Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ANTAGONISTS OF BMP9, BMP10, ALKI AND OTHER ALKI LIGANDS,
AND USES THEREOF
RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. 119(e) of United States
provisional application Serial No. 61/053,644, filed May 15, 2008, the entire
contents of which are incorporated by reference herein. This application is
also a
continuation-in-part of United States application Serial No. 11/982,738, filed
November 2, 2007, which in turn claims the benefit under 35 U.S.C. 119(e) of
United States provisional application Serial No. 60/856,592, filed November 2,
2006.
BACKGROUND
Angiogenesis, the process of forming new blood vessels, is critical in many
normal and abnormal physiological states. Under normal physiological
conditions,
humans and animals undergo angiogenesis in specific and restricted situations.
For
example, angiogenesis is normally observed in wound healing, fetal and
embryonic
development and formation of the corpus luteum, endometrium and placenta.
Undesirable or inappropriately regulated angiogenesis occurs in many
disorders, in which abnormal endothelial growth may cause or participate in
the
pathological process. For example, angiogenesis participates in the growth of
many
tumors. Deregulated angiogenesis has been implicated in pathological processes
such as rheumatoid arthritis, retinopathies, hemangiomas, and psoriasis. The
diverse
pathological disease states in which unregulated angiogenesis is present have
been
categorized as angiogenesis-associated diseases.
Both controlled and uncontrolled angiogenesis are thought to proceed in a
similar manner. Capillary blood vessels are composed primarily of endothelial
cells
and pericytes, surrounded by a basement membrane. Angiogenesis begins with the
erosion of the basement membrane by enzymes released by endothelial cells and
leukocytes. The endothelial cells, which line the lumen of blood vessels, then
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protrude through the basement membrane. Angiogenic factors induce the
endothelial cells to migrate through the eroded basement membrane. The
migrating
cells form a "sprout" protruding from the parent blood vessel, where the
endothelial
cells undergo mitosis and proliferate. Endothelial sprouts merge with each
other to
form capillary loops, creating the new blood vessel.
Agents that inhibit angiogenesis have proven to be effective in treating a
variety of disorders. AvastinTM (bevacizumab), a monoclonal antibody that
binds to
Vascular Endothelial Growth Factor (VEGF), has proven to be effective in the
treatment of a variety of cancers. MacugenTM, an aptamer that binds to VEGF
has
proven to be effective in the treatment of neovascular (wet) age-related
macular
degeneration. Antagonists of the SDF/CXCR4 signaling pathway inhibit tumor
neovascularization and are effective against cancer in mouse models (Guleng et
al.
Cancer Res. 2005 Jul 1;65(13):5864-71). The isocoumarin 2-(8-hydroxy-6-
methoxy-1-oxo-1 H-2-benzopyran-3-yl) propionic acid (NM-3) has completed phase
1 clinical evaluation as an orally bioavailable angiogenesis inhibitor. NM-3
directly
kills both endothelial and tumor cells in vitro and is effective in the
treatment of
diverse human tumor xenografts in mice (Agata et al. Cancer Chemother
Pharmacol.
2005 Dec;56(6):610-4.). Thalidomide and related compounds have shown
beneficial effects in the treatment of cancer, and although the molecular
mechanism
of action is not clear, the inhibition of angiogenesis appears to be an
important
component of the anti-tumor effect (see, e.g., Dredge et al. Microvasc Res.
2005
Jan;69(1-2):56-63). The success of TNF-alpha antagonists in the treatment of
rheumatoid arthritis is partially attributed to anti-angiogenic effects on the
inflamed
joint tissue (Feldmann et al. Annu Rev Immunol. 2001;19:163-96). Anti-
angiogenic
therapies are widely expected to have beneficial effects on other inflammatory
diseases, particularly psoriasis. Although many anti-angiogenic agents have an
effect on angiogenesis regardless of the tissue that is affected, other
angiogenic
agents may tend to have a tissue-selective effect.
It is desirable to have additional compositions and methods for inhibiting
angiogenesis. These include methods and compositions which can inhibit the
unwanted growth of blood vessels, either generally or in certain tissues
and/or
disease states.
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SUMMARY
In part, the present disclosure presents a characterization of an activin-like
kinase I (ALK1)-mediated regulatory system, including its ligands BMP9 and
BMP10, and the role of this system in angiogenesis. In certain aspects, the
disclosure provides antagonists of ALK-1 ligands and the use of such
antagonists as
anti-angiogenic agents. Additionally, the disclosure provides antagonists of
ALK-1
itself, and the use of such antagonists as anti-angiogenic agents. As
described
herein, ALKI is a receptor for the GDF5 group of ligands, which includes GDF6
and GDF7, and also for the BMP9 group of ligands, which includes BMPIO. This
disclosure demonstrates that signaling mediated by ALKI and the ligands
described
above is involved in angiogenesis in vivo, and that inhibition of this
regulatory
system has a potent anti-angiogenic effect. With respect to BMP9 and BMP10 in
particular, these data are in contrast to the published literature, which
identify BMP9
and BMP10 as potent anti-angiogenic agents that signal through ALKI. See,
e.g.,
Scharpfenecker et al., J Cell Sci. 2007 Mar 15;120(Pt 6):964-72; David et al.
Blood
Mar 1;109(5):1953-61; 2007; David et al., Circ Res. 2008 Apr 25;102(8):914-22.
Thus, in certain aspects, the disclosure provides antagonists of the ALKI
regulatory
system, including antagonists of the receptor or one or more of the ligands,
for use in
inhibiting angiogenesis. In certain aspects, the disclosure provides
antagonists of
ALKI ligands for the treatment of cancers, particularly multiple myeloma,
melanoma, lung cancer, rheumatoid arthritis, and disorders associated with
pathological angiogenesis in the eye.
