Note: Descriptions are shown in the official language in which they were submitted.
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MORPHOGEN-INDUCED ENHANCEMENT OF FERTILITY
FIELD OF THE INVENTION
The invention relates generally to methods and compositions for the modulation
of
human fertility. More particularly, the present invention relates to methods
and compositions
the to enhance fertility, methods and compositions to delay the onset or
alleviate symptoms of
menopause, and methods and compositions to decrease fertility.
BACKGROUND OF THE INVENTION
Morphogens are members of the TGF-(3 superfamily that perform essential
physiological functions in morphogenesis and organogenesis. Morphogens are
expressed in a
tissue-specific manner in many different cell types during embryonic and adult
life in both
vertebrates and invertebrates. The importance of morphogens in regulating
crucial events in
morphogenesis, organogenesis, and cytodifferentiation has been clearly
established from
studies of morphogen-deficient animals.
Morphogens, also referred to as osteogenic proteins (OPs) or bone morphogenic
proteins (BMPs), are generally classified as a subgroup of the TGF-(3
superfamily of growth
factors. Hogan, Genes c~ Development 10: 1580-1594 (1996). Members of the
morphogen
family of proteins include the mammalian osteogenic protein-1 (OP-l, also
known as BMP-7,
and the Drosoplzila homolog 60A), osteogenic protein-2 (OP-2, also known as
BMP-8),
osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the
Drosophila homolog dpp), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-
5,
BMP-6 and its murine homolog Vgr-1, BMP-9, BMP-10, BMP-11, BMP-12, GDF3 (also
known as Vgr2), GDFB, GDF9, GDF10, GDFl l, GDF12, BMP-13, BMP-14, BMP-15,
GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7
(also
known as CDMP-3), the Xenopus homolog Vgl and NODAL, UNIVIN, SCREW, ADMP, and
NEURAL.
Members of this family encode secreted polypeptides that share common
structural
features. The mature form of such proteins results from processing through a
"pro-form" to
yield a mature polypeptide chain competent to dimerize and containing a
carboxyl terminal
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active domain of approximately 97-106 amino acids. All members share a
conserved pattern
of cysteines in this domain and the active form of these proteins can be
either a
disulfide-bonded homodimer of a single family member or a heterodimer of two
different
members. See, e.g., Massague, Auuu. Rev. Cell Biol. 6: X97 (1990); Sampath et
u1., J. Biol.
Chem. 265: 13198 (1990). See also, U.S. Patents 5,011,691, and 5,266,683;
Ozkaynak et al.,
EMBO J. 9: 2085-2093 ( 1990), Wharton et al., Proc. Natl. Acad. Sci USA 88: 92
14-9218
(1991); Ozkaynak, J. Biol. Chem. 267: 25220-25227 (1992); Celeste et al.,
Proc. Natl. Acad.
Sci USA 87: 9843-9847 (1991); Lyons et al., Proc. Natl. Acad. Sci USA 86: 554-
4558 (1989).
These disclosures describe the amino acid and DNA sequences, as well as the
chemical and
physical characteristics, of morphogens. See also, Wozney et al., Science 242:
1528-1534
(1988); PCT application WO 93/00432; Padgett et al., Nature 325: 81-84 (1987);
and Weeks,
Cell ~l: 861-867 (1987).
The biological effects of morphogens are mediated by specific cell surface
receptors.
morphogen receptors exist as two subtypes, the type I receptors and the type
II receptors. Both
types of morphogen receptors are structurally similar and both types possess
intrinsic
serine/threonine kinase activity. Two type I morphogen receptors, BMPR-IA (or
ALK-3) and
BMPR-IB (or ALK-6), have been identified. One type II morphogen receptor, BMPR-
II, has
also been identified. Individually, either type I morphogen receptors or type
II morphogen
receptors can bind morphogen as a ligand with low affinity. However, both
receptor types are
necessary to achieve high affinity binding and ligand-mediated signal
transduction. After the
ligand-receptor complex is formed, the type II receptor phosphorylates and
activates the type I
receptor. The type I receptor then triggers downstream events in the morphogen-
signaling
pathway.
Although much is known about the cellular function and biological importance
of
morphogen signaling in a many embryonic and adult tissues, the role of
morphogen signaling
in the reproductive system is poorly understood. Thus far, no experimental
evidence has
shown that morphogen action is available in reproductive cells for any
species. Messenger
ribonucleic acids (mRi'~lAs) encoding BMP-2, BMP-3, BMP-3b, BMP-6 and BMP-15
have
been identified in manunalian ovaries and expression of BMP-6 and BMP-15 has
been
localized to the oocytes by in situ hybridization. However, nothing is known
about the
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expression of receptors for morphogens or the functional consequences of
morphogen receptor
activation in the ovary.
Two hormones known to be important for the regulation of human fertility are
the
follicle stimulating hormone (FSH) and the luteinizing hormone (LH), each
produced by the
anterior hypothalmus in the brain. FSH stimulates ovarian follicle growth in
females (a
physiological process that depends on the hormone estrogen) and
spermatogenesis in males.
LH stimulates ovulation and luteinization of ovarian follicles in females (a
physiological
process that depends on the hormone progesterone) and testosterone secretion
in males.
Together, FSH and LH stimulate sex hormone release and regulate the hormonal
balance of
estrogen and progesterone. All four of these hormones are necessary for the
development of
the ovarian follicle, the female reproductive organ in which an oocyte (egg
cell) is surrounded
by one or more layers of granulosa cells, as well as other cells. In the final
stage of ovarian
differentiation, a cavity forms in the ovary follicle; the ovarian follicle is
then termed a
Graafian follicle. A major concept in ovarian physiology is that FSH-dependent
ovarian
follicle growth is marked by increasing synthesis of estrogen, but not
progesterone, in the cells
of the developing ovarian follicle. This transformation of the mature ovarian
follicle and its
theca interna into a corpus luteum after ovulation, and the formation of
luteal tissue is termed
luteinization. It has been known for many years that a "luteinization
inhibitor" plays an
important role in inhibiting follicular progesterone production, but the
molecular nature of the
"luteinization inhibitor" remains a mystery.
Thus, there is a need in the art for the identification of the long sought
"luteinization
inhibitor" in Graafian follicles during follicular growth and development and
the medical use
of the "luteinization inhibitor" in the treatment of human fertility.
SUMMARY OF THE INVENTION
The invention provides a method for increasing fertility, by providing a
"luteinization
inhibitor" to a female subject in the form of a therapeutically effective
amount of a morphogen
pharmaceutical. The morphogen is a peptide having an amino acid sequence
selected from a
sequence: (1) having at least 70% homology with the C-terminal seven-cysteine
skeleton of
human OP-1. amino acids 330-431 of SEQ ID NO: 2; (2) having greater than 60%
amino acid
sequence identity with said C-terminal seven-cysteine skeleton of human OP-1;
(3) defined by
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SEQ ID NO: 5; (4) defined by SEQ ID NO: 6; (6) defined by SEQ ID NO: 7; (7)
defined by
SEQ ID NO: 8; or (8) defined by OPX, SEQ ID NO: 4. In one embodiment, the
therapeutically effective amount is nanomolar. The administration of the
morphogen induces
estrogen synthesis by the ovary of the subject, and can also attenuate
progesterone synthesis by
the ovary of the subject. The subject receiving the morphogen can have healthy
ovary
follicles, atretic ovary follicles, or both.
The invention also provides a method for alleviating symptoms of menopause or
for
delaying the onset of menopause, in which a therapeutically effective amount
of a morphogen
is administered to the subject. The morphogen induces ovarian follicle growth,
which is
marked by increasing ovarian synthesis of estrogen The administration of the
morphogen can
also attenuate progesterone synthesis by the ovary of the subject. Morphogens
can thus be
used to prolong promote follicular growth over the duration of a women's life
when she can
have menstrual cycles. Because menstruation results from a balance of hormonal
factors,
those factors that promote follicular growth can be used to delay the onset of
menstruation.
1 S The invention further provides a method for contraception. The method
reduces
ovarian follicular growth, and thus ovulation and production of hormones in
the ovary, by
administering to a subject a compound which interferes with the binding of the
morphogen
and its receptor on oocytes or ovarian granulosa cells .is administered.
Treating a subject with
the compound results in a subdued ovulation and an increased luteinization of
the follicles,
decreasing the fertility of the woman receiving the compound. In one
embodiment, the
compound that interferes the binding of the morphogen to its receptor is an
anti-morphogen
antibody. In another embodiment, the compound that interferes the binding of
the morphogen
to its receptor is an anti-receptor antibody. In yet another embodiment, the
compound that
interferes the binding of the morphogen to its receptor is a morphogen
receptor antagonist.
As used herein, the terms "morphogen," "bone morphogen," "osteogenic protein,"
"OP," "bone morphogenic protein," "BMP," "morphogenic protein" and
"morphogenetic
protein" all embrace the class of proteins typified by human osteogenic
protein 1 (hOP-1).
