGT GAG GGG GAG 1488
Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu
320 325 330
45 TGC TCC TTC CCA CTG GAC TCC TGC ATG MT GCC ACC MC CAC GCC ATC 1536
Cys Ser Phe Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala Ile
335 340 345
W O 94/10203 P ~ /US93/10~20
21 ~ 759~
- 129 -
CTG CAG TCC CTG GTG CAC CTG ATG AAG CCA M C GCA GTC CCC AAG GCG 1584
Leu Gln Ser Leu Val His Leu Met Lys Pro Asn Ala Val Pro Lys Ala
- 350 355 360 365
5 TGC TGT GCA CCC ACC AAG CTG AGC GCC ACC TCT GTG CTC TAC TAT GAC 1632
Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp
370 375 380
AGC AGC M C AAC GTC ATC CTG CGC AAA GCC CGC AAC ATG GTG GTC AAG 1680
10 Ser Ser Asn Asn Val Ile Leu Arg Lys Ala Arg Asn Net Val Val Lys
385 390 395
GCC TGC GGC TGC CAC T GAGTCAGCCC GCCCAGCCCT ACTGCAG 1723
Ala Cys Gly Cys His
400
(2) INFORNATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 402 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) NOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Net Thr Ala Leu Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys
301 5 10 15
Ala Leu Gly Gly Gly Gly Pro Gly Leu Arg Pro Pro Pro Gly Cys Pro
Gln Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Val Gln Arg Glu Ile
35 40 45
Leu Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Pro Pro
50 55 60
Ala Ala Ser Arg Leu Pro Ala Ser Ala Pro Leu Phe Net Leu Asp Leu
65 70 75 80
Tyr His Ala Met Ala Gly Asp Asp Asp Glu Asp Gly Ala Pro Ala Glu
4585 90 95
Arg Arg Leu Gly Arg Ala Asp Leu Val Met Ser Phe Val Asn Met Val
100 105 110
W O 94/10203 PCT/US93/10520
21~75~8
- 130 -
Glu Arg Asp Arg Ala Leu Gly His Gln Glu Pro His Trp Lys Glu Phe
115 120 125
Arg Phe Asp Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Thr Ala Ala
130 135 140
Glu Phe Arg Ile Tyr Lys Val Pro Ser Ile His Leu Leu Asn Arg Thr
145 150 155 160
Leu His Val Ser Het Phe Gln Val Val Gln Glu Gln Ser Asn Arg Glu
165 170 175
Ser Asp Leu Phe Phe Leu Asp Leu Gln Thr Leu Arg Ala Gly Asp Glu
180 185 190
Gly Trp Leu Val Leu Asp Val Thr Ala Ala Ser Asp Cys Trp Leu Leu
195 200 205
Lys Arg His Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Glu Asp
210 215 220
Gly His Ser Val Asp Pro Gly Leu Ala Gly Leu Leu Gly Gln Arg Ala
225 230 235 240
Pro Arg Ser Gln Gln Pro Phe Val Val Thr Phe Phe Arg Ala Ser Pro
245 250 255
Ser Pro Ile Arg Thr Pro Arg Ala Val Arg Pro Leu Arg Arg Arg Gln
260 265 270
Pro Lys Lys Ser Asn Glu Leu Pro Gln Ala Asn Arg Leu Pro Gly Ile
275 280 285
Phe Asp Asp Val His Gly Ser His Gly Arg Gln Val Cys Arg Arg His
290 295 300
Glu Leu Tyr Val Ser Phe Gln Asp Leu Gly Trp Leu Asp Trp Val Ile
305 310 315 320
Ala Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ser Phe
325 330 335
Pro Leu Asp Ser Cys Met Asn Ala Thr Asn His Ala Ile Leu Gln Ser
340 345 350
Leu Val His Leu Met Lys Pro Asn Ala Val Pro Lys Ala Cys Cys Ala
355 360 365
W O 94/10203 PCT/US93/10520
-- 21 ~ 7~ 8
- 131 -
Pro Thr Lys Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn
370 375 380 ~-
Asn Val Ile Leu Arg Lys Ala Arg Asn Met Val Val Lys Ala Cys Gly
385 390 395 400
Cys His
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1926 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(vi) ORIGINAL SOURCE:
(A) ORGANISM: HURTnAF.