In certain aspects, the disclosure provides polypeptides comprising a ligand
binding portion of the extracellular domain of ALKI ("ALKI ECD polypeptides")
for use in inhibiting angiogenesis. While not wishing to be bound to any
particular
mechanism of action, it is expected that such polypeptides act by binding to
ligands
of ALKI and inhibiting the ability of these ligands to interact with ALKI as
well as
other receptors. In certain embodiments, an ALKI ECD polypeptide comprises an
amino acid sequence that is at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or
100% identical to the sequence of amino acids 22-118 of the human ALKI
sequence
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of SEQ ID NO: 1. An ALK I ECD polypeptide may be used as a small monomeric
protein or in a dimerized form (e.g., expressed as a fusion protein),
particularly for
local administration into tissues such as the eye. An ALKI ECD may also be
fused
to a second polypeptide portion to provide improved properties, such as an
increased
half-life or greater ease of production or purification. Fusions to an Fc
portion of an
immunoglobulin or linkage to a polyoxyethylene moiety (e.g., polyethylene
glycol)
may be particularly useful to increase the serum half-life of the ALKI ECD
polypeptide in systemic administration (e.g., intravenous, intraarterial and
intra-
peritoneal administration). As demonstrated herein, a systemically
administered
ALKI-Fc polypeptide has a potent anti-angiogenic effect in the eye and also
provides positive effects in murine models of rheumatoid arthritis and various
tumors. In certain embodiments, an ALKI-Fc fusion protein comprises a
polypeptide having an amino acid sequence that is at least 70%, 80%, 90%, 95%,
96%, 97%, 98%, 99% or 100% identical to the sequence of amino acids 22-118 of
SEQ ID NO: 1, which polypeptide is fused, either with or without an
intervening
linker, to an Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion
protein binds to GDF5, GDF7, BMP9 and/or BMP10 with a KD of less than 1 x 10.7
M and/or binds to TGF(3-1 with a KD of greater than 1 x 10-6. An Fc portion
may be
selected so as to be appropriate to the organism. Optionally, the Fc portion
is an Fc
portion of a human IgG 1. In a preferred embodiment, the ALK 1-Fc fusion
protein
comprises amino acids 22-118 of SEQ ID NO:1. Optionally, the ALKI-Fc fusion
protein comprises the amino acid sequence of SEQ ID NO: 3. Optionally, the
ALK1-Fc fusion protein is the protein produced by expression of the nucleic
acid of
SEQ ID NO:4 in a mammalian cell line, particularly a Chinese Hamster Ovary
(CHO) cell line. ALKI-ECD polypeptides may be formulated as a pharmaceutical
preparation that is substantially pyrogen free. The pharmaceutical preparation
may
be prepared for systemic delivery (e.g., intravenous, intraarterial or
subcutaneous
delivery) or local delivery (e.g., to the eye).
In certain aspects, the disclosure identifies difficulties in developing
relatively homogeneous preparations of ALKI-Fc fusion protein for use in a
therapeutic setting. As described herein, the ALK1-Fc fusion protein tends to
aggregate into higher order multimers. The disclosure provides solutions to
these
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difficulties and therefore provides pharmaceutical preparations comprising
ALKI-Fc
fusion proteins wherein such preparations are at least 85%, 90%, 95%, 96%,
97%,
98% or 99% composed of dimeric ALK I -Fc fusion protein. Therefore, in certain
aspects, the disclosure provides pharmaceutical preparations comprising an
ALKI-
Fc fusion protein comprising: a polypeptide having an amino acid sequence that
is at
least 97% identical to the sequence of amino acids 22-118 of SEQ ID NO:1,
which
polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the
ALKI-
Fc fusion protein binds to GDF5, GDF7, BMP9 and/or BMP10 with a KD of less
than 1 x 10-7 M and/or binds to TGF(3-1 with a KD of greater than 1 x 10-6 and
wherein at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the ALKI-Fc fusion
protein is present in a dimeric form. The Fc portion of the ALK1-Fc fusion
protein
may be an Fc portion of a human IgG 1. The ALK 1-Fc fusion protein may
comprise
the amino acid sequence of SEQ ID NO: 3. The ALKI-Fc fusion protein may be
produced by expression of the nucleic acid of SEQ ID NO:4 in a mammalian cell
line, particularly a Chinese Hamster Ovary (CHO) cell line. Such
pharmaceutical
preparations may formulated so as to be appropriate for administration to the
eye,
particularly by injection. The disclosed pharmaceutical preparations may be
used
for a variety of therapeutic purposes described herein, including inhibiting
angiogenesis, treating a tumors, treating rheumatoid arthritis, and treating
ocular
disorders associated with angiogenesis.
The ALKI-Fc pharmaceutical preparations and preparations comprising
antibodies directed to ALKI or one or more ligands of ALKI (e.g., BMP9 and/or
BMP10) may be used in conjunction with a second agent that inhibits
angiogenesis,
such as a VEGF antagonist (e.g., Avastin, sorafenib, and VEGF receptor traps).
In certain aspects, the disclosure provides methods for inhibiting
angiogenesis in a mammal by administering any of the ALKI ECD polypeptides
described generally or specifically herein. In one embodiment, a method
comprises
administering to the mammal an effective amount of an ALKI-Fc fusion protein,
wherein the ALKI Fc fusion protein comprises a polypeptide having an amino
acid
sequence that is at least 90% identical to the sequence of amino acids 22-118
of SEQ
ID NO:1, which polypeptide is fused to an Fc portion of an immunoglobulin, and
wherein the ALK1-Fc fusion protein binds to TGFP-I with a KD of greater than 1
x
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10-6. Optionally, the ALKI-Fc fusion protein binds to one or more ALKI ligands
selected from the group consisting of. GDF5, GDF6, GDF7, BMP9 and BMPIO.
Optionally, the ALKI-Fc fusion protein has a sequence of SEQ ID NO:3. The
ALKI ECD polypeptide may be delivered locally (e.g., to the eye) or
systemically
(e.g., intravenously, intraarterially or subcutaneously). In a particular
embodiment,
the disclosure provides a method for inhibiting angiogenesis in the eye of a
mammal
by administering an ALKI-Fc protein to the mammal at a location distal to the
eye,
e.g. by systemic administration.
In certain aspects, the disclosure provides antibodies that bind to ALKI,
particularly an epitope situated in the extracellular domain, amino acids 22-
118 of
SEQ ID NO:1, and inhibit the binding of ALKI to at least one ALKI ligand
selected
from the group consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. Based on
the affinity of these ligands for ALKI, an antibody may bind with a KD of less
than
5 x 10-8 M, and optionally between 5 x 10"8 and 1 x 10-10. An antibody with
affinity
within this range would be expected to inhibit signaling by one or more of
GDF5,
GDF6, and GDF7 while having less effect on signaling by BMP9 and BMP10. Such
an antibody preferably inhibits angiogenesis stimulated by at least one ALKI
ligand
selected from the group consisting of. GDF5, GDF6, and GDF7. While not wishing
to be bound to a particular mechanism, it is expected that such antibodies
will act by
inhibiting ALKI activity directly, which should be contrasted to the activity
of an
ALKI-Fc fusion protein, which is expected to inhibit the activity of ALKI
ligands.
An anti-ALKI antibody is not expected to interfere with the ability of GDF5,
GDF6,
GDF7, BMP9 or BMP10 to signal through alternative receptor systems, such as
the
BMPRIa, BMPRIb and BMPRII complexes. However, an anti-ALKI antibody is
expected to interfere with the ability of low affinity ligands for ALKI (e.g.,
TGF-(3,
which is generally recognized as triggering significant signaling events
through
ALK-1 even though binding is relatively weak) to signal through ALKI, even
though an ALKI ECD may not bind to or inhibit such low affinity ligands. An
antibody may bind to the ALK I polypeptide with a KD of less than 1 x 10"10 M.