Nucleotide and amino acid sequences for hOP-1 are provided in SEQ ID NOS: 1
and 2,
respectively. For ease of description, hOP-1 is considered a representative
morphogen. It will
be appreciated that OP-1 is merely representative of the TGF-(3 subclass of
true tissue
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morphogens and is not intended to limit the description. Other known and
useful morphogens
include, but are not limited to, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6,
BMP-8,
BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-l~. GDF-1, GDF-2, GDF-3, GDF-5,
GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-1 l, GDF-12, 60A, NODAL, UNIVIN,
SCREW, ADMP, and NEURAL, and morphogenically-active amino acid variants of any
thereof.
In specific embodiments, useful morphogens include those sharing the conserved
seven
cysteine skeleton, and sharing at least 70% amino acid sequence homology
(similarity), within
the C-terminal seven-cysteine skeleton of human OP-1, residues 330-431 of SEQ
ID NO: 2
(hereinafter referred to as the presently-preferred reference sequence). In
another embodiment,
the invention encompasses use of biologically active species (phylogenetic)
variants of any of
the morphogenic proteins recited herein, including conservative amino acid
sequence variants,
proteins encoded by degenerate nucleotide sequence variants, and
morphogenically-active
proteins sharing the conserved seven cysteine skeleton as defined herein and
encoded by a
DNA sequence competent to hybridize under standard stringency conditions to a
DNA
sequence encoding a morphogenic protein disclosed herein, including, without
limitation,
OP-1 or BMP-2 or BMP-4. Presently, however, the preferred reference sequence
is that of
residues 330-431 of SEQ ID NO: 2 (OP-1).
In still another embodiment, morphogens useful in methods and compositions of
the
invention are defined as morphogenically-active proteins having any one of the
generic
sequences defined herein, including OPX (SEQ ID NO: 3) and Generic Sequences 7
and 8
(SEQ ID NOS: 5 and 6, respectively), or Generic Sequences 9 and 10 (SEQ ID
NOS: 7 and 8,
respectively). OPX encompasses the observed variation between the known
phylogenetic
counterparts of the osteogenic OP-1 and OP-2 proteins, and is described by the
amino acid
sequence presented herein below and in SEQ ID NO: 3. Generic Sequence 9 is a
97 amino
acid sequence containing the C-terminal six cysteine skeleton observed in hOP-
1 (residues
335-431 of SEQ ID NO: 2) and wherein the remaining residues encompass the
observed
variation among OP-l, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8,
BMP-9, BMP-10, BMP-11, BMP-1~, GDF-1. GDF-3, GDF-5, GDF-6, GDF-7,
GDF-8, GDF-9, GDF-10, GDF-11, 60A, UNIVIN, NODAL, DORSALIN, NEURAL,
SCREW and ADMP. That is, each of the non-cysteine residues is independently
selected from
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the corresponding residue in this recited group of known, naturally-sourced
proteins. Generic
Sequence 10 is a 102 amino acid sequence which includes a five amino acid
sequence added to
the N-terminus of the Generic Sequence 9 and defines the seven cysteine
skeleton observed in
hOP-1 (330-431 SEQ ID NO: 2). Generic Sequences 7 and 8 are 97 and 102 amino
acid
sequences, respectively, containing either the six cysteine skeleton (Generic
Sequence 7) or the
seven cysteine skeleton (Generic Sequence 8) defined by hOP-1 and wherein the
remaining
non-cysteine residues encompass the observed variation among OP-1, OP-2, OP-3,
BMP-2,
BMP-3, BMP-4, 60A, dpp, Vgl, BMP-S, BMP-6, Vgr-1, and GDF-1.
Of particular interest herein are morphogens which, when provided to a
specific tissue
of a mammal, induce tissue-specific morphogenesis or maintain the normal state
of
differentiation and growth of that specific tissue. In preferred demonstrative
embodiments, the
present morphogens induce the formation of vertebrate (e.g., avian or
mammalian) body
tissues, such as but not limited to nerve, eye, bone, cartilage, bone marrow,
ligament, tooth
dentin, periodontium, liver, kidney, lung, heart, or gastrointestinal lining.
The present
demonstrations can be carried out in the context of developing embryonic
tissue, or at an
aseptic, unscarred wound site in post-embryonic tissue. Methods of identifying
such
morphogens, or morphogen receptor agonists, are known in the art and include
assays for
compounds which induce morphogen-mediated responses (e.g., induction of
endochondral
bone formation, induction of differentiation of metanephric mesenchyme, and
the like). In a
currently preferred demonstrative embodiment, morphogens of the present
invention, when
implanted in a mammal in conjunction with a matrix permissive of bone
morphogenesis, are
capable of inducing a developmental cascade of cellular and molecular events
that culminates
in endochondral bone formation. See U.S. Patent 4,968,590 and Sampath, et at.,
Proc. Natl.
Acad. Sci USA 80: 659 1-6595 (1983), the disclosures of which are incorporated
herein by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 (panels 1-A through 1-M) are a tabular alignment of the amino acid
sequences of
various naturally-occurring morphogens with a preferred reference sequence of
human OP-l,
residues 330-431 of SEQ ID NO: 1.
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FIG. 2 is a tabular presentation of alternative amino acids for "Xaa"
positions in
generic sequences SEQ ID NOS: 5, 6, and 9 that represent amino acid variations
in known
morphogens.
FIG. 3 is a tabular presentation of alternative amino acids for "Xaa"
positions in
generic sequences SEQ ID NOS: 5, 6, and 9 that represent amino acid variations
in known
morphogens.
FIG. 4 is a tabular presentation of alternative amino acids for "Xaa"
positions in
generic sequences SEQ ID NOS: 7, 8, and 10 that represent amino acid
variations in known
morphogens.
FIG. 5 is a set of graphs showing the effects of morphogens on estrogen and
progesterone production by granulosa cells. Granulosa cells (5 x 10~ viable
cells/well/200 ~1)
were cultured for 48 hours (hr) in serum-free medium containing
androstenedione (1 pM), and
either no additions (control), BMP-4 (3, 10, or 30 ng/ml), BMP-7 (3, 10, or 30
ng/ml), FSH
(0.1, 0.3, 1, 3, or 10 ng/ml) or their combination. After culture, estrogen
levels (Panels A and
C) and progesterone levels (Panels B and D) in the conditioned media were
measured by
radioimmunoassay.
FIG. 6 is a set of graphs showing the time course effect of BMP-7 on estrogen
and
progesterone production by granulosa cells. Granulosa cells (5 x 10~ viable
cells/well/200 ~1)
were cultured for 48 hr in serum-free medium containing androstenedione (1
~M), and FSH
(3 ng/ml) in the absence or presence of BMP-7 (30 ng/ml). After culture,
estrogen levels
(Panel A) and progesterone levels (Panel B) in the conditioned media were
measured by
radioimmunoassay.
FIG. 7 is a set of graphs showing that FSH/3Luc expression is increased by
morphogens
and activin.
FIG. 8 is a set of graphs showing dose-dependent inhibition of FSH(3Luc
expression
using rabbit anti mOP-11.
FIG. 9 is a set of bar graphs showing the neutralizing effects of mOP-1 and
activin
antibodies.
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DETAILED DESCRIPTION OF THE INVENTION
A. General
The invention provides methods for increasing fertility by inhibiting
luteinization in
the ovarian follicles. Luteinization can be an important part of a healthy
menstrual cycle when
induced by the luteinization hormone (LH), an increase in the levels of which
occur normally
prior to ovulation. However, luteinization is a complex differentiation
process involving the
interaction of extrinsic and intraovarian factors. Undesirable luteinization
caused by a low
production of hormones by ovarian cells can result in a lowered fertility. In
one embodiment,
the invention provides a method for increasing fertility, by providing a
"luteinization inhibitor"
to a female subject in the form of a morphogen pharmaceutical.
It is well known that an insufficiency in ovarian follicular growth can cause
a
decreased fertility. Daly, Fertil. Steril. 51(1):51-7 (1989). Reduced
fertility can be due to an
inappropriate luteinization, in which the luteal phase defect is associated
with either an
impaired follicular growth or an abnormal surge in LH levels. Ayabe et al.,
Fertil. Steril.
61 (4):652-6 (1994); Lewinthal et al., Fertil. Steril. 46(5):833-9 (1986).
The administration of morphogens can increase fertility by several mechanisms.
First,
morphogens can act directly on the cells of the ovary to increase ovarian
synthesis of estrogen.
Second, morphogens act on the pituitary to increase synthesis of FSH. FSH then
binds to
granulosa cells in the ovaries, stimulating estrogen production. High FSH
levels also advance
the preovulatory stage of the dominant follicle in the early follicular phase
of the cycle.
Furthermore, FSH controls follicle development in women at the recruitment-
selection stage.
Messinis et al., Hum Reprod 5(2):153-6 (1990). Third, morphogens can act on
ovarian cells to
potentiate the effects of FSH. Recent findings from the study of the
regulation of follicular
development show that the potentiating effect of various growth factors on
ovarian sensitivity
to FSH. Lunenfeld et al., Baillieres Clin Obstet Gynaecol 4(3):473-89 (1990).