(F) TISSUE TYPE: EMBRYO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 93..1289
(D) OTHER INFORMATION: /function= "OSTEOGENIC PROTEIN"
/product= "mOP2-PP n
/note= nmOP2 cDNA"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
GCCAGGCACA GGTGCGCCGT ~l~GlC~lCC CC~l~lGGCG TCAGCCGAGC CCGACCAGCT 60
ACCAGTGGAT GCGCGCCGGC TG MM GTCCG AG ATG GCT ATG CGT CCC GGG CCA 113
Met Ala Met Arg Pro Gly Pro
CTC TGG CTA TTG GGC CTT GCT CTG TGC GCG CTG GGA GGC GGC CAC GGT 161
Leu Trp Leu Leu Gly Leu Ala Leu Cys Ala Leu Gly Gly Gly His Gly
10 15 20
CCG CGT CCC CCG CAC ACC TGT CCC CAG CGT CGC CTG GGA GCG CGC GAG 209
Pro Arg Pro Pro His Thr Cys Pro Gln Arg Arg Leu Gly Ala Arg Glu
25 30 35
CGC CGC GAC ATG CAG CGT G M ATC CTG GCG GTG CTC GGG CTA CCG GGA 257
Arg Arg Asp Met Gln Arg Glu Ile Leu Ala Val Leu Gly Leu Pro Gly
WO 94/10203 PCI/US93/10520
~14759~ ~
-- 132 --
CGG CCC CGA CCC CGT GCA CM CCC GCC GCT GCC CGG CAG CCA GCG TCC 305
Arg Pro Arg Pro Arg Ala Gln Pro Ala Ala Ala Arg Gln Pro Ala Ser
60 65 70
GCG CCC CTC TTC ATG TTG GAC CTA TAC CAC GCC ATG ACC GAT GAC GAC 353
Ala Pro Leu Phe Met Leu Asp Leu Tyr His Ala Het Thr Asp Asp Asp
75 80 85
GAC GGC GGG CCA CCA CAG GCT CAC TTA GGC CGT GCC GAC CTG GTC ATG 401
lO Asp Gly Gly Pro Pro Gln Ala His Leu Gly Arg Ala Asp Leu Val Met
go 95 100
AGC TTC GTC MC ATG GTG GM CGC GAC CGT ACC CTG GGC TAC CAG GAG 449
Ser Phe Val Asn Met Val Glu Arg Asp Arg Thr Leu Gly Tyr Gln Glu
105 110 115
CCA CAC TGG MG GM TTC CAC m GAC CTA ACC CAG ATC CCT GCT GGG 497
Pro His Trp Lys Glu Phe His Phe Asp Leu Thr Gln Ile Pro Ala Gly
120 125 130 135
GAG GCT GTC ACA GCT GCT GAG TTC CGG ATC TAC MA GM CCC AGC ACC 545
Glu Ala Val Thr Ala Ala Glu Phe Arg Ile Tyr Lys Glu Pro Ser Thr
140 145 150
25 CAC CCG CTC MC ACA ACC CTC CAC ATC AGC ATG TTC GM GTG GTC CM 593
His Pro Leu Asn Thr Thr Leu His Ile Ser Met Phe Glu Val Val Gln
155 160 165
GAG CAC TCC MC AGG GAG TCT GAC TTG TTC m TTG GAT CTT CAG ACG 641
30 Glu His Ser Asn Arg Glu Ser Asp Leu Phe Phe Leu Asp Leu Gln Thr
170 175 180
CTC CGA TCT GGG GAC GAG GGC TGG CTG GTG CTG GAC ATC ACA GCA GCC 689
Leu Arg Ser Gly Asp Glu Gly Trp Leu Val Leu Asp Ile Thr Ala Ala
185 190 195
AGT GAC CGA TGG CTG CTG MC CAT CAC MG GAC CTG GGA CTC CGC CTC 737
Ser Asp Arg Trp Leu Leu Asn His His Lys Asp Leu Gly Leu Arg Leu
200 205 210 215
TAT GTG GM ACC GCG GAT GGG CAC AGC ATG GAT CCT GGC CTG GCT GGT 785
Tyr Val Glu Thr Ala Asp Gly His Ser Met Asp Pro Gly Leu Ala Gly
220 225 230
45 CTG CTT GGA CGA CM GCA CCA CGC TCC AGA CAG CCT TTC ATG GTA ACC 833
Leu Leu Gly Arg Gln Ala Pro Arg Ser Arg Gln Pro Phe Met Val Thr
235 240 245