An
antibody with affinity within this range would be expected to inhibit
signaling by
BMP9 or BMPI O. Such an antibody preferably inhibits binding of BMP9 and
BMP10 to ALKI. Notably, based on the data disclosed herein, an antibody that
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binds relatively poorly to ALKI may inhibit TGF(3 binding to ALKI while
failing to
inhibit the tighter binding ligands such as GDF5 or BMP9. The antibodies
described
herein are preferably recombinant antibodies, meaning an antibody expressed
from a
nucleic acid that has been constructed using the techniques of molecular
biology,
such as a humanized antibody or a fully human antibody developed from a single
chain antibody. Fv, Fab and single chain antibodies are also included within
the
term "recombinant antibody". Antibodies may also be polyclonal or non-
recombinant monoclonal antibodies (including human or murine forms, as well as
human antibodies obtained from transgenic mice). Antibodies and ALKI-ECD
polypeptides may be formulated as a pharmaceutical preparation that is
substantially
pyrogen free. The pharmaceutical preparation may be prepared for systemic
delivery (e.g., intravenous, intraarterial or subcutaneous delivery) or local
delivery
(e.g., to the eye). Antibodies described in WO 2007/040912 may be useful in
the
various methods described herein.
In certain aspects, the disclosure provides methods for inhibiting
angiogenesis in a mammal by administering to the mammal an effective amount of
an antibody that binds to an ALKI polypeptide, described herein either
generally or
specifically. An antibody useful for this purpose may bind to the
extracellular
domain of ALKI (e.g., bind to a polypeptide consisting of amino acids 22-118
of
SEQ ID NO:1) or another portion of ALKI. The antibody may bind to a
polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the
binding of at least one ALKI ligand selected from the group consisting of.
GDF5,
GDF6, GDF7, BMP9 and BMP10. The antibody may bind to the ALKI polypeptide
with a KD of less than 5 x 10-8 M, and optionally between 5 x 10-8 and 1 x
1010. The
antibody may inhibit angiogenesis stimulated by at least one ALKI ligand
selected
from the group consisting of. GDF5, GDF6 and GDF7. An antibody that
selectively
inhibits signaling mediated by GDF5, GDF6, or GDF7 relative to signaling by
BMP9 or BMP10 may be used as a selective inhibitor of angiogenesis that occurs
in
tissues where GDF5, GDF6, or GDF7 are localized: primarily bone or joints. The
antibody may bind to the ALKI polypeptide with a KD of less than I x 10"10 M.
The
antibody may inhibit the binding of ALKI to an ALKI ligand, wherein the ALKI
ligand is selected from the group consisting of. BMP9 and BMPIO. The anti-ALKI
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antibody may be delivered locally (e.g., to the eye) or systemically (e.g.,
intravenously, intraarterially or subcutaneously). In a particular embodiment,
the
disclosure provides a method for inhibiting angiogenesis in the eye of a
mammal by
administering an anti-ALKI antibody. In another particular embodiment, the
disclosure provides a method for treating patients with multiple myeloma. In a
particular embodiment, the disclosure provides a method for inhibiting
angiogenesis
in disorders that are associated with pathological angiogenesis as a
consequence of
multiple pro-angiogenic factors, such as VEGF, PDGF and/or FGF.
In certain aspects, the disclosure provides antibodies that bind to an ALKI
ligand disclosed herein and inhibit the binding of the ALKI ligand to ALKI.
While
not wishing to be bound to any particular mechanism, it is expected that
antibodies
that bind to ALKI ligands will have effects that are similar in nature to ALKI
ECD
polypeptides, because both types of agent bind to the ligands rather than the
receptor
itself. In certain embodiments, the antibody binds to a ligand selected from
the
group consisting of GDF5, GDF6 and GDF7. The antibody may bind to the ALKI
ligand with a KD of less than 5 x 10-8 M. The antibody may be selected for
inhibition of angiogenesis stimulated by the ALKI ligand. A CAM assay is an
appropriate assay system for selection of desirable antibodies. Such
antibodies are
preferably recombinant antibodies, and may be formulated as a pharmaceutical
preparation that is substantially pyrogen free. The pharmaceutical preparation
may
be prepared for systemic delivery (e.g., intravenous, intraarterial or
subcutaneous
delivery) or local delivery (e.g., to the eye).
In certain aspects, the disclosure provides antibodies that bind to an ALKI
ligand and inhibit the binding of the ALKI ligand to ALKI, wherein the ALK1
ligand is selected from the group consisting of BMP9 and BMP10. Notably, as
shown herein, a neutralizing anti-BMP9 antibody inhibits angiogenesis in vivo.
Additionally, as demonstrated herein, BMP-10 stimulates angiogenesis while an
antagonist of BMP-10 inhibits angiogenesis. The antibody may bind to the ALKI
ligand with a KD of less than I x 1010 M. Such antibodies are preferably
recombinant antibodies, and maybe formulated as a pharmaceutical preparation
that
is substantially pyrogen free. The pharmaceutical preparation may be prepared
for
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systemic delivery (e.g., intravenous, intraarterial or subcutaneous delivery)
or local
delivery (e.g., to the eye).
In certain aspects, the disclosure provides methods for inhibiting
angiogenesis in a mammal, the method comprising, administering to the mammal
an
effective amount of an antibody that binds to an ALKI ligand and inhibits the
binding of the ALKI ligand to ALKI, wherein the ALKI ligand is selected from
the
group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10. The antibody may
inhibit angiogenesis stimulated by at least one ALKI ligand selected from the
group
consisting of. GDF5, GDF6 and GDF7.
Members of the BMP/GDF family, including BMP9, BMP10, GDF5, GDF6
and GDF7 bind to a type I and a type II receptor in order to form a functional
signaling complex. The binding sites for these receptors are different.
Accordingly,
in certain embodiments, an antibody that binds to an ALKI ligand and inhibits
the
ligand to ALKI is an antibody that binds at or near the type I receptor
binding site of
the ligand.
In certain aspects, the disclosure provides methods for inhibiting
angiogenesis in a mammal by administering other inhibitors of the ALKI
signaling
system disclosed herein. Such inhibitors may include nucleic acids (e.g.,
antisense
or RNAi constructs) that decrease the production of ALKI, GDF5, GDF6, GDF7,
BMP9 or BMP10). A variety of affinity binding reagents can also be used, such
as
aptamers, random peptides, protein scaffolds that can be modified to allow
binding
to selected targets (examples of such scaffolds include anticalins and FNIII
domains); in each case, an affinity binding reagent would be selected for the
ability
to disrupt the ALKI regulatory system disclosed herein, either by disrupting
the
ALKI-ligand interaction or by inhibiting the signaling that occurs after
binding.