As shown
below, administration of morphogens can act in a more direct manner to
potentiate the effects
of FSH. Finally, morphogens can act on ovarian cells to increase synthesis of
FSH.
In one embodiment, the invention provides a method for alleviating symptoms
menopause. Menopause results from the ovaries decreasing their production of
the sex
hormones estrogen and progesterone. The drop in estrogen levels causes the
most common
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symptoms during menopause. Current methods for treating menopause include
estrogen
replacement therapies, taken in the form of oral tablets, skin patches, or
injections. Estrogen
circulates through the body to reduce the short-term changes of menopause. The
combination
therapy of estrogen plus progesterone is called hormone replacement therapy.
As an
alternative to estrogen replacement therapies or hormone replacement
therapies, estrogen
receptor modulators are used to treat menopause.
The invention provides a method of using morphogens to induce FSH-dependent
ovarian follicle growth, which is marked by increasing ovarian synthesis of
estrogen. The
invention thus provides another treatment, which can be an alternative to or a
supplement for
estrogen replacement therapies. Morphogens can be used to prolong promote
follicular growth
over the duration of a women's life when she can have menstrual cycles.
Because
menstruation results from a balance of hormonal factors, those factors that
promote follicular
growth can be used to delay the onset of menstruation. By promoting healthy
follicular
growth, the method of the invention also results in increased ovarian levels
of estrogen.
The invention also provides a method of contraception. Treating a female
subject with
a compound that prevents the binding of morphogen to a morphogen receptor on
oocytes or
ovarian granulosa cells will result in a subdued ovulation and an increased
luteinization of the
follicles. It is well known in the art that a woman's fertility is reduced as
her ovulation
diminishes, or when her ovaries' production of estrogen and other sex hormones
decreases.
The invention provides a method for reducing ovarian follicular growth and
thus
ovulation and production of hormones in the ovary, by administering compounds
which
interfere with morphogen activity in promoting follicular growth early in the
menstrual cycle.
This decreases the fertility of the woman receiving the compounds that prevent
the binding of
morphogen to BMP receptor.
In the present invention, the cellular sites of expression of the morphogen
type IA,
type IB, and type II receptors (BMPR-IA, BMPR-IB, BMPR-II) mRNAs and BMP-4 and
BMP-7 mRNAs were characterized in the rat ovary, establishing for the first
time the existence
of a functional morphogen ligand-receptor system in the ovary of any species.
The genes
encoding morphogens and the genes encoding the family of morphogen receptors
are
expressed in a cell-type-specific manner. The co-expression of BMP-4 and BMP-7
mRNAs
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by the theca cells indicates the coordinate regulation of these two morphogens
during ovarian
follicle development. The expression of both morphogens is stage-specific in
the cycle of
folliculogenesis, being very high in healthy follicles but barely detectable
in follicles
undergoing atresia. This pattern of expression shows that the coordinate
expression of these
two morphogens is subject to different patterns of regulation during follicle
development and
atresia.
Tissue culture results demonstrate that morphogens elicit key biological
responses in
granulosa cells. Two types of responses were observed. First, BMP-4 and BMP-7
both
caused a time-dependent and dose-dependent amplification FSH-induced estrogen
production
and a time-dependent and dose-dependent attenuation of FSH-induced
progesterone
production. The ED;° of the morphogen responses (~10 ng/ml or ~3 x
10'°M) is equivalent to
the reported Kd of the morphogen receptor (2.54 x 10'° M). This
strongly suggests that
morphogen responses are mediated by cell surface receptors and are
physiological. Second,
granulosa cells are more sensitive to FSH-action after morphogen treatment.
This effect
appears to be coupled only to estrogen production. The interaction of
nanomolar amounts of
BMP-4 and BMP-7 with morphogen receptors causes marked stimulatory effects on
FSH-induced estrogen production and inhibitory effects on FSH-induced
progesterone
production, respectively. Thus, morphogens appear to influence FSH signaling
pathways to
promote estrogen production, and decrease progesterone production.
Growth hormone and insulin-like growth factor-I (IGF-I) are potent stimulators
of
BMP-4 mRNA levels in human dental pulp fibroblasts cultured in vitYO. IGF-I is
a potent
stimulator of rat theca cell function. Also, IGF-I expression is strong and
weak in healthy and
atretic follicles respectively. Thus, IGF-I may be a physiological stimulus
for morphogens
expression during ovarian follicle growth.
B. Biochemical, Structural and Functional Properties of Bone Morphogenic
Proteins
In its mature, native form, natural-sourced morphogen is a glycosylated dimer,
typically having an apparent molecular weight of about 30-36 kDa as determined
by
SDS-PAGE. When reduced, the 30 kDa protein gives rise to two glycosylated
peptide
subunits having apparent molecular weights of about 16 kDa and 18 kDa. In the
reduced state,
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the protein has no detectable osteogenic activity. The unglycosylated protein,
which also has
osteogenic activity, has an apparent molecular weight of about 27 kDa. When
reduced, the 27
kDa protein gives rise to two unglycosylated polypeptide chains, having
molecular weights of
about 14 kDa to 16 kDa. Typically, the naturally-occurring morphogens are
translated as a
precursor, having an N-terminal signal peptide sequence typically less than
about 30 residues,
followed by a "pro" domain that is cleaved to yield the mature C-terminal
domain. The signal
peptide is cleaved rapidly upon translation, at a cleavage site that can be
predicted in a given
sequence using the method of Von Heijne, Naicleic Acids Res. 14: 4683-4691
(1986). The pro
domain typically is about three times larger than the fully processed mature C-
terminal
domain.
Morphogens useful herein include any known naturally-occurring native proteins
including allelic, phylogenetic counterpart and other variants thereof,
whether
naturally-occurring or biosynthetically produced (e.g., including "muteins" or
"mutant
proteins"), as well as new, osteogenically active members of the general
morphogenic family
of proteins.
Particularly useful sequences include those comprising the C-terminal 97 or
102 amino
acid sequences of dpp (from Drosophila), Vgl (from Xenopus), Vgr-1 (from
mouse), the OP-1
and OP-2 proteins (see U.S. Patents 5,011,691 and 5,266,683; Ozkaynak et al.,
EMBO J. 9:
2085-2093 ( 1990)), as well as the proteins referred to as BMP-2, BMP-3, BMP-4
(see
WO 88/0020, U.S. Patent 5,013,649 and WO 91/18098), BMP-5 and BMP-6 (see
WO 90/11366, PCT/U590/01630), BMP-8 and BMP-9. Other proteins useful in the
practice
of the invention include active forms of OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-
4, BMP-5,
BMP-6, BMP-9, GDF-5, GDF-6, GDF-7, dpp, Vgl, Vgr, 60A protein, GDF-1, GDF-3,
GDF-5,
GDF-6, GDF-7, BMP-10, BMP-11, BMP-13, BMP-15, LTNIVIN, NODAL, SCREW, ADMP or
MURAL and amino acid sequence variants thereof. In one currently preferred
embodiment,
morphogens include any one of: OP-1, OP-2, OP-3, BMP-2, BMP-4, BMP-5, BMP-6,
BMP-9,
and amino acid sequence variants and homologs thereof, including species
homologs, thereof.
Publications disclosing OP-1 and OP-2 sequences, as well as their chemical and
physical
properties, include U.S. Patents 5,011,691 and 5,266,683, incorporated by
reference herein.
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In preferred embodiments, morphogens for use in methods of the invention
include
proteins having at least 70% homology with the amino acid sequence of the C-
terminal
seven-cysteine skeleton of human OP-l, SEQ ID NO: 2, and having the ability to
induce
endochondral bone formation in the Reddi and Sampath assay described herein.
Compounds
that meet these requirements are considered functionally equivalent to a known
response
morphogen. To determine whether a candidate amino acid sequence is
functionally equivalent
to a reference morphogen, the candidate sequence and the reference sequence
are aligned. The
first step for performing an alignment is to use an alignment tool, such as
the dynamic
programming algorithm described in Needleman et al., .l. Mol. Biol. 48: 443
(1970), and the
Align Program, a commercial software package produced by DNAstar, Inc. the
teachings of
which are incorporated by reference herein. After the initial alignment is
made, it is then
refined by comparison to a multiple sequence alignment of a family of related
proteins, such as
those shown in FIG. 1A through 1M, which is a multiple sequence alignment of a
family of
known morphogens, including hOP-1. Once the alignment between the candidate
and
1 S reference sequences is made and refined, a percent homology score is
calculated. The
individual amino acids of each sequence are compared sequentially according to
their
similarity to each other.
Similarity factors include similar size, shape and electrical charge. One
particularly
preferred method of determining amino acid similarities is the PAM250 matrix
described in
Dayhoff et al., 5 ATLAS OF PROTEIN SEQUENCE AND STRUCTURE 345-352 (1978 &
Supp.),
incorporated by reference herein. A similarity score is first calculated as
the sum of the
aligned pairwise amino acid similarity scores. Insertions and deletions are
ignored for the
purposes of percent homology and identity. Accordingly, gap penalties are not
used in this
calculation. The raw score is then normalized by dividing it by the geometric
mean of the
scores of the candidate compound and the seven cysteine skeleton of hOP-1. The
geometric
mean is the square root of the product of these scores. The normalized raw
score is the percent
homology.