W O 94/10203 PCT/US93/10520
21 ~ 7~98
- 133 -
TTC TTC AGG GCC AGC CAG AGT CCT GTG CGG GCC CCT CGG GCA GCG AGA 881
Phe Phe Arg Ala Ser Gln Ser Pro Val Arg Ala Pro Arg Ala Ala Arg
~ 250 255 260
5 CCA CTG M G AGG AGG CAG CCA AAG MM ACG AAC GAG CTT CCG CAC CCC 929
Pro Leu Lys Arg Arg Gln Pro Lys Lys Thr Asn Glu Leu Pro His Pro
265 270 275
M C MM CTC CCA GGG ATC m GAT GAT GGC CAC GGT TCC CGC GGC AGA 977
10 Asn Lys Leu Pro Gly Ile Phe Asp Asp Gly His Gly Ser Arg Gly Arg
280 285 290 295
GAG GTT TGC CGC AGG CAT GAG CTC TAC GTC AGC TTC CGT GAC CTT GGC 1025
Glu Val Cys Arg Arg His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly
300 305 310
TGG CTG GAC TGG GTC ATC GCC CCC CAG GGC TAC TCT GCC TAT TAC TGT1073
Trp Leu Asp Trp Val Ile Ala Pro Gln Gly Tyr Ser Ala Tyr Tyr Cys
315 320 325
GAG GGG GAG TGT GCT TTC CCA CTG GAC TCC TGT ATG AAC GCC ACC M C1121
Glu Gly Glu Cys Ala Phe Pro Leu Asp Ser Cys Het Asn Ala Thr Asn
330 335 340
25 CAT GCC ATC TTG CAG TCT CTG GTG CAC CTG ATG M G CCA GAT GTT GTC 1169
His Ala Ile Leu Gln Ser Leu Val His Leu Met Lys Pro Asp Val Val
345 350 355
CCC AAG GCA TGC TGT GCA CCC ACC MM CTG AGT GCC ACC TCT GTG CTG 1217
30 Pro Lys Ala Cys Cys Ala Pro Thr Lys Leu Ser Ala Thr Ser Val Leu
360 365 370 375
TAC TAT GAC AGC AGC M C M T GTC ATC CTG CGT AAA CAC CGT AAC ATG 1265
Tyr Tyr Asp Ser Ser Asn Asn Val Ile Leu Arg Lys His Arg Asn Met
380 385 390
GTG GTC M G GCC TGT GGC TGC CAC TGAGGCCCCG CCCAGCATCC TGCTTCTACT 1319
Val Val Lys Ala Cys Gly Cys His
395
ACCTTACCAT CTGGCCGGGC CC~lClCCAG AGGcArAAAc CCTTCTATGT TATCATAGCT 1379
cAr~AcAGG&& C M TGGGAGG CCCTTCACTT CCCCTGGCCA CllC~l~CTA AAATTCTGGT 1439
CTTTCCCAGT lC~ lCC TTCAl~GG~l llCG&GGCTA TCACCCCGCC CTCTCCATCC 1499
TCCTACCCCA AGCATAr.ACT GM TGCACAC AGCATCCCAG AGCTATGCTA ACTGAGAGGT 1559
W O 94/10203 PCT/US93/lOS20
2147~98
- 134 -
~l~GG~lCAG CACTG M GGC CCACATGAGG M GACTGATC CTTGGCCATC CTCAGCCCAC 1619
M TGGC MM T TCTGGATGGT cTAArAAGGc CCTGG M TTC TA M CTAGAT GATCTGGGCT 1679
CTCTGCACCA TTCATTGTGG CAGTTGGGAC AT m TAGGT ATAArArJAçA ÇATAÇACTTA 1739
GATC M TGCA lCG~l~lACT CCTTG MM TC AGAGCTAGCT TGTTAr.AAAA AGM TCAGAG 1799
CCAGGTATAG CGGTGCATGT CATT M TCCC AGCGCT MM G AGACArAr,AC AGGAG M TCT 1859
CTGTGAGTTC M GGCCACAT Ar~AAAr~AGcc l~l~lCGGGA GCAGGAAAAA AAAAAAAAAC 1919
GG M TTC 1926
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 399 amino acids
(B) T~E: amino acid
(D) TOPOLOGY: linear
(ii) HOLECULE TYPE: protein
25(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Ala Met Arg Pro Gly Pro Leu Trp Leu Leu Gly Leu Ala Leu Cys
1 5 10 15
Ala Leu Gly Gly Gly His Gly Pro Arg Pro Pro His Thr Cys Pro Gln