In a further embodiment, the disclosure describes the role of DAN as a
regulator of the ALKI regulatory system. As shown herein, DAN binds to the
GDF5 group of ligands but fails to bind to the BMP9 group of ligands. Thus,
DAN
is expected to inhibit angiogenesis mediated by GDF5, GDF6 or GDF7 but not
angiogenesis mediated by BMP9 or BMPIO. DAN may therefore be used as a
selective agent for inhibiting angiogenesis in the bone or joints, where the
GDF5
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group of proteins is primarily expressed. Thus, in certain embodiments the
disclosure provides DAN proteins for use as anti-angiogenic agents in the
context of
bone or joint angiogenesis, including rheumatoid arthritis and cancers that
involve
the bone or joints (e.g., multiple myeloma and bone metastases). A DAN protein
will generally bind to one or more ALKI ligands selected from the group
consisting
of. GDF5, GDF6 and GDF7, while having relatively poor binding to BMP9 or
BMPIO. A DAN protein may comprise an amino acid sequence that is at least 70%,
80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of amino
acids corresponding to amino acids 17-180 of SEQ ID NO:10 (mature human DAN)
or amino acids 21-125 of SEQ ID NO:10 (conserved cysteine knot domain of DAN).
A DAN protein may also be encoded by a nucleic acid that comprises a sequence
the
complement of which hybridizes under stringent hybridization conditions to
nucleotides 153-467 of SEQ ID NO:1I or a variant of nucleotides 153-467 of SEQ
ID NO: I I that has the same coding sequence (a "silent" variant, such as a
variant
containing one or more alterations at a wobble position in the triplet code),
or to
nucleotides 93-635 of SEQ ID NO: I I or a silent variant thereof. In certain
aspects,
the DAN protein is a fusion protein, such as an Fc fusion protein. While DAN
is
expected to be particularly useful for the inhibition of angiogenesis in bone
and
joints (including tumors located in the bone or joints, such as multiple
myeloma and
bone metastases), it may also be useful in other contexts, such as in a tumor
located
elsewhere, or in the eye.
According to another aspect of the invention, a method of inhibiting
angiogenesis in a mammal is provided. The method comprises administering to
the
mammal an effective amount of an antagonist of BMP9 and/or BMP10. In some
embodiments, the antagonist is an antibody that binds to BMP9 and/or BMPIO.
The
antibody may be a polyclonal, monoclonal, and chimeric or a humanized
antibody.
The antagonist may be a Fd, Fv, Fab, F(ab'), F(ab)2, or F(ab')2 fragment,
single
chain Fv (scFv), diabody, triabody, tetrabody, minibody or a peptibody. In
some
embodiments the antagonist is an aptamer (peptide or nucleic acid). Given the
overlapping effects of antagonists of BMP9 and BMP10, as demonstrated herein,
the
disclosure provides for antagonists of both BMP9 and BMP10, such as antibodies
that cross-react and thus antagonize both proteins effectively (e.g., affinity
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nM or less than 1 nM for both BMP9 and BMP10). Another example is ALK-1-
Fc, which binds to both BMP9 and BMP10 and inhibits the activities of both
ligands.
The antagonist may be an inhibitor of BMP9 and/or BMP10 expression. In
5 some embodiments the antagonist is a nucleic acid that inhibits BMP9 and/or
BMP10 expression. The nucleic acid may be an antisense or RNAi nucleic acid.
The antagonist may be a non-antibody protein that binds to BMP9 and/or
BMP 10. The antagonist may be a member of a GDF Trap family. Examples of the
GDF Trap family include but are not limited to follistatin, FLRG, noggin and
10 gremlin.
In some embodiments, the antagonist is a polypeptide that comprises an
amino acid sequence was selected from a library of amino acid sequences by a
method comprising a step of detecting binding to BMP9 and BMP10.
In certain aspects, the disclosure provides methods for treating rheumatoid
arthritis in a mammal, the method comprising, administering to a mammal that
has
rheumatoid arthritis an effective amount of an agent selected from the group
consisting of, an ALK1 ECD protein; an antibody that binds to an ALK1 ligand
and
inhibits the binding of the ALK1 ligand to ALK1, wherein the ALK1 ligand is
selected from the group consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; an
antibody that binds to an ALK1 polypeptide consisting of amino acids 22-118 of
SEQ ID NO:1 and inhibits the binding of at least one ALK1 ligand selected from
the
group consisting of. GDF5, GDF6, GDF7, BMP9 and BMP10; and a DAN
polypeptide.
In certain aspects the disclosure provides methods for treating a tumor in a
mammal. Such a method may comprise administering to a mammal that has a tumor
an effective amount of an agent selected from the group consisting of. an ALK1
ECD protein; an antibody that binds to an ALK1 ligand and inhibits the binding
of
the ALKI ligand to ALK1, wherein the ALK1 ligand is selected from the group
consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; an antibody that binds to an
ALKI polypeptide consisting of amino acids 22-118 of SEQ ID NO:I and inhibits
the binding of at least one ALK1 ligand selected from the group consisting of.
GDF5, GDF6, GDF7, BMP9 and BMPIO; and a DAN polypeptide. A method may
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further comprise administering a second agent that inhibits angiogenesis. A
tumor
may be a tumor that is associated with bone, such as a leukemia, a bone marrow
tumor, a multiple myeloma or bone metastases, such as those commonly
associated
with breast or prostate cancer. A tumor may be a melanoma or lung cancer
tumor.
A tumor may also be one that utilizes multiple pro-angiogenic factors, such as
a
tumor that is resistant to anti-VEGF therapy.
In certain aspects the disclosure provides ophthalmic formulations. Such
formulations may comprise an agent selected from the group consisting of: an
ALKI
ECD protein; an antibody that binds to an ALKI ligand and inhibits the binding
of
the ALKI ligand to ALKI, wherein the ALKI ligand is selected from the group
consisting of GDF5, GDF6, GDF7, BMP9 and BMP10; an antibody that binds to an
ALKI polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits
the binding of at least one ALKI ligand selected from the group consisting of:
GDF5, GDF6, GDF7, BMP9 and BMP10; and a DAN polypeptide.
In certain aspects, the disclosure provides methods for treating an
angiogenesis related disease of the eye. Such methods may comprise
administering
systemically or to said eye a pharmaceutical formulation comprising: an
effective
amount of an agent selected from the group consisting of. an ALKI ECD protein;
an
antibody that binds to an ALKI ligand and inhibits the binding of the ALKI
ligand
to ALKI, wherein the ALKI ligand is selected from the group consisting of
GDF5,
GDF6, GDF7, BMP9 and BMPIO; an antibody that binds to an ALKI polypeptide
consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the binding of at
least one ALKI ligand selected from the group consisting of. GDF5, GDF6, GDF7,
BMP9 and BMP10; and a DAN polypeptide.