In an alternative preferred embodiment, a functionally-equivalent morphogen
sequence
shares at least 60% amino acid identity with a reference sequence. That is,
any 60% of the
aligned amino acids are identical to the corresponding amino acids in the
reference sequence.
Any one or more of the naturally-occurring or biosynthetic morphogens
disclosed herein may
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be used as a reference sequence to determine whether a candidate sequence
falls within the
morphogen family. In a preferred embodiment, the reference sequence is the C-
terminal
seven-cysteine skeleton sequence of human OP-1 as shown in SEQ ID NO: 2.
Examples of
conservative substitutions for use in the above calculations include the
substitution of one
amino acid for another with similar characteristics, e.g., substitutions
within the following
groups are well-known: (a) valine, glycine; (b) glycine, alanine; (c) valine,
isoleucine, leucine;
(d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) seine,
threonine; (g) lysine,
arginine, methionine; and (h) phenylalanine, tyrosine. The term "conservative
variant" or
conservative variation" also includes the use of a substituted amino acid in
place of an
unsubstituted parent amino acid in a given polypeptide chain, provided that
antibodies having
binding specificity for the resulting substituted polypeptide chain also have
binding specificity
(i.e., "crossreact" or "immunoreact" with) the unsubstituted or parent
polypeptide.
In a preferred embodiment, morphogens useful in the resent invention are
defined by a
generic amino acid sequence that represents variations in known morphogens.
For example,
SEQ ID NOS: 4 and 5 encompass observed variations between preferred
morphogens,
including OP-1, OP-2, OP-3, CBMP-2A, CBMP-2B, BMP-3, 60A, dpp, Vgl, BMP-S, BMP-
6,
Vgr-1, and GDF-1. SEQ ID NO: S includes all of SEQ ID NO: 4, and also includes
at its
N-terminus the five amino acid sequence of SEQ ID NO: 8. 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 (SEQ ID NOS: S and 6, respectively), and
alternative
amino acids for variable positions within the sequence. Positions that allow
for alternative
amino acids are represented by "Xaa". FIG. 2 shows the alternative amino acids
for each
"Xaa" position in SEQ ID NOS: 5, 6 and 9. For example, refernng to SEQ ID NO:
6 and
FIG. 2, the "Xaa" at position 2 may be a tyrosine or a lysine. The generic
sequences provide
an appropriate cysteine skeleton for inter- or intramolecular disulfide
bonding, and contain
certain critical amino acids likely to influence the tertiary structure of the
proteins. In addition,
the "Xaa" at position 36 in SEQ ID NO: 5, or at position 41 in SEQ ID NO: 6,
may be an
additional cysteine, thereby encompassing the morphogenically-active sequences
of OP-2 and
OP-3.
In another embodiment, useful morphogens include those defined by SEQ ID NOS:
7
or 7, which are composite amino acid sequences of the following morphogens:
human OP-l,
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human OP-2, human OP-3, human BMP-2, human BMP-3, human BMP-4, human BMP-5,
human BMP-6, human BMP-8, human BMP-9, human BMP-10, human BMP-11, DrosoplZila
60A, Xenopus Vg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-S), human CDMP-2
(mouse GDF-6, human BMP-13), human CDMP-3 (mouse GDF-7, human BMP-12), mouse
GDF-3, human GDF-l, mouse GDF-1, chicken DORSALIN, Drosophila dpp, Dr-osophila
SCREW, mouse NODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10,
human GDF-11, mouse GDF-11, human BMP-15, and rat BMP-3b. SEQ ID NO: 8
includes
all of SEQ ID NO: 7 and also includes at its N-terminus the five amino acid
sequence of SEQ
ID NO: 10. SEQ ID NO: 7 accommodates the C-terminal six-cysteine skeleton, and
SEQ ID
NO: 8 accommodates the seven-cysteine skeleton. Positions that allow for
alternative amino
acids are represented by "Xaa". FIG. 4 shows the alternative amino acids for
each "Xaa"
position in SEQ ID NOS: 7, 8 and 10.
As noted above, certain preferred morphogen sequences useful in this invention
have
greater than 60% identity, preferably greater than 65% identity, with the
amino acid sequence
defining the preferred reference sequence of hOP-1. These particularly
preferred sequences
include allelic and phylogenetic variants of the OP-1 and OP-2 proteins,
including the
Drosophila 60A protein, as well as the closely related proteins BMP-5, BMP-6
and Vgr-1.
Accordingly, in certain particularly preferred embodiments, useful morphogens
include
proteins comprising the generic amino acid sequence SEQ ID NO: 3 (referred to
herein as
"OPX"), which defines the seven-cysteine skeleton and accommodates the
homologies
between several identified variants of OP-1 and OP-2. Positions that allow for
alternative
amino acids are represented by "Xaa". FIG. 4 shows the alternative amino acids
for each
"Xaa" position in SEQ ID NO: 3.
In still another preferred embodiment, useful morphogens include those having
an
amino acid sequence encoded by a polynucleotide that hybridizes under high
stringency
conditions with DNA or RNA encoding a reference morphogen. Standard stringency
conditions are well characterized in standard molecular biology texts. See
generally,
MOLECULAR CLONING: A LABORATORY MANUAL, (Sambrook et al., eds., 1989); DNA
CLONING, Vol. I & II (D.N. Glover ed., 1985); OLIGONUCLEOTIDE SYNTHESIS (M.J.
Gait ed.,
1984); NUCLEIC ACID HYBRIDIZATION (B. D. Hames and S.J. Higgins eds., 1984);
B. Perbal, A
PRACTICAL GUIDE TO MOLECULAR CLONING (1984).
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In another embodiment, morphogens useful in the invention include the soluble
complex form comprising a mature morphogen dimer linked to a morphogen pro
domain or a
solubility-eWancing fragment thereof. A solubility-enhancing fragment is any N-
terminal or
C-terminal fragment of a morphogen pro domain that forms a complex with the
mature
morphogen diner and increases the solubility of the morphogen diner.
Preferably, the soluble
complex comprises a morphogen diner and two pro domain peptides. Morphogen
soluble
complex is described in published application WO 94/03600, incorporated by
reference herein.
In yet another embodiment, useful morphogens include biologically active
biosynthetic
constructs, including novel biosynthetic morphogens and chimeric proteins
designed using
sequences from two or more known morphogens. See U.S. Patent 5,011,691,
incorporated by
reference herein (e.g., COP-l, COP-3, COP-4, COP-5, COP-7, and COP-16).
C. Formulations and Methods of Treatment
Compositions of the present invention (i. e., comprising a molecules capable
of
releasing morphogen inhibition administered, alone or in combination with a
morphogen) may
be administered by any route which is compatible with the particular molecules
and, when
included, with the particular morphogen. Thus, as appropriate, administration
may be oral or
parenteral, including intravenous and intraperitoneal routes of
administration. In addition,
administration may be by periodic injections of a bolus of the composition, or
may be made
more continuous by intravenous or intraperitoneal administration from a
reservoir which is
external (e.g., an i.v. bag) or internal (e.g., a bioerodable implant, or a
colony of implanted,
morphogen-producing cells).
Therapeutic compositions of the present invention may be provided to an
individual by
any suitable means, directly (e.g., locally, as by injection, implantation or
topical
administration to a tissue locus) or systemically (e.g., parenterally or
orally). Where the
composition is to be provided parenterally, such as by intravenous,
subcutaneous,
intramolecular, ophthalmic, intraperitoneal, intramuseular, buccal, rectal,
vaginal, intraorbital,
intracerebral, intracranial, intraspinal, intraventricular, intrathecal,
intracisternal, intracapsular,
intranasal or by aerosol administration, the composition preferably comprises
part of an
aqueous or physiologically compatible fluid suspension or solution. Thus, the
carrier or
vehicle is physiologically acceptable so that in addition to delivery of the
desired composition
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to the patient, it does not otherwise adversely affect the patient's
electrolyte and/or volume
balance. The fluid medium for the agent thus can comprise normal physiologic
saline (e.g.,
9.8~% aqueous NaCI, 0.15 M, pH 7-7.4).
For morphogens, association of the mature morphogen dimer with a morphogen pro
domain results in the pro form of the morphogen which typically is more
soluble in
physiological solutions than the corresponding mature form. In fact,
endogenous morphogens
are thought to be transported (e.g., secreted and circulated) in the mammalian
body in this
form. This soluble form of the protein can be obtained from culture medium of
morphogen-secreting mammalian cells, e.g., cells transfected with nucleic acid
encoding and
competent to express the morphogen. Alternatively, a soluble species can be
formulated by
complexing the mature, morphogenically-active polypeptide dimer (or an active
fragment
thereof) with a morphogen pro domain polypeptide or a solubility-enhancing
fragment thereof.