20 25 30
Arg Arg Leu Gly Ala Arg Glu Arg Arg Asp Met Gln Arg Glu Ile Leu
35 40 45
Ala Val Leu Gly Leu Pro Gly Arg Pro Arg Pro Arg Ala Gln Pro Ala
50 55 60
Ala Ala Arg Gln Pro Ala Ser Ala Pro Leu Phe Het Leu Asp Leu Tyr
4065 70 75 80
His Ala Met Thr Asp Asp Asp Asp Gly Gly Pro Pro Gln Ala His Leu
Gly Arg Ala Asp Leu Val Met Ser Phe Val Asn Met Val Glu Arg Asp
100 105 110
WO 94/10203 PCI/US93/10520
21 ~ 7$9~
-- 135 --
Arg Thr Leu Gly Tyr Gln Glu Pro His Trp Lys Glu Phe His Phe Asp
115 120 125
Leu Thr Gln Ile Pro Ala Gly Glu Ala Val Thr Ala Ala Glu Phe Arg
130 135 140
Ile Tyr Lys Glu Pro Ser Thr His Pro Leu Asn Thr Thr Leu His Ile
145 150 155 160
10 Ser Met Phe Glu Val Val Gln Glu His Ser Asn Arg Glu Ser Asp Leu
165 170 175
Phe Phe Leu Asp Leu Gln Thr Leu Arg Ser Gly Asp Glu Gly Trp Leu
180 185 190
Val Leu Asp Ile Thr Ala Ala Ser Asp Arg Trp Leu Leu Asn His His
195 200 205
Lys Asp Leu Gly Leu Arg Leu Tyr Val Glu Thr Ala Asp Gly His Ser
210 215 220
Met Asp Pro Gly Leu Ala Gly Leu Leu G'y Arg Gln Ala Pro Arg Ser
225 230 235 240
25 Arg Gln Pro Phe Met Val Thr Phe Phe Arg Ala Ser Gln Ser Pro Val
245 250 255
Arg Ala Pro Arg Ala Ala Arg Pro Leu Lys Arg Arg Gln Pro Lys Lys
260 265 270
Thr Asn Glu Leu Pro His Pro Asn Lys Leu Pro Gly Ile Phe Asp Asp
275 280 285
Gly His Gly Ser Arg Gly Arg Glu Val Cys Arg Arg His Glu Leu Tyr
290 295 300
Val Ser Phe Arg Asp Leu Gly Trp Leu Asp Trp Val Ile Ala Pro Gln
305 310 315 320
40 Gly Tyr Ser Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asp
325 330 335
Ser Cys Met Asn Ala Thr Asn His Ala Ile Leu Gln Ser Leu Val His
340 345 350
Leu Met Lys Pro Asp Val Val Pro Lys Ala Cys Cys Ala Pro Thr Lys
355 360 365
W O 94/10203 PCT/US93/10520
-
2147S98
- 136 -
Leu Ser Ala Thr Ser Val Leu Tyr Tyr Asp Ser Ser Asn Asn Val Ile
370 375 380
Leu Arg Lys His Arg Asn Met Val Val Lys Ala Cys Gly Cys His
385 390 395
(2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQu~N~ CHARACTERISTICS:
(A) LENGTH: 6418 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1..6361
(D) OTHER INFORMATION: /note= nHOP-2 genomic sequence"
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1..837
(D) OTHER INFORMATION: /note= "E~ON ONE"
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 884..885
(D) OTHER INFORMATION: /note= "A Gap Occurs Between
Positions 884 and 885 in this Sequence"
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1088..1277
(D) OTHER INFORMATION: /note= "E~ON TUO"
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1350..1814
(D) OTHER INFORMATION: /note= "E~ON THREE"
(ix) FEATURE:
(A) NAME/KEY: misc feature