In each instance, an agent described herein may be administered in
conjunction with a second agent that inhibits angiogenesis. Where it is
desirable to
inhibit angiogenesis of a tumor, the agent may be administered in conjunction
with a
second agent that has an anti-cancer effect, such as a chemotherapeutic agent
or a
biologic anti-cancer agent.
The disclosure also provides an ophthalmic pharmaceutical formulation
comprising an ALKI-Fc fusion protein having an amino acid sequence that is at
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least 97% identical to the sequence of amino acids 22-118 of SEQ ID NO:1,
which
polypeptide is fused to an Fc portion of an immunoglobulin, and wherein the
ALK1-
Fc fusion protein binds to GDFS, GDF7, BMP9 and/or BMP10 with a KD of less
than 1 x 10"7 M and/or binds to TGF(3-1 with a KD of greater than 1 x 10-6 .
In one
embodiment, the fusion protein has the sequence of SEQ ID NO: 3. In one
embodiment, the Fc portion is from human IgGI. In one embodiment, the fusion
protein is produced by expression of the nucleic acid of SEQ ID NO:4 in a
mammalian cell line. In one embodiment, the cell line is Chinese Hamster Ovary
cell line. The formulation may further comprise one or more of the following
medicaments: pegaptanib, ranibizumab, or a glucocorticoid. In one embodiment,
the
formulation is substantially pyrogen free.
The application also provides for an ophthalmic pharmaceutical formulation
comprising an antibody that binds to an ALK1 polypeptide consisting of amino
acids 22-118 of SEQ ID NO:1 and inhibits the binding of at least one ALKI
ligand
selected from the group consisting of. GDF5, GDF6, GDF7, BMP9 and BMP10. In
one embodiment, the antibody inhibits angiogenesis stimulated by at least one
ALK1 ligand selected from the group consisting of. GDF5, GDF6 and GDF7. In one
embodiment, the antibody binds to the ALK1 polypeptide with a KD of less than
5 x
10"8 M. In another embodiment, the antibody binds to the ALK1 polypeptide with
a
KD of less than 1 x 10"10 M. In one embodiment, the antibody inhibits
angiogenesis
stimulated by GDF5, GDF6, GDF7, BMP9, or BMPIO. The formulation may further
comprise one or more of the following medicaments: pegaptanib, ranibizumab, or
a
glucocorticoid. In one embodiment, the formulation is substantially pyrogen
free.
In certain aspects, the disclosure provides for an ophthalmic pharmaceutical
formulation comprising an antibody that binds to an ALKI ligand disclosed
herein
and inhibits the binding of the ALKI ligand to ALKI. In certain embodiments,
the
antibody binds to a ligand selected from the group consisting of GDF5, GDF6
and
GDF7. The antibody may bind to the ALK1 ligand with a KD of less than 5 x 10"8
M. The antibody may be selected for inhibition of angiogenesis stimulated by
the
ALKI ligand. A CAM assay is an appropriate assay system for selection of
desirable antibodies. As shown herein, an antibody to BMP9 inhibits VEGF-
mediated angiogenesis in a CAM assay. Such antibodies are preferably
recombinant
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antibodies. The formulation may further comprise one or more of the following
medicaments: pegaptanib, ranibizumab, or a glucocorticoid. In one embodiment,
the
formulation is substantially pyrogen free.
The application also provides methods of treating an angiogenesis related
disease of the eye comprising administering to said eye an ophthalmic
pharmaceutical formulation comprising an ALKI-Fc fusion protein comprising: a
polypeptide having an amino acid sequence that is at least 97% identical to
the
sequence of amino acids 22-118 of SEQ ID NO:1, which polypeptide is fused to
an
Fc portion of an immunoglobulin, and wherein the ALK1-Fc fusion protein binds
to
GDF5, GDF7, BMP9 and/or BMPIO with a KD of less than I x 10"' M and/or binds
to TGF(3-1 with a KD of greater than 1 x 10-6'. In one embodiment, the fusion
protein
has the sequence of SEQ ID NO: 3. In one embodiment, the Fc portion is from
human IgG 1. In one embodiment, the fusion protein is produced by expression
of
the nucleic acid of SEQ ID NO:4 in a mammalian cell line. In one embodiment,
the
cell line is Chinese Hamster Ovary cell line. The formulation may further
comprise
one or more of the following medicaments: pegaptanib, ranibizumab, or a
glucocorticoid. In one embodiment, the formulation is substantially pyrogen
free.
The application also provides methods of treating an angiogenesis related
disease of the eye comprising administering to said eye an ophthalmic
pharmaceutical formulation comprising an antibody that binds to an ALKI
polypeptide consisting of amino acids 22-118 of SEQ ID NO:1 and inhibits the
binding of at least one ALKI ligand selected from the group consisting of.
GDF5,
GDF6, GDF7, BMP9 and BMP10. In one embodiment, the antibody inhibits
angiogenesis stimulated by at least one ALKI ligand selected from the group
consisting of. GDF5, GDF6 and GDF7. In one embodiment, the antibody binds to
the ALKI polypeptide with a KD of less than 5 x 10-8 M. In another embodiment,
the
antibody binds to the ALK 1 polypeptide with a KD of less than I x 1010 M. In
one
embodiment, the antibody inhibits angiogenesis stimulated by GDF5, GDF6, GDF7,
BMP9, or BMP10. The formulation may further comprise one or more of the
following medicaments: pegaptanib, ranibizumab, or a glucocorticoid. In one
embodiment, the formulation is substantially pyrogen free.
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In one embodiment of the disclosed methods, the angiogenesis related
disease is selected from the group consisting of a tumor, a tumor that is
resistant to
anti-VEGF therapy, a multiple myeloma tumor, a tumor that has metastasized to
the
bone, joint or bone inflammation, rheumatoid arthritis, diabetic retinopathy,
retinopathy of prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, and retrolental fibroplasias.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the amino acid sequence for the human Activin Like Kinase
1, ALKI (SEQ ID NO:1). Single underlining shows the predicted extracellular
domain. Double underlining shows the intracellular domain. The signal peptide
and
the transmembrane domain are not underlined.
Figure 2 shows the nucleic acid sequence of a human ALKI cDNA (SEQ ID
NO:2). The coding sequence is underlined. The portion encoding the
extracellular
domain is double underlined.
Figure 3 shows an example of a fusion of the extracellular domain of human
ALKI to an Fc domain (SEQ ID NO:3). The hALKI-Fc protein includes amino
acids 22-120 of the human ALKI protein, fused at the C-terminus to a linker
(underlined) and an IgGI Fc region.