Solubility-enhancing pro domain fragments can be any N-terminal, C-terminal or
internal
fragment of the pro region of a member of the morphogen family that complexes
with the
mature polypeptide dimer to enhance stability and/or dissolubility of the
resulting noncovalent
or convalent complex. Typically, useful fragments are those cleaved at the
proteolytic site
Mg-Xaa-Xaa-Mg. A detailed description of soluble complex forms of morphogenic
proteins,
including how to make, test and use them, is described in WO 94/03600. In the
case of OP-1,
useful pro domain polypeptide fragments include the intact pro domain
polypeptide (residues
30-292) and fragments 48-292 and 158-292, all of SEQ ID NO: 2. Another
molecule capable
of enhancing solubility and particularly useful for oral administrations, is
casein. For example,
addition of 0.2% casein increases solubility of the mature active form of OP-1
by 80%. Other
components found in milk and/or various serum proteins may also be useful.
Useful solutions for parenteral administration may be prepared by any of the
methods
well known in the pharmaceutical art, described, for example, in IZEMINGTON's
PHARMACEUTICAL SctENCES (Gennaro, A., ed.), Mack Pub., 1990. Formulations of
the
therapeutic agents of the invention may include, for example, polyalkylene
glycols such as
polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and
the like.
Formulations for direct administration, in particular, may include glycerol
and other
compositions of high viscosity to help maintain the agent at the desired
locus. Biocompatible,
preferably bioresorbable, polymers, including, for example, hyaluronic acid,
collagen,
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tricalcium phosphate, polybutyrate, lactide, and glycolide polymers and
lactide/glycolide
copolymers, may be useful excipients to control the release of the agent in
vivo. Other
potentially useful parenteral delivery systems for these agents include
ethylene-vinyl acetate
copolymer particles, osmotic pumps, implantable infusion systems, and
liposomes.
S Formulations for inhalation administration contain as excipients, for
example, lactose, or may
be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether,
glycocholate
and deoxycholate, or oily solutions for administration in the form of nasal
drops, or as a gel to
be applied intranasally. Formulations for parenteral administration may also
include
glycocholate for buccal administration, methoxysalicylate for rectal
administration, or cutric
acid for vaginal administration. Suppositories for rectal administration may
also be prepared
by mixing the molecule capable of releasing morphogen inhibition (alone or in
combination
with a morphogen) with a non-irritating excipient such as cocoa butter or
other compositions
which are solid at room temperature and liquid at body temperatures.
Formulations for topical administration to the skin surface may be prepared by
dispersing the molecule capable of releasing morphogen inhibition (alone or in
combination
with a morphogen) with a dermatologically acceptable carrier such as a lotion,
cream,
ointment or soap. Particularly useful are carriers capable of forming a film
or layer over the
skin to localize application and inhibit removal. For topical, administration
to internal tissue
surfaces, the agent may be dispersed in a liquid tissue adhesive or other
substance known to
enhance adsorption to a tissue surface. For example, hydroxypropylcellulose or
fibrinogen/thrombin solutions may be used to advantage. Alternatively, tissue-
coating
solutions, such as pectin-containing formulations may be used.
Where the composition is intended for use as a therapeutic for disorders of
the CNS, an
additional problem must be addressed: overcoming the blood-brain barrier, the
brain capillary
wail structure that effectively screens out all but selected categories of
substances present in
the blood, preventing their passage into the brain. The blood-brain barrier
can be bypassed
effectively by direct infusion of the molecule capable of releasing morphogen
inhibition (alone
or in combination with a morphogen) into the brain, or by intranasal
administration or
inhalation of formulations suitable for uptake and retrograde transport by
olfactory neurons.
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D. Bioassay of Osteogenic Activity: Endochondral Bone Formation and
Related Properties
The art-recognized bioassay for bone induction described by Sampath and Reddi,
Proc.
Natl. Acad. Sci. USA 80: 659 1-6595 (1983) and U.S. Patent 4,968,590), the
disclosures of
which are incorporated by reference herein, are useful to establish the
efficacy of a given
device or formulation. Briefly, the assay consists of depositing test samples
in subcutaneous
sites in recipient rats under ether anesthesia. A vertical incision (1 cm) is
made under sterile
conditions in the skin over the thoracic region, and a pocket is prepared by
blunt dissection. In
certain circumstances, approximately 25 mg of the test sample is implanted
deep into the
pocket and the incision is closed with a metallic skin clip. The heterotropic
site allows for the
study of bone induction without the possible ambiguities resulting from the
use of orthotopic
sites.
The sequential cellular reactions occurnng at the heterotropic site are
complex. The
mufti-step cascade of endochondral bone formation includes: binding of fibrin
and fibronectin
to implanted matrix, chemotaxis of cells, proliferation of fibroblasts,
differentiation into
chondroblasts, cartilage formation, vascular invasion, bone formation,
remodeling, and bone
marrow differentiation.
Successful implants exhibit a controlled progression through the stages of
protein-induced endochondral bone development including: (1) transient
infiltration by
polymorphonuclear leukocytes on about day one; (2) mesenchymal cell migration
and
proliferation on about days two and three; (3) chondrocyte appearance on about
days five and
six; (4) cartilage matrix formation on about day seven; (5) cartilage
calcification on about day
eight; (6) vascular invasion, appearance of osteoblasts, and formation of a
new bone on about
days nine and ten; (7) appearance of osteoblastic and bone remodeling on about
days twelve to
eighteen; and (8) hematopoietic bone marrow differentiation in the ossicle on
about day
twenty-one. The time course of this process varies according to the matrix.
Histological sectioning and staining is preferred to determine the extent of
osteogenesis
in the implants. Staining with toluidine blue or hemotoxylin/eosin clearly
demonstrates the
ultimate development of endochondral bone. Twelve day bioassays are sufficient
to determine
whether bone inducing activity is associated with the test sample.
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Additionally, alkaline phosphatase activity can be used as a marker for
osteogenesis.
The enzyme activity can be determined spectrophotometrically after
homogenization of the
excised test material. The activity peaks at 9-10 days in vivo and thereafter
slowly declines.
Samples showing no bone development by histology should have no alkaline
phosphatase
activity under these assay conditions. The assay is useful for quantitation
and obtaining an
estimate of bone formation very quickly after the test samples are removed
from the rat. For
example, samples containing morphogen at several levels of purity have been
tested to
determine the most effective dose/purity level, in order to seek a formulation
that could be
produced on an industrial scale. The results as measured by alkaline
phosphatase activity level
and histological evaluation can be represented as "bone forming units". One
bone-forming
unit represents the amount of protein that is needed for half maximal bone
forming activity on
day 12. Additionally, dose curves can be constructed for bone inducing
activity in vivo at each
step of a purification scheme by assaying various concentrations of protein.
Accordingly, the
skilled artisan can construct representative dose curves using only routine
experimentation.
Example 1 A Functionally Active Morphogen System in the Ovary
1.1 1u situ hybridization of BMP-4 and BMP-7, and type IA, type IB, and
type II morphogen receptors in adult rat ovaries
In situ hybridization was used to determine the localization and level of
expression of
morphogens and morphogen receptors in adult rat ovaries. Probes to the
morphogens, BMP-4
and BMP-7, and probes to the type IA, type IB, and type II morphogen receptors
were used.
Reagents and supplies
The recombinant proteins, Xenopus BMP-4, human BMP-7, and human activin-A,
were prepared as previously described by Kubo et al., Biol. Reprod. 58: 712-
718 (1998).
Ovine FSH (NIDDK-oFSH-S l, 4453 IU/mg) was supplied by the National Hormone
and
Pituitary Program of the NIDDK (Rockville, MD). McCoy's 5a medium, Medium 199,
and
dinucleotide triphosphates were purchased from Gibco BRL (Grand Island, NY).
Cell culture
plates were purchased from Falcon (Lincoln Park, NJ). Reagents for RT-PCR were
obtained
from Perkin Elmer (Foster City, CA).
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Cell culture
Twenty-three day old Sprague-Dawley rats (Harlan Industries, Indianapolis, IN)
were
implanted with silastic capsules containing 10 mg of diethylstilbestrol (DES)
to increase
granulosa cell number. Ovaries were removed and the granulosa cells isolated
and cultured as
previously described by Erickson and Hsueh, Endocrinology 102, 1275-1282
(1978).
Granulosa cells (5 x 10~ viable cells) were pipetted into 96-well culture
plates containing
200 :1 (final volume) of tissue culture medium (McCoy's Sa Medium containing
100 U/ml
penicillin, 100 mg/ml streptomycin sulfate, 2 mM L-glutamine, and 1 ~M
androstenedione).
Granulosa cells were cultured for up to 48 hr at 37~C in water-saturated
atmosphere containing
5% CO, in air with the indicated concentrations of FSH, BMP-4, BMP-7, or
activin-A. After
culture, the levels of progesterone and estrogen in the media were measured by
radioimmunoassay as previously described by Wang et al., JBiol Chem 254, 11330-
11336
(1979).