(B) LOCATION: 1834..1835
(D) OTHER INFORMATION: /note= "A Gap Occurs Between
Positions 1834 and 1835 in this Sequence"
W O 94/10203 PCT/US93/10520
`- 21 ~ 7~98
- 137 -
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 1883..2077
(D) OTHER INFORMATION: /note= "E~ON FOUR"
(ix) FEATURE:
(A) NANE/KEY: exon
(B) LOCATION: 2902..2981
(D) OTHER INFORNATION: /note= "E~ON FIVE"
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 3507..3617
(D) OTHER INFORMATION: /note= "EgON SI~"
(ix) FEATURE:
(A) NAME/KEY: exon
(B) LOCATION: 6116..6361
(D) OTHER INFORNATION: /note= "E~ON SEVEN"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GGAATTCCGG CCACAGTGGC GCCGGCAGAG CAGGAGTGGC TGGAGGAGCT GTGGTTGGAG 60
CAGGAGGTGG CACGGCAGGG CTGGAGGGCT CCCTATGAGT GGCGGAGACG GCCCAGGAGG 120
CGCTGGAGCA ACAGCTCCCA CACCGCACCA AGCGGTGGCT GCAGGAGCTC GCCCATCGCC 180
CCTGCGCTGC TCGGACCGCG GCCACAGCCG GACTGGCGGG TACGGCGGCG ACAGACGGAT 240
TGGCCGAGAG TCCCAGTCCG cAr~AGTAr~cc CCGGC~lCGA GGCGGTGGCG TCCGCGTCCT 300
CTCGTCCAGG AGcrArJrAcA GGTGTCGCGC GGCGGGCCGT CCAGGGACCG CGCTGAGGCC 360
GCGGTCGCCC GTCCCGCCCC GCCCCGCCGC CCGCCGCCCG CCGAGCCCAG CCTCCTTGCC 420
~lCGGGGCGT CCCCAGGCCC lGG~lCGGCC GCGGAGCCGA TGCGCGCCCG CTGAGCGCCC 480
CAGCTGAGCG CCCCCGGCCT GCCATGACCG CG~-lCCCCGG CCCGCTCTGG CTCCTGGGCC 540
TGGCGCTATG CGCGCTGGGC GGGGGCGGCC CCGGCCTGCG ACCCCCGCCC GGCTGTCCCC 600
AGCGACGTCT GGGCGCGCGC GAGCGCCGGG ACGTGCAGCG CGAGATCCTG GCGGTGCTCG 660
GGCTGCCTGG GCGGCCCCGG CCCCGCGCGC CACCCGCCGC CTCCCGGCTG CCCGCGTCCG 720
CGCCGCTCTT CATGCTGGAC CTGTACCACG CCATGGCCGG CGACGACGAC GAGGACGGCG 780
W O 94/10203 PCT/US93/10520
2147S9~
- 138 -
CGCCCGCGGA GCGGCGCCTG GGCCGCGCCG ACCTGGTCAT GAG~-llC~ll AACATGGGTG 840
AGTGCGGCGC CCGCGCGGGG ACCCTCGGAG TAAACTGGCT GCAGCTGCAG GGCC~ l 900
GGCTCTACAC cccGGrAccA AGCCTGGAAC AAACG m GC ACTAAATGAA GCCGGCCCCA 960
CCCAGGCCTC CClGGGlCCG CTCCACCTTG A~ lGGGT GGCTGGGGGC GGTGGCTCAC 1020
ACCAGCTCTG CCCC~lCCAG AGCCCGAGCC ATTCTGAGTG CCAGCCCAGC GCTGC m GT 1080
CTTCTAGTGG AGCGAGACCG TGCC~lGGGC CACCAGGAGC CCCATTGGAA GGAGTTCCGC 1140
m GACCTGA CCCAGATCCC GGCTGGGGAG GCGGTCACAG CTGCGGAGTT CCGGATTTAC 1200
AAGGTGCCCA GCATCCACCT GCTCAACAGG ACCCTCCACG TCAGCATGTT CCAGGTGGTC 1260
CAGGAGCAGT CC M CAGGTG C~llCCCC-ll GGCCCGGGlG CCCACCTAAC CCCCCACCTC 1320
ACAGTCTCAT GGTCAAGGCA GCCCAGCAGG GAGlCGlGGl GGGTGAAAGA GAGCCTCAAA 1380
GATGGGAAGG ATGCTTGGCC CGAGGCCCTG CA~l~lGGGA AGAGCCCCAG TGACAATCCT 1440
GACTTCAAGT CCCTGCCCTC CATCCTGCTG TGGGGACTTG GACATGGTCA CTGAGACTCA 1500
GlllCCCCAT GTGTACACCT ~l~lGGGCTG AGGCAATGAG ATGAGGCTCA GAAGGGCGCA 1560
GCCAGAGTCA GGTGGGAGAC GClCCGGlGA CAGCCCCCAG CGGGCCCTGG AGACACGGAG 1620