Figure 4 shows the nucleic acid sequence for expression of the hALKI-Fc
polypeptide of SEQ ID NO:3. The encoded amino acid sequence is also shown.
The leader sequence is cleaved such that Asp 22 is the N-terminal amino acid
of the
secreted protein.
Figure 5 shows the anti-angiogenic effect of murine ALKI-Fc ("RAP") and
human ALKI-Fc ("ACE") in an endothelial cell tube forming assay. All
concentrations of RAP and ACE reduced the level of tube formation in response
to
Endothelial Cell Growth Supplement (ECGF) to a greater degree than the
positive
control, Endostatin.
Figure 6 shows the angiogenic effect of GDF7 in a chick chorioallantoic
membrane (CAM) assay. The GDF7 effect is comparable to that of VEGF.
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Figure 7 shows the anti-angiogenic effect of the human ALK 1-Fc fusion in
the CAM assay. hALK1-Fc inhibits angiogenesis stimulated by VEGF, FGF and
GDF7.
Figure 8 shows comparative anti-angiogenic effects of murine ALKI-Fc
(mALK I-Fc), hALK 1-Fc, a commercially available anti-ALK 1 monoclonal
antibody (Anti-ALKI mAb) and a commercially available, neutralizing anti-VEGF
monoclonal antibody. The anti-angiogenic effect of the ALKI-Fc constructs is
comparable to the effects of the anti-VEGF antibody.
Figure 9 shows the anti-angiogenic effects of hALKI-Fc and the anti-VEGF
antibody in vivo. hALK1-Fc and anti-VEGF had comparable effects on
angiogenesis in the eye as measured by the mouse corneal micropocket assay.
Figure 10 shows the effects of mALKI-Fc in the murine collagen-induced
arthritis (CIA) model of rheumatoid arthritis. The graph shows mean group
arthritic
scores determined during the 42 day observation period in the collagen-induced
male DBA/I arthritic mice. RAP-041 is mALKI-Fc. AvastinTM is the anti-VEGF
antibody bevacizumab.
Figure 11 shows resolution of hALK I-Fc (SEQ ID NO: 3) and an hALK 1-Fc
fusion protein from R&D Systems (Minneapolis, MN) by Superose 12 10/300 GL
Size Exclusion column (Amersham Biosciences, Piscataway, NJ). The R&D
Systems material contains approximately 13% aggregated protein, as shown by
the
peaks on the left hand side of the graph, as well as some lower molecular
weight
species. The material of SEQ ID NO:3 is greater than 99% composed of dimers of
the appropriate molecular size.
Figure 12 shows fluorescent signal from luciferase-expressing Lewis lung
cancer (LL/2-luc) cells in mice treated with PBS (circles) and mALK1-Fc
(squares).
Tumor cells were injected into the tail vein and treatment (PBS or 10mg/kg
mALKI-Fc IP, twice weekly) was initiated on the day of cell administration.
PBS-
treated mice were sacrificed on day 22 as being moribund. The treatment and
control groups each consisted of seven animals (n=7).
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Figure 13 shows the effect of recombinant human BMP9 ("rhB9") and a
commercially available anti-BMP9 monoclonal antibody ("mabB9") on VEGF-
mediated angiogenesis in the CAM assay. Intriguingly, both BMP9 and anti-BMP9
treatment inhibit VEGF-mediated angiogenesis.
Figure 14 shows the effects of mALK1-Fc on an orthotopic xenograft model
using the MDA-MB-231 cell line, a cell line derived from ER- breast cancer
cells.
At a dose of 30 mg/kg, the mALKI-Fc has a significant growth-delaying effect
on
the xenograft tumor.
Figure 15 shows the effects of hALKI-Fc on an orthotopic xenograft model
using the MCF7 cell line, a cell line derived from ER+ breast cancer cells. At
a dose
of 10 or 30 mg/kg, the hALK1-Fc has a significant growth-delaying effect on
the
xenograft tumor.
Figure 16 shows the ability of hALK I -Fc to inhibit by more than 80% the
transcriptional reporter activity induced by BMP10 in a cell-based assay.
Figure 17 shows the effects of a BMP9/10 antagonist or hALKI-Fe on
VEGF- or BMP 10-stimulated angiogenesis in a chick chorioallantoic membrane
(CAM) assay. Data are means SEM; ***, p < 0.0001. BMP9/10 antagonist
reduced both VEGF-stimulated angiogenesis and BMPIO-stimulated angiogenesis
by more than 50%, and effects of similar magnitude were obtained with hALKI-
Fc.
Figure 18 shows a dose-dependent inhibition of BMPIO-stimulated
angiogenesis by a BMP9/10 antagonist in a chick chorioallantoic membrane (CAM)
assay. Data are means f SEM; * *, p < 0.01.
Figure 19 shows an alignment of the mature portions of the human BMP9
(SEQ ID NO:12) and BMP10 (SEQ ID NO:13) proteins. Regions of identity are
shown with asterisks.
DETAILED DESCRIPTION
1. Overview
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ALK1 is a type I cell-surface receptor for the TGF- 0 superfamily of ligands
and is also known as ACVRLI and ACVRLKI. ALK1 has been implicated as a
receptor for TGF-01, TGF- (33 and BMP-9 (Marchuk et al., Hum Mol Genet. 2003;
Brown et al., J Biol Chem. 2005 Jul 1;280(26):25111-8).
In mice, loss-of-function mutations in ALK1 lead to a variety of
abnormalities in the developing vasculature (Oh et al., Proc. Natl Acad. Sci.
USA
2000, 97, 2626-263 1; Urness et al., Nat. Genet. 2000, 26, 328-33 1).
In humans, loss-of-function mutations in ALKI are associated with
hereditary hemorrhagic telangiectasia (HHT, or Osler-Rendu-Weber syndrome), in
which patients develop arteriovenous malformations that create direct flow
(communication) from an artery to a vein (arteriovenous shunt), without an
intervening capillary bed. Typical symptoms of patients with HHT include
recurrent
epistaxis, gastrointestinal hemorrhage, cutaneous and mucocutaneous
telangiectases,
and arteriovenous malformations (AVM) in the pulmonary, cerebral, or hepatic
vasculature.
Recent publications from David et al. (Blood. 2007 Mar 1;109(5):1953-61.)
and Scharpfenecker et al. (J Cell Sci. 2007 Mar 15;120(Pt 6):964-72) conclude
that
BMP9 and BMP10 activate ALK1 in endothelial cells, and that the consequence of
this activation is to inhibit endothelial cell proliferation and migration.