Construction of probe plasmids
Total RNA from 27-day old rat ovaries was prepared. Single-stranded cDNA was
synthesized by reverse transcriptase and then subjected to PCR as described
previously by
Shimasaki et al., JBiol Chem 266, 10646-10653 (1991). To design primers for
PCR, DNA
sequences of rat BMP-4 and BMPR-IA were obtained from GenBank. PCR primers for
rat
BMP-7, BMPR-IB, and BMPR-II were designed by choosing the homologous DNA
sequence
regions between human and mouse homologues of BMP-7, BMPR-IB and BMPR-II cDNAs
which were available from GenBank. Specifically, these primers are derived
from the cDNA
clones at nucleotides 737-757 and 1181-1200 (accession number of the cDNA
clone is
222607) for BMP-4 (Chen et al., Biochem. Biophys. Acta 1174, 289-292 (1993));
nucleotides
497-514 and 865-882 (accession number X56906) for BMP-7 (Ozkaynak et al.,
Biochem
Biophys Res Commun 179, 116-123 (1991)); nucleotides 441-460 and 876-895
(accession
number D38082) for BMPR-IA (Takeda et al., Biochem Biophys Res Commun 204, 203-
209
( 1994)); nucleotides 528-547 and 965-984 (accession number U89326) for BMPR-
IB; and
nucleotides 525-S44 and 895-904 (accession number AF003942) for BMPR-II. These
primers
were selected from different exons of the corresponding genes to discriminate
PCR products
that might arise from possible chromosome DNA contaminants. PCR was performed
under
the following conditions: 35 cycles, annealing at 50~C for 30 sec; extension
at 72~C for 30 sec;
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denaturation at 94 C for 30 sec. All PCR products were cloned into pBluescript
SK+ plasmid
and their DNA sequences confirmed.
In situ hybridization
ha situ hybridizations were performed as previously described by Nakatani et
al.,
Endocrinology 129, 1521-1529 (1991 ), with minor modifications. Eight
consecutive sections
(8 Vim) were cut from each ovary and mounted onto poly-L-lysine-coated glass
slides. The
sections were digested with proteinase K, acetylated, washed and dehydrated.
Each antisense
and sense cRNA probe was prepared by means of in vitro transcription using T3
or T7 RNA
polymerase. Hybridization was carried out with the 3'S-labeled RNA probe (4-6
x 106
cpm/ml) in a solution containing 50% (vol/vol) deionized formamide, 0.3 M
NaCI, 10 mM
Tris (pH 8.2), 1 mM EDTA, 0.05% yeast tRNA, 10 mM dithiothreitol, 1 x
Denhardt's solution
and 10% dextran sulfate. Hybridization solution (20 l~l) was placed over each
section and
covered with a 60 x 22 mm acid washed, siliconized coverslip. Coverslips were
sealed with
liquid DPX. Sections were hybridized for 16 hr at 58-60°C in a
humidified chamber. After
hybridization, the sections were treated with ribonuclease A and washed in 15
mM
NaCI/1.5 mM sodium citrate at 60-62°C for 30 min. Dehydrated slides
were exposed to X-ray
film for several days. After adequate X-ray film images were obtained, the
ovary sections
were treated with xylene, rinsed in 100% ethanol, air dried, and then coated
with Kodak
NTB-2 liquid autoradiograph emulsion. Slides were exposed for four weeks at
4°C in a
desiccated dark box. After exposure, the slides were developed (Kodak D19, 3.5
min, 14°C),
rinsed briefly in distilled water and fixed. After washing in distilled water
for 1 hr, slides were
lightly counterstained with hematoxylin and eosin. After an autoradiography
and
counterstaining, the sections were analyzed microscopically. The in situ
hybridizations were
performed at least two times for each morphogen ligand and receptor using one
ovary from six
different animals in each experiment
Results
The mRNAs for BMP-4, BMP-7, and the BMPR-IA, BMPR-IB, and BMPR-II
receptors were expressed in a tissue-specific manner in the adult rat ovary.
BMP-7 mRNA
was present in the theca interstitial cells of healthy Graafian (dominant)
follicles, but was
undetectable in other ovary cell types. Hybridization with the control sense
BMP-7 cRNA
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probe showed a nonspecific background signal; this was true for the other
control sense probes
used in these experiments. BMP-4 mRNA was also expressed strongly in the theca
cells of the
dominant Graafian follicles, being present in both the theca interstitial and
theca externa cells.
A weak but variable BMP-4 signal was observed in some corpora lutea and
surface epithelial
cells. BMP-4 mRNA was not detectable in the other ovarian cell types.
The mRNAs for BMPR-IA and BMPR-IB are widely expressed in the rat ovary, with
the strongest hybridization signals being observed in the granulosa cells and
oocytes of
developing follicles. The intensity of the signals for BMPR-IB were higher
than those for
BMPR-IA. Hybridization signals for BMPR-II were most intense in the granulosa
cells of all
growing follicles (healthy and atretic) after the secondary stage. The BMPR-II
message was
weakly expressed in some corpora lutea. A weak BMPR-II signal was observed in
growing
oocytes of primary follicles (those with a single layer of cuboidal granulosa
cells), but none
was observed in oocytes in late pre-antral and Graafian follicles. No BMPR-II
signal above
background was observed in the other ovary cell types.
1 S Conchcsions
Morphogens are expressed strongly in the ovary, being prominent in thecal
cells. High
levels of morphogen receptor expression found in granulosa cells. Morphogen
receptor
(BMPR-IA, BMPR-IB, and BMPR-II) mRNAs are uniformly expressed at high levels
in all
granulosa cells in all follicles, healthy as well as atretic, suggesting that
the granulosa cells are
important targets for morphogen signaling. These results suggest a paracrine
role for
morphogens in regulating ovarian follicle growth.
1.2 Effects of BMP-4 and BMP-7 on basal and FSH-stimulated estrogen
and progesterone production by granulosa cells.
These observations obtained in Example 1.1 suggest a paracrine role for
morphogens
whereby morphogens produced by thecal cells interact with morphogen receptors
in the
granulosa cells to regulate biological responses by a paracrine mechanism. To
assess this
potential paracrine role, the effects of two morphogens, BMP-4 and BMP-7, were
assessed on
basal and FSH-stimulated estrogen and progesterone production in primary
cultures of rat
granulosa cells grown in serum-free medium.
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When granulosa cells were cultured for 48 hr in the absence of FSH, there was
no
detectable estrogen or progesterone in the medium. As illustrated in FIG. 5,
treatment with
B~~iP-4 or BMP-7 did not significantly affect these baseline levels.
FSH markedly increased estrogen and progesterone production in a dose
dependent
mariner (EDSO for FSH stimulated E= 0.52 ~ 0.10; P= 1.19 ~ 0.13). As
illustrated in FIG. 5A
and C, BMP-4 and BMP-7 significantly modified the levels of FSH-stimulated
estrogen and
progesterone production. The levels of FSH-induced estrogen production were
significantly
increased (~2 to 3-fold) by both morphogens and the effects were dose-
dependent (EDS° for
BMP-4 = 89 ~ 0.4 ng/ml; ED;~ for BMP-7 = 11.0 ~ 1.0 ng/ml). Besides increasing
the
magnitude of the estrogen levels, BMP-4 and BMP-7 also caused a significant
increase
(~1.5-fold; p<0.05) in FSH sensitivity with respect to estrogen production. In
contrast to the
positive effects on estrogen production, both BMP-4 and BMP-7 caused marked
decreases
(approximately 60%) in FSH-induced progesterone production. See FIG. 5B and D.
These
effects on FSH-induced progesterone production were dose dependent
(EDS° for BMP-4 =
10.9 ~ 1.5 ng/ml; EDS° for BMP-7 = 11.6 ~ 3.2 ng/ml).
1.3 Time course of BMP-7 effects on FSH-stimulated estrogen and
progesterone production by granulosa cells
In order to assess the time course of the effects of morphogens on FSH-induced
estrogen and progesterone production, granulosa cells were incubated in the
presence of
BI\~iP-7 and FSH for a period of 72 hr and the levels of estrogen and
progesterone were
determined every 12 hr.
After a 24 hr lag phase, FSH induced a progressive increase in estrogen
production
which reached high levels after 72 hr of incubation. The 24 hr lag phase
reflects the time
needed for FSH to induce P4SOAROMATASE activity. Richards, Endocf~. Rev 15,
725-751 (1994).
As depicted in FIG. 6, co-treatment of FSH with a saturating dose of BMP-7 (30
ng/ml)
further increased the levels of estrogen (~2-fold) at each time point, but
produced no change in
the rate of FSH-induced estrogen accumulation tln-oughout the 72 hr. By
contrast, BMP-7
decreased the rate of FSH-stimulated progesterone accumulation by
approximately 12 hr. The
data in FIG. 6 illustrate that BMP-7 completely inhibited the FSH stimulation
of progesterone
at 24 hr and then continued to suppress progesterone levels (>50%) at 48 and
72 hr of culture.
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These findings suggest that BMP-7 suppresses both the maximal and relative
rates of
FSH-induced progesterone production.