GCAGCTGTGC CGGCCGCCGG TTAATTGTTC m CATGTCC ACAGGGGAGT CTGACTTGTT 1680
C-lllllGGAT CTTCAGACGC TCCGAGCTGG ArACr.AGGGC lGG~-lG~lGC TGGATGTCAC 1740
AGCAGCCAGT GACTGCTGGT TGCTGAAGCG Tr-AcAAGr~Ac CTGGGACTCC GCCTCTATGT 1800
GGAGACTGAG GACGGTGAGG ~lGGGGCl~l GCAGCTGCAG AGCCACTGCC CGTGAGTGAC 1860
CCCl~l~lCC lll~ lC AGGGCACAGC GTGGATCCTG GCCTGGCCGG CCTGCTGGGT 1920
CAACGGGCCC CACGCTCCCA ACAGCC m C GTGGTCACTT TCTTCAGGGC CAGTCCGAGT 1980
CCCATCCGCA CCCClCGGGC AGTGAGGCCA CTGAGGAGGA GGCAGCCGAA GAAAAGCAAC 2040
GAGCTGCCGC AGGCCAACCG ACTCCCAGGG Al~lllGGlG AGGGlCGGGC AGGCTGGGGC 2100
GAGGCTGTGG ~l~lClGGCT GAGAGAGGCA GGGCr.AGAAC CAAGTGGTGG CCCAGAGCCC 2160
AGAGCCTCAG GCTAGGTCGG TTCAAGCTGA CGGCCACTCT CCAGCCACCT TTCCTGACAC 2220
W O 94/10203 PCr/US93/10520
-- 21 g 75~
- 139 -
CAl~llGGCC CTGATGCACC CTGGTGACCG GCACTCCGAG GC~l~lC-ClG G~ lCCClG 2280
CTGCCAG M G l~lCC~ C TCCCCCTGGC lC~lCCGG~l ClllClCAGG AGCCTCCTTC 2340
AGM TCAGCT GCCCCllCCC TGGGAGCCGC AGCCCCTCAT GACCTGCGGT TGTGCCTGGG 2400
CACCl~lGGA lCClCGGllG CTTATGCGAT lll~lCCCCA ACTGGCC M G CTTCAGGATC 2460
AGGGACAGGC CTGACCC M C CCCGTGCCCT C~llCCCAGG GAGTCGGCCC TTGACTGGCC 2520
16~1C~lGAG CCACTTG M C CTCGGG M TG G61~1GGCAG GAGAGGGTGG GCTGGAGTCA 2580
CAGGG~l~lC cAr~A~AGGAG GAGGCACAGG ATGGCCGAGG GTCCTGCTGG GCTGTTTACT 2640
GGAGCATA M GATGCTCATA GGCTGAAGGA CAGGGGAGGA CTGGGCACAG TGTCACTCTA 2700
GCCATTGGGA GCCATGGCAG GCTTCTGAGC TGGGTCATGG TAC M GCAGA GTTCCAGGGA 2760
TGGGC m AT GAGCCA M TG ~lllC~l~lC ATTCATTTAT TT~A~AAATG TGCTCATCAG 2820
GGCATCCCCC ACCCl6~1AC CCCATAGTAG cTGrArAcAG CAGGAACCCC AGAAAAGACC 2880
TTGCCCCTTC TGTCCCTGCA GATGACGTCC ACGGCTCCCA CGGCCGGCAG GTCTGCCGTC 2940
GGCACGAGCT CTACGTCAGC TTCCAGGACC TCGGCTGGCT GGT M TTGCT GACTCTCCTT 3000
6~ A M TGACAATCAC CACCTGTAGA TCAG M GTGA ATCTGCAGGG AGGACATAr.A 3060
ATCATGGTGA CTTCAATTTT CTTATGTATT lllll~llC1 61~1111CCA A~llll~lAA 3120
AGTr~Ar~AATA TGGTGAGA M GGGllll~ll 611~ 11G 1~111111~1 11111111 M 3180
A M CCCATGA AAATGAAGAC TG M TC M CC M CT M GCTG TCAGCATTGC CGCAGGGTAA 3240
CTGAGACCTC CCTGCATTGG CTACGACTGC AG~l~lGGGA G~l~lGGGCA GGGGAGGGCC 3300
GGCTGGGGAG GGCCGGCTGG Gr~Ar~GGGAcA CAAAGTG M G AlGGGG611~ TTGGGCCTGA 3360
GClC~lGCCC AGCCllllCC GCCGGGGllC CTGGGTGGAT TC M GCCTCT TGGGGGAGAC 3420
GCGCTGCAGG GcTGr~AGrAT GGGClll~GG CCCTGAGGCT rAGGr~AGGAG CACATGGATG 3480
GGACTCACCT l~-lCCCllGC CCCCAGGACT GGGTCATCGC TCCCC M GGC TACTCGGCCT 3540
ATTACTGTGA GGGGGAGTGC lCCllCCCAC TGGACTCCTG CATGAATGCC ACCAACCACG 3600
CCATCCTGCA GTCCCTGGTC GATACCGTCG CCCATCCTGC CCAGCCCCCT GGTGGAGGCC 3660
W O 94/10203 PCT/US93/10520
21~7~98
- 140 -
CTGCAr,AGAG GG~ G~lC CAGCCAGCCG GGAGGCAGTG AGGCCACCTG CTCCATGTCT 3720