These effects
are directly opposed to those of pro-angiogenic factors such as VEGF. Thus,
these
publications conclude that BMP9 and BMP10 are themselves anti-angiogenic
factors, and further, that ALK 1 activation has an anti-angiogenic effect. By
contrast,
the present disclosure demonstrates that antagonists, rather than agonists, of
BMP9
and BMP10 have anti-angiogenic effects.
The disclosure relates to the discovery that polypeptides comprising a
portion of the extracellular domain of ALK1 ("ALK 1 ECD polypeptides") may be
used to inhibit angiogenesis in vivo, including VEGF-independent angiogenesis
and
angiogenesis that is mediated by multiple angiogenic factors, including VEGF,
FGF
and PDGF. In part, the disclosure provides the identity of physiological, high
affinity ligands for ALK1 and demonstrates that ALK1 ECD polypeptides inhibit
angiogenesis. The data demonstrate that an ALK 1 ECD polypeptide can exert an
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anti-angiogenic effect even in the case where the ALKI ECD polypeptide does
not
exhibit meaningful binding to TGF-p31. Moreover, ALKI ECD polypeptides inhibit
angiogenesis that is stimulated by many different pro-angiogenic factors,
including
VEGF, FGF, and GDF7. Thus, the disclosure provides a description of an ALKI
regulatory system, in which ALKI is a receptor for the GDF5 group of ligands,
which includes GDF6 and GDF7, and also for the BMP9 group of ligands, which
includes BMP10, with different affinities for the two groups of ligands. It
was
demonstrated that antagonists of the ALKI signaling pathway are effective
against
tumors or cancers including, for example, breast, lung, estrogen receptor
positive
(ER+), estrogen receptor negative (ER-), bone, tumor or cancer metastasized to
bone, melanoma, multiple myeloma (see, for example W02008/057461). Further,
the disclosure demonstrates that signaling mediated by ALKI and the ligands
described above is pro-angiogenic in vivo, and that inhibition of this
regulatory
system has a potent anti-angiogenic effect in vivo. Thus, in certain aspects,
the
disclosure provides antagonists of the ALKI regulatory system, including
antagonists of the receptor or one or more of the ligands, for use in
inhibiting
angiogenesis, including both VEGF-dependent angiogenesis and VEGF-independent
angiogenesis. However, it should be noted that antibodies directed to ALKI
itself
are expected to have different effects from an ALKI ECD polypeptide. A pan-
neutralizing antibody against ALKI (one that inhibits the binding of all
strong and
weak ligands) would be expected to inhibit the signaling of such ligands
through
ALKI but would not be expected to inhibit the ability of such ligands to
signal
through other receptors (e.g., BMPR1a, BMPRIb, BMPRII in the case of GDF5-7
and BMP9-10 and TBRI and TBRII in the case of TGFp3). On the other hand, an
ALKI ECD polypeptide would be expected to inhibit all of the ligands that it
binds
to tightly, including, for a construct such as that shown in the Examples,
GDF5-7
and BMP9-10, but would not affect ligands that it binds to weakly, such as TGF-
p3.
So, while a pan-neutralizing antibody against ALKI would block BMP9 and TGF-p3
signaling through ALKI it would not block BMP9 and TGF-p3 signaling through
another receptor, and while an ALKI ECD polypeptide may inhibit BMP9 signaling
through alI receptors (even receptors other than ALK I) it would not be
expected to
inhibit TGF-p3 signaling through any receptor, even ALKI.
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Proteins described herein are the human forms, unless otherwise specified.
Genbank references for the proteins are as follows: human GDF5, CAA56874;
human GDF6, AAH43222; human GDF7, NP_878248; human BMP9, Q9UK05;
human BMPIO, 095393; human DAN, BAA92265. ALK1 sequences are set forth
in Figures 1-5.
Human Dan amino acid sequence (SEQ ID NO:10) (Genbank BAA92265):
MLRVLVGAVL PAMLLAAPPP INKLALFPDK SAWCEAKNIT QIVGHSGCEA KSIQNRACLG
QCFSYSVPNT FPQSTESLVH CDSCMPAQSM WEIVTLECPG HEEVPRVDKL VEKILHCSCQ
ACGKEPSHEG LSVYVQGEDG PGSQPGTHPH PHPHPHPGGQ TPEPEDPPGA PHTEEEGAED
The mature Dan protein is expected to correspond to amino acids 17-180.
The conserved cysteine knot domain of Dan corresponds to amino acids 21-125
(underlined).
Human Dan cDNA sequence (SEQ ID NO: 11) (Genbank BC012037):
gccgagcctc ctggggcgcc cgggcccgcg acccccgcac ccagctccgc aggaccggcg
ggcgcgcgcg ggctctggag gccacgggca tgatgcttcg ggtcctggtg ggggctgtcc
tccctgccat gctactggct gccccaccac ccatcaacaa gctggcactg ttcccagata
agagtgcctg gtgcgaagcc aagaacatca cccagatcgt gggccacagc ggctgtgagg
ccaagtccat ccagaacagg gcgtgcctag gacagtgctt cagctaaagc gtccccaaca
ccttcccaca gtccacagag tccctggttc actgtgactc ctgcatgcca gcccagtcca
tgtgggagat tgtgacgctg gagtgcccgg gccacgagga ggtgcccagg gtggacaagc
tggtggagaa gatcctgcac tgtagctgcc aggcctgcgg caaggagcct agtcacgagg
ggctgagcgt ctatgtgcag ggcgaggacg ggcggggatc ccagcccggc acccaccctc
acccccatcc ccacccccat cttggcgggc agacccctga gcccgaggac ccccctgggg
ccccccacac agaggaagag ggctgtgagg actgaggccc ccccaactct tcctcccctc
tcatccccct gtggaatgtt gggtctcact ctctggggaa gtcaggggag aagctgaagc
ccccctttgg cactggatgg acttggcttc agactcggac ttgaatgctg cccggttgcc
atggagatct gaaggggcgg ggttagagcc aagctgaaca atttaatata ttcaagagtg
gggggaggaa gaagaggtct tcagggCtct ttttttgggg ggggggtggt ctcttcctgt
ctggcttcta gagatgtgcc tgtgggaggg ggaggaagtt ggctgagcca ttgagtgctg
ggggaggcca ttcaagatgg cctgaatcgg gctaaggtcc ctgggggtgc agatggtact
gctgaggtcc cgggcttagt gtgagcatct tgccagcctc aggcttgagg gagggctggg
ctagaaagac cactggcaga aacaggaggc tccggcccca caggtttccc caaggcctct
caccccactt cccatctcca gggaagcgtc gccccaatgg cactgaagtg gccctccctc
agcggagggg tttgggagtc aggcctgggc aggaccctgC tgactcgtgg cgcgggagct
gggagccagg ctctccgggc ctttctctgg cttccttggc ttgcctggtg ggggaagggg
aggaggggaa gaaggaaagg gaagagtctt ccaaggccag aaggaggggg acaacccccc
aagaccatcc ctgaagacga gcatccccct cctctccctg ttagaaatgt tagtgccccg
cactgtgccc caagttctag gccccccaga aagctgtcag agccggccgc cttctcccct
ctcccaggga tgctctttgt aaatatcgga tgggtgtggg agtgaggggt tacctccctc
gccccaaggt tccagaggcc ctaggcggga tgggctcgct gaacctcgag gaactccagg
acgaggagga catgggactt gcgtggacag tcagggttca cttgggctct ctctagctcc
ccaattctgc ctgcctcctc cctcccagct gcactttaac cctagaaggt gggaacctgg
ggggagggac agggcaggcg ggcccatgaa gaaagcccct cgttgcccag cactgtctgc
gtctgctctt ctgtgcccag ggtggctgcc agcccactgc ctcctgcctg gggtggcctg
gccctcctgg ctgttgcgac gcgggcttct ggagcttgtc accattggac agtctccctg
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atggaccctc agtcttctca tgaataaatt ccttcaacgc caaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaa
The coding sequence for DAN precursor corresponds to nucleic acids 93-
635. The coding sequence for the mature DAN protein corresponds to nucleic
acids
141-632. The coding sequence for the conserved cysteine knot portion of DAN
corresponds to nucleic acids 153-467.