Conclusions
The observation that BMPR-IA, BMPR-IB, and BMPR-II expression is strongest in
the
granulosa cells suggests that these cells are important targets for morphogen
signaling.
Morphogen receptor mRNAs are uniformly expressed at high levels in all
granulosa cells in all
follicles, healthy as well as atretic. These results implicate morphogens in
normal follicle
development and in the events leading to follicle death by apoptosis.
It is well established that the mechanism by which FSH stimulates estrogen and
progesterone production involves the induction of the expression of specific
steroidogenic
genes. Evidence that SMAD proteins mediate morphogen signaling and cyclic AMP
mediates
FSH signaling suggests the differential regulation might cross-talk between
these different
signaling pathways. A possible mechanism for morphogen enhancement of estrogen
production can be that the FSH signaling pathway receives positive regulatory
inputs through
1 S SMAD proteins that lead to increases in P4SOAROMATAS~ activity. A possible
mechanism for
morphogen inhibition of progesterone production can be that SMAD proteins are
negative
regulator for the FSH signals that induce enzymes in the progesterone
biosynthetic pathway,
including StAR (steroidogenic acute regulatory protein), P450SCC (side-chain
cleavage) or
(3-hydroxysteroid dehydrogenase.
Example 2 Morphogen Enhancement of FSH Expression in Pituitary
2.1 Stimulation of FSH(3LUC activity by morphogen and activin
Pituitary cells from transgenic mice harboring FSH(3LUC were dispersed and
cultured
for two days before treatments. The FSH(3LUC construct contains an ovine FSH(3
promoter
driving a luciferase gene and functions in several transgenic mouse lines.
Luciferase was
expressed only in the pituitary and was regulated as if it were FSH(3 itself.
Therefore,
luciferase activity seems to reflect normal FSH expression.
Cells were pretreated with follistatin (follicle stimulating hormone
suppressing protein;
FSP) (250 ng/ml). Follistatin suppresses the release of by FSH and LH.
Follistatin was
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withdrawn and OP-l, BMP-6, or activin-A was added. Four hours after addition
of a
morphogen or activin-A, the cells were harvested and assayed for luciferase
activity. The data
are representative of >3 individual experiments. As illustrated in FIG. 7, OP-
1, BMP-6, and
activin-A enhanced FSH expression in these mouse pituitary cell lines.
The morphogen effect on FSH expression was dose-dependent. Cultured pituitary
cells
from FSH(3LUC transgenic mice were treated with rabbit anti-mOP-1 (0.1 to 10
~tl/ml) or
follistatin (250 ng/ml) for 24 hr. After a 24 hr incubation, luciferase
activity was measured.
As depicted in FIG. 8, the anti-mOP-1 antibody produced a dose-dependent
decrease in FSH
expression, as measured by luciferase activity.
2.2 FSH(3LUC activity is inhibited by anti-morphogen antibodies but not by
anti-activin antibodies
In order to assess the specificity of the enhanced FSH expression by a
morphogen and
activin observed Example 2.1, the cultured pituitary cells from FSH(3Luc
transgenic mice were
treated for 24 hr with antibodies to mOP-1, activin-A, or activin-B and then
assayed for
luciferase activity.
As depicted in FIG. 9, the rabbit and sheep anti-mOP-1 antibodies were both
capable of
blocking the FSH expression in mouse pituitary cultures. However, neither anti-
activin-A
antisera nor anti-activin-B antisera significantly inhibit FSH(3LUC in the
mouse pituitary
system. FSH(3LUC activity was mildly blocked by monoclonal antibody 1B12 at
100 ~g/ml
(weak inhibition) but not by the 1263 monoclonal antibody
These results suggest that morphogens are the primary drive of FSH synthesis
in the
pituitary. Further, because anti-OP-1 blocks FSH(3LUC expression almost as
well as
follistatin, follistatin may inhibit FSH expression by binding and
inactivating morphogens or
morphogen receptors.
Example 3 Morphogen Regulation of Follicle-Stimulating Hormone
Follicle-stimulating hormone (FSH) is produced in pituitary gonadotropes as an
a,/(3
heterodimer, and synthesis of the (3 subunit is the rate-limiting step in
overall FSH production.
Synthesis of FSH(3 is regulated by activin and inhibin, both of which are
members of the
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transforming growth factor (3 (TGF(3) superfamily. Bone morphogenetic proteins
(BMPs) also
belong to the TGF(3 family and are multifunctional growth factors involved in
many aspects of
tissue development and morphogenesis including regulation of FSH action in the
ovary.
Using primary pituitary cell cultures derived from transgenic mice which carry
the
ovine FSH~3 promoter linked to a luciferase reporter gene (oFSH[3Luc), BMP-7
or BMP-6 was
found to stimulate oFSH(3Luc expression by 6-fold. Also transient expression
of the
oFSH~3Luc in a transformed gonadotrope cell line, L~3T2, was induced 4-fold by
BMP-7 or
BMP-6 treatment. Both BMP-7 and BMP-6 increased FSH secretion from L(3T2
cells,
demonstrating for the first time that a functional BMP system is present in
gonadotropes. Two
neutralizing antibodies to BMP-7, which cross-react with BMP-6 but not with
activin A,
decreased the basal expression of oFSH(3Luc in transgenic mouse pituitary
cultures by 80-
90%, suggesting an autocrine or paracrine role for BMP-7 or BMP-6 in FSH
synthesis.
Neither bio-neutralizing antibody to activin A or activin B decreased basal
oFSH(3Luc
expression significantly. Furthermore, mRNAs for BMP-7 and BMP-6 were detected
in
mouse pituitaries using RT-PCR. These results indicate that BMP-7 and BMP-6
can function
as FSH stimulators and may be significant physiological factors maintaining
basal FSH
expression.
Example 4 Morphogen and GnRH Regulation of FSH[3
Expression of follicle-stimulating hormone (FSH) depends on gonadotropin
releasing
hormone (GnRH), and part of this regulation is thought to occur directly and
selectively at
FSH~3 transcription. Although difficult, it has been shown that GnRH can
induce FSH(3
transcription by 2- to 3-fold in vivo and in tissue culture of primary
gonadotropes or non-
gonadotropes fortified with GnRH receptors.
Studies with non-gonadotropes have identified two highly conserved AP-1 sites
in the
proximal promoter (-120 by and -83 bp) of the ovine FSH(3 gene as being
important for
induction by GnRH. To study the significance of these AP-1 sites in primary
gonadotropes,
transgenic mice were produced that express luciferase under control of 4.7 kb
of the ovine
FSH[3 promoter (oFSH[3LUC) with or without functioning AP-1 sites (-120/-83).
Luciferase
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was expressed in these mice (+/- AP-1 sites) only in the pituitary and
regulated ih vivo as if it
were FSH(3, itself (+/- AP-1 sites). Using static cultures of pituitaries from
these mice, it was
possible to show a 2- to 3-fold stimulation of luciferase expression by GnRH
in wild-type
cultures, but not in cultures expressing the AP-1 mutant oFSH(3LUC.
These results link the AP-1 sites to GnRH induction of FSH(3 transcription.
However,
since expression of oFSH(3LUC (mutant or wild-type) reflected normal FSH(3
expression in all
transgenic mouse lines, it appears that direct transcriptional regulation of
FSH(3 by GnR_H_ in
vivo may be relegated to subtle changes that are likely to be important but
not yet understood.
Our studies on GnRH eventually led to a focus on activin and other
Morphogensthat had larger
effects on FSH(3 transcription than GnRH. These studies indicated that activin
A, as well as
BMP6 and BMP7 could stimulate FSH(3 transcription 8- tol2-fold. Future studies
will define
activin/BMP response elements) and use transgenic technology to determine the
physiological
relevance of activin/BMPs to reproductive function.
In conclusion, these studies, taken together with those of others, suggest
that GnRH
regulates FSH expression primarily at a global level of gonadotrope "well
being" rather than at
the micro-management level of specifically altering FSH(3 transcription. By
contrast,
Morphogens more likely to influence FSH(3 directly and selectively at the
transcriptional level.
Support came from NIH grant HD 34863 and gifts of BMP reagents from Dr. P.L.
Kaplan &
Dr. D.M. Bosukonda, Creative Biomolecules, Inc., Hopkinton, MA 01748
The foregoing description has been presented only for the purposes of
illustration and
is not intended to limit the invention to the precise form disclosed, but by
the claims appended
hereto. In the specification and the appended claims, the singular forms
include plural
references, unless the context clearly dictates otherwise. All patents and
publications cited in
this specification are incorporated by reference.