CGGGGC m G TCTGCACAGA GTCAGTM CG TCGCT M CTT CCCACAGCTC TGCAGG M CT 3780
GGlC~lCATA CAGCCACACT ACTA~ACATA GACCCACACC CAAACACGGA CACACGTG M 3840
CAGTCGCGTA TCATGCCTGT TCTATGCACT r.AACAAACTC CTGTGGGACA CTTACACACC 3900
TGCGTGCGGC GCTCAGAGGC ACAr,CACATG AAACArATGT GTACACTGTG TGGGGGCTGT 3960
GTGATCTTAA CACACGGGCC CCCGAGTACG CTGGCAAGTC TGACCGCCCG TGATATGTGC 4020
GCACAGTGTG TGGGGTGTGC GTGTGCATCA CCCACCTGTG CCGCACCACA GGTAGG M GC 4080
TTCTAGATGG TGTGGCTCTC M C~llllGG ~lllllCCCG CA~lllCl~l CTTGGCTGTC 4140
1~1~1111~1 CTGGATCCCC TGGCTTTTGA TGCCGllGGl GTCTGGGGCA ACCTTAAAGG 4200
ArAAAAGCAG GCTTCTGATG GGATCACTGG TGCTGCTCAC CACTGAGTGC lC~ 4260
GCGGATTCTG GCACCGAGGC llCCll~lAG M GTTTTTAC CTAGAATCCC AGTTCCTGGT 4320
ATTGCACAGC CTTATGTTTT CCTCTTAGGA GGTTC M CGG TGAlGCCllG ATCAGGCGCA 4380
25 GTGGCTCACC CTGT M TCGC AGCArACr.AG CCCAGM GTT cAArAcAAGc CTGAGCM CA 4440
CAGCAAAACC Cl~lCl~l M AATAAAAATT AAAACArAr,A CACACACACA CACArACACA 4500
CACACACGTG CGCACA~AAT GCCll~ l GAGAGG MM G M ATTACC M AAGCTGCTCT 4560
GAGCCTATGA TAATACTTCC m CTGGGCA GTCA M TGGT GTTTGCTGGA CACCCTGGAG 4620
CCAlClC~-ll GGAAAGGCCC AGGGGTGATG AGGAGCTCCG lCGGG~l æ C CTGGCCAGCA 4680
35 CC m ATGCC Gl~lGGll~-l CACAGCTGCA l~l~lGGGAG GTACATGGGA AGGTGACTGC 4740
ACCTGCGCTC CTGGACTCCA l~-lCC'l~lGC CCTTGCCCCT GCCC~lCACG TGC M CTAGA 4800
GTGAGTGCTC ACAGCCTACA GGGCAGCAAA CAGGCACTGT GCTCTAGGGG AGG~l~lCGG 4860
TGGGCACAGA AGCAAACCAA CCGTGGAGTT GACACCTCCT GTGAGGAAGA GCAGACGAGC 4920
CGTGCCGTCA GTGGAGTGAG ACTGGGCCCA GCTCTCCACA CAArJrAGGGG CACGTCAGCA 4980
45 GCTGGAGGAG G M TGTTCCA r~AAGr,AGCAA GTGC M GGCC CTAArArAGG AGCAGGCTGG 5040
CCCT M GTTC AGGGCAGGGG Ar~r~A~AGGGG CTGGGTGCAG TGM GGGGAG GAGAGTGGAG 5100
W O 94/10203 PCT/US93/10520
2~ 759~ ~
- 141 -
GGAGGTGATC CGGGGlGATA GGCCAGCTCC CGTAGCCTGG GTTCCCTGGG M GAGGGTGG 5160
ATTTTATTCC M GC M CCCC AGAGGCTGTC AGAGGl~llC AGcAAAr~AGT ~lC~ GlC 5220
TGCGTCACCC TccAGAAGr~A C~ lGGC TTGGGGAGGT CGCGGGAGTG G M GGCAGAG 5280
GAGCAGGGGA TGAGTGAGGG CTG~l~lG~l CACCTGGCAG GTGATGGCAG CTCGACTGGG 5340
CAGGlGGlCC GAGGCAGCAC GGAGGTGGAG GTTGAGCCAG GGGCTGCTCT CAGGG M GGG 5400
AGGAGGCG M AGGAGTCATC CAGGAGGCCT CCCAGGCGGG AGCTATGATG TCAGGGCGGG 5460
AGG M TTCTA TGTTCCACTG AGGCCTCATT AGACCCCC M GTGCAG M GT GGG M GGGGA 5520
GCAGGATCCG CAAGTCTGGA GTTcArAAr~A GAGGTCCAAG CTGAGCCAGG GGAGTGGAGA 5580
GGTGCGGGCC M TGCAGGGC CTTG M GTGC TGAGGGCGGA TCGAGTCCTC TGGGAG M GG 5640
AGCAGCACAG GAGAGGGGGC GAGGCTGGCT CCCAGAGCCT GGGGAGGr~AG GCAGGTGTGG 5700
GGAGGCAGAG CllGGGGGGG TCTG M GGGC TATAA~.AAr,A CAGTGGTCCT TCCAGGTTCC 5760
CCCTTGGACC TCACT M GGG r-ACAAACCTG GCCATGAGGT l~-lC~llCCC ATTATCCCCA 5820