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SEQUENCE LISTING
<110> Sampath, Kuber
<120> Morphogen-Induced Enhancement of Fertility
<130> CBM-1 00960-501
<140> 09/561,171
<141> 2000-04-30
<150> 60/131,721
<151> 1999-04-30
<160> 10
<170> PatentIn Ver. 2.0
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Glu Val His Ser Ser Phe Ile His Arg Arg Leu Arg Ser Gln Glu Arg
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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
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c
ccg cgc ccg cac ctc cag ggc aag cac aac tcg gca ccc atg ttc atg 297
Pro Arg Pro His Leu Gln Gly Lys His Asn Ser Ala Pro Met Phe Met
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ctg gac ctg tac aac gcc atg gcg gtg gag gag ggc ggc ggg ccc ggc 345
Leu Asp Leu Tyr Asn Ala Met Ala Val Glu Glu Gly Gly Gly Pro Gly
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ggc cag ggc ttc tcc tac ccc tac aag gcc gtc ttc agt acc cag ggc 393
Gly Gln Gly Phe Ser Tyr Pro Tyr Lys Ala Val Phe Ser Thr Gln Gly
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ccc cct ctg gcc agc ctg caa gat agc cat ttc ctc acc gac gcc gac 441
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-1-
SUBSTITUTE SHEET (RULE26)
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120 125 130
atggtcatg agcttcgtc aacctc gtggaacat gacaaggaa ttcttc 489
MetValMet SerPheVal AsnLeu ValGluHis AspLysGlu PhePhe
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cacccacgc taccaccat cgagag ttccggttt gatctttcc aagatc 537
HisProArg TyrHisHis ArgGlu PheArgPhe AspLeuSer LysIle
150 155 160
ccagaaggg gaagetgtc acggca gccgaattc cggatctac aaggac 585
ProGluGly GluAlaVal ThrAla AlaGluPhe ArgIleTyr LysAsp
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tacatccgg gaacgcttc gacaat gagacgttc cggatcagc gtttat 633
TyrIleArg GluArgPhe AspAsn GluThrPhe ArgIleSer ValTyr
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caggtgctc caggagcac ttgggc agggaatcg gatctcttc ctgctc 681
GlnValLeu GlnGluHis LeuGly ArgGluSer AspLeuPhe LeuLeu
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gacagccgt accctctgg gcctcg gaggagggc tggctggtg tttgac 729
AspSerArg ThrLeuTrp AlaSer GluGluGly TrpLeuVal PheAsp
215 220 225
atcacagcc accagcaac cactgg gtggtcaat ccgcggcac aacctg 777
IleThrAla ThrSerAsn HisTrp ValValAsn ProArgHis AsnLeu
230 235 240
ggcctgcag ctctcggtg gagacg ctggatggg cagagcatc aacccc 825
GlyLeuGln LeuSerVal GluThr LeuAspGly GlnSerIle AsnPro
245 250 255
aagttggcg ggcctgatt gggcgg cacgggccc cagaacaag cagccc 873
LysLeuAla GlyLeuIle GlyArg HisGlyPro GlnAsnLys GlnPro
260 265 270 275
ttcatggtg getttcttc aaggcc acggaggtc cacttccgc agcatc 921
PheMetVal AlaPhePhe LysAla ThrGluVal HisPheArg SerIle
280 285 290
cggtccacg gggagcaaa cagcgc agccagaac cgctccaag acgccc 969
ArgSerThr GlySerLys GlnArg SerGlnAsn ArgSerLys ThrPro
295 300 305
aagaaccag gaagccctg cggatg gccaacgtg gcagagaac agcagc 1017
LysAsnGln GluAlaLeu ArgMet AlaAsnVal AlaGluAsn SerSer
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agcgaccag aggcaggcc tgtaag aagcacgag ctgtatgtc agcttc 1065
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cgagacctg ggctggcag gactgg atcatcgcg cctgaaggc tacgcc 1113
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SUBSTITUTE SHEET (RULE 26)
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ccg gaa acg gtg ccc aag ccc tgc tgt gcg ccc acg cag ctc aat gcc 1257
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tac aga aac atg gtg gtc cgg gcc tgt ggc tgc cac tagctcctcc 1351
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tgtgagagta ttaggaaaca tgagcagcat atggcttttg atcagttttt cagtggcagc 1531
atccaatgaa caagatccta caagctgtgc aggcaaaacc tagcaggaaa aaaaaacaac 1591
gcataaagaa aaatggccgg gccaggtcat tggctgggaa gtctcagcca tgcacggact 1651
cgtttccaga ggtaattatg agcgcctacc agccaggcca cccagccgtg ggaggaaggg 1711
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-3-
SUBSTITUTE SHEET (RULE 26)
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Met His Val Arg Ser Leu Arg Ala Ala Ala Pro His Ser Phe Val Ala
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SUBSTITUTE SHEET (RULE 26)
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SUBSTITUTE SHEET (RULE 26)
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Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro Glu
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Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn
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SUBSTITUTE SHEET (RULE 26)
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SUBSTITUTE SHEET (RULE 26)
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Tyr Lys Asp Tyr Ile Arg Glu Arg Phe Asp Asn Glu Thr Phe Arg Ile
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Phe Leu Leu Asp Ser Arg Thr Leu Trp Ala Ser Glu Glu Gly Trp Leu
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Val Phe Asp Ile Thr Ala Thr Ser Asn His Trp Val Val Asn Pro Arg
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Lys Gln Pro Phe Met Val Ala Phe Phe Lys Ala Thr Glu Val His Phe
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Arg Ser Ile Arg Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn Arg Ser
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SUBSTITUTE SHEET (RULE 26)
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Gly Tyr Ala Ala Tyr'Tyr Cys Glu Gly Glu Cys Ala Phe Pro Leu Asn
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Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro Thr Gln
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<210> 4
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<212> PRT
<213> Artificial Sequence
<220>
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<222> (1)..(102)
<223> OPX; where each Xaa is independently selected from
a group of one or more specified amino acids as
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<400> 4
Cys Xaa Xaa His Glu Leu Tyr Val Xaa Phe Xaa Asp Leu Gly Trp Xaa
-9-
SUBSTITUTE SHEET (RULE 26)
Val Ser Phe
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1 5 10 15
Asp Trp Xaa Ile Ala~Pro Xaa Gly Tyr Xaa Ala Tyr Tyr Cys Glu Gly
20 25 30
Glu Cys Xaa Phe Pro Leu Xaa Ser Xaa Met Asn Ala Thr Asn His Ala
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Ile Xaa Gln Xaa Leu Val His Xaa Xaa Xaa Pro Xaa Xaa Val Pro Lys
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Xaa Ala Cys Gly Cys His
100
<210> 5
<211> 97
<212> PRT
<213> Artificial Sequence
<220>
<221> PEPTIDE
<222> (1) . . (97)
<223> Generic-Seq-7; wherein each Xaa is independently
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amino acids as defined in the specification
-10-
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<400> 5
Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa Xaa Xaa Xaa Xaa Xaa
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20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala Xaa Xaa Xaa Xaa Xaa
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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Xaa Pro
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Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa
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Xaa
<210> 6
<211> 102
<212> PRT
<213> Artificial Sequence
<220>
<221> PEPTIDE
-11-
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<222> (1) . . (102)
<223> OPX; wherein each Xaa is independently selected
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as defined in the specification
<400> 6
Cys Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Trp Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Ala Xaa Tyr Cys Xaa Gly
20 25 30
Xaa Cys Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn His Ala
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Cys Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Val Xaa Leu Xaa Xaa Xaa Xaa Xaa Met Xaa Val
85 90 95
Xaa Xaa Cys Xaa Cys Xaa
100
<210> 7
<211> 97
<212> PRT
<213> Artificial Sequence
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<220>
<221> PEPTIDE
<222> (1)..(97)
<223> Generic-Seq-9; wherein each Xaa is independently
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amino acids as defined in the specification
<400> 7
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa
20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Pro
50 55 60
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa
65 70 75 80
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Cys
85 90 95
Xaa
-13-
SUBSTITUTE SHEET (RULE 26)
CA 02371695 2001-10-25
WO 00/66620 PC'T/US00/11501
<210> 8
<211> 102
<212> PRT
<213> Artificial Sequence
<220>
<221> PEPTIDE
<222> (1)..(102)
<223> Generic-Seq-10; wherein each Xaa is independently
selected from a group of one or more specified
amino acids as defined in the specification
<400> S
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10 15
Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Gly
20 25 30
Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
50 55 60
Xaa Xaa Cys Xaa Pro Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa Xaa
65 70 75 90
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95
Xaa Xaa Cys Xaa Cys Xaa
-14-
SUBSTITUTE SHEET (RULE 26)
CA 02371695 2001-10-25
WO 00/66620 PCT/US00/11501
loo
<210> 9
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<221> PEPTIDE
<222> (1) . . (5)
<223> wherein each Xaa is independently selected from a
group of one or more specified amino acids as
defined in the specification
<400> 9
Cys Xaa Xaa Xaa Xaa
1 5
<210> 10
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<221> PEPTIDE
<222> (1) . . (5)
<223> wherein each Xaa is independently selected from a
group of one or more specified amino acids as
defined in the specification
-1$-
SUBSTITUTE SHEET (R ULE 26)
CA 02371695 2001-10-25
WO 00/66620 PCT/US00/11501
<400> 10
Cys Xaa Xaa Xaa Xaa'
- 16-
SUBSTITUTE SHEET (RULE 26)