GGAGG M GTC TGAGCCCTTG GCCTGGGACT CGAGGCCCCT CATTAGTGCC CTGCCCACCT 5880
GCCCCACACC CTGGGGCTGC CATGTATCCC TCCCTGGGCA CTGTGGGCAC CACAGCTCCC 5940
GCTCCCAGAG CTCTCAGGGC TGCTCTTATT CCTGTT M TA ATTCTTATTA TTGTGCTGCT 6000
CCCATGTGGC TTGGAGATGG CCAGGGCAGG GAGCAGGTGG AGCTGGGGCG GGCTAGGTGG 6060
GTCCTCAGAG GAGGCCACTG GCTCATGCCC CTGC~l~lGC lCCCll U lG GCCAGGTGCA 6120
CCTGATG M G cr-AAAcGcAG TCCCC M GGC GTGCTGTGCA CCCACC M GC TGAGCGCCAC 6180
~ lG~lC TACTATGACA GCAGC M C M CGTCATCCTG CGC M GCACC GC M CATGGT 6240
GGTC M GGCC TGCGGCTGCC ACTGAGTCAG CCCGCCCAGC CCTACTGCAG CCACC~ l 6300
CATCTGGATC GGGCCCTGCA GAGGCAG MM ACCCTTAAAT GCTGTCACAG CTC M GCAGG 6360
AGTGTCAGGG GCCCTCACTC TCTGTGCCTA ~llC~ lCA GG~ lGGl CClll~lC 6418
W O 94/10203 PCT/US93/10520
~4~ 5 ~8 142 -
(2) INFORNATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 97 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) ~OLECULE TYPE: protein
(ix) FEATURE:
(A) NAME/KEY: Protein
(B) LOCATION: 1..97
(D) OTHER INFORNATION: /label= Generic-Seq-7
/note= "wherein each gaa is independently selected
from a group of one or more specified amino acids
as defined in the specification. n
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Leu gaa gaa gaa Phe gaa gaa gaa Gly Trp gaa gaa gaa Xaa gaa Xaa
1 5 10 15
Pro gaa gaa gaa gaa Ala gaa Tyr Cys gaa Gly gaa Cys gaa Xaa Pro
20 25 30
Xaa gaa gaa gaa gaa gaa gaa gaa Asn His Ala gaa Xaa Xaa Xaa Xaa
35 40 45
Xaa gaa gaa gaa gaa gaa Xaa gaa gaa gaa gaa gaa Cys Cys Xaa Pro
gaa gaa gaa gaa gaa gaa gaa gaa Leu gaa gaa ~aa ~aa gaa gaa gaa
65 70 75 80
Val gaa Leu gaa gaa gaa gaa gaa Het gaa Val gaa gaa Cys gaa Cys
85 90 95
gaa
W O 94/10203 PCT/US93/10520
- 21 ~ 7598
- 143 -
(2) INFORNATION FOR SEQ ID NO:13:
(i) S~Qu~C~ CHARACTERISTICS:
(A) LENGTH: 102 amino acids
(B) m E: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) NOLECULE m E: protein
(ix) FEATURE:
(A) NANE/KEY: Protein
(B) LOCATION: 1..102
(D) OTHER INFORNATION: /label= Generic-Seq-8
/note= "wherin each Xaa is independently selected
from a group of one or more specified amino acids
as defined in the specification."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Cys gaa gaa gaa gaa Leu gaa gaa gaa Phe gaa gaa gaa Gly Trp gaa
1 5 10 15
gaa ~aa Xaa gaa gaa Pro gaa Xaa Xaa gaa Ala gaa Tyr Cys gaa Gly
gaa Cys gaa gaa Pro gaa gaa gaa gaa gaa gaa gaa Xaa Asn His Ala
35 40 45
gaa gaa gaa gaa gaa gaa gaa Xaa gaa gaa gaa Xaa Xaa gaa Xaa Xaa
50 55 60
gaa Cys Cys gaa Pro gaa gaa Xaa gaa gaa gaa gaa gaa Leu gaa gaa
65 70 75 80
Xaa gaa gaa ~aa gaa Val gaa Leu gaa ~aa gaa ~aa Xaa Met gaa Val
85 90 95
gaa gaa Cys gaa Cys gaa
100