The terms used in this specification generally have their ordinary meanings
in the art, within the context of this disclosure and in the specific context
where each
term is used. Certain terms are discussed in the specification, to provide
additional
guidance to the practitioner in describing the compositions and methods
disclosed
herein and how to make and use them. The scope or meaning of any use of a term
will be apparent from the specific context in which the term is used.
2. Soluble ALK1 Polypeptides
Naturally occurring ALK 1 proteins are transmembrane proteins, with a
portion of the protein positioned outside the cell (the extracelluar portion)
and a
portion of the protein positioned inside the cell (the intracellular portion).
Aspects of
the present disclosure encompass polypeptides comprising a portion of the
extracellular domain of ALKI.
In certain embodiments, the disclosure provides "ALK1 ECD polypeptides".
The term "ALK1 ECD polypeptide" is intended to refer to a polypeptide
consisting
of or comprising an amino acid sequence of an extracellular domain of a
naturally
occurring ALK1 polypeptide, either including or excluding any signal sequence
and
sequence N-terminal to the signal sequence, or an amino acid sequence that is
at
least 33 percent identical to an extracellular domain of a naturally occurring
ALK 1
polypeptide, and optionally at least 60%, at least 70%, at least 80%, at least
85%, at
least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or
100% identical to the sequence of an extracellular domain of a naturally
occurring
ALK1 polypeptide, as exemplified by the cysteine knot region of amino acids 34-
95
of SEQ ID NO:1 or the cysteine knot plus additional amino acids at the N- and
C-
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termini of the extracellular domain, such as amino acids 22-118 of SEQ ID NO.
1.
Likewise, an ALK1 ECD polypeptide may comprise a polypeptide that is encoded
by nucleotides 100-285 of SEQ ID NO:2, or silent variants thereof or nucleic
acids
that hybridize to the complement thereof under stringent hybridization
conditions
(generally, such conditions are known in the art but may, for example, involve
hybridization in 50% v/v formamide, 5x SSC, 2% w/v blocking agent, 0.1% N-
lauroylsarcosine, 0.3% SDS at 65 C overnight and washing in, for example,
5xSSC
at about 65 C ). Additionally, an ALK1 ECD polypeptide may comprise a
polypeptide that is encoded by nucleotides 64-384 of SEQ ID NO:2, or silent
variants thereof or nucleic acids that hybridize to the complement thereof
under
stringent hybridization conditions (generally, such conditions are known in
the art
but may, for example, involve hybridization in 50% v/v formamide, 5x SSC, 2%
w/v blocking agent, 0.1 % N-lauroylsarcosine, 0.3% SDS at 65 C overnight and
washing in, for example, 5xSSC at about 65 C ). The term "ALK1 ECD
polypeptide" accordingly encompasses isolated extracellular portions of ALKI
polypeptides, variants thereof (including variants that comprise, for example,
no
more than 2, 3, 4, 5 or 10 amino acid substitutions, additions or deletions in
the
sequence corresponding to amino acids 22-118 of SEQ ID NO:I and including
variants that comprise no more than 2, 3, 4, 5, or 10 amino acid
substitutions,
additions or deletions in the sequence corresponding to amino acids 34-95 of
SEQ
ID NO: 1), fragments thereof and fusion proteins comprising any of the
preceding,
but in each case preferably any of the foregoing ALK 1 ECD polypeptides will
retain
substantial affinity for one or more of GDF5, GDF6, GDF7, BMP9 or BMP10. The
term "ALK1 ECD polypeptide" is explicitly intended to exclude any full-length,
naturally occurring ALK1 polypeptide. Generally, an ALK1 ECD polypeptide will
be designed to be soluble in aqueous solutions at biologically relevant
temperatures,
pH levels and osmolarity.
As described above, the disclosure provides ALK 1 ECD polypeptides
sharing a specified degree of sequence identity or similarity to a naturally
occurring
ALK1 polypeptide. To determine the percent identity of two amino acid
sequences,
the sequences are aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or nucleic acid
sequence
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for optimal alignment and non-homologous sequences can be disregarded for
comparison purposes). The amino acid residues at corresponding amino acid
positions are then compared. When a position in the first sequence is occupied
by
the same amino acid residue as the corresponding position in the second
sequence,
then the molecules are identical at that position (as used herein amino acid
"identity"
is equivalent to amino acid "homology"). The percent identity between the two
sequences is a function of the number of identical positions shared by the
sequences,
taking into account the number of gaps, and the length of each gap, which need
to be
introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity and
similarity between two sequences can be accomplished using a mathematical
algorithm. (Computational Molecular Biology, Lesk, A. M., ed., Oxford
University
Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith,
D.
W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data,
Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,
1994;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987;
and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton
Press, New York, 1991).
In one embodiment, the percent identity between two amino acid sequences
is determined using the Needleman and Wunsch (J Mol. Biol. (48):444-453
(1970))
algorithm which has been incorporated into the GAP program in the GCG software
package (available at http://www.gcg.com). In a specific embodiment, the
following
parameters are used in the GAP program: either a Blosum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of
1, 2, 3,
4, 5, or 6. In yet another embodiment, the percent identity between two
nucleotide
sequences is determined using the GAP program in the GCG software package
(Devereux, J., et al., Nucleic Acids Res. 12(1):387 (1984)) (available at
http://www.gcg.com). Exemplary parameters include using a NWSgapdna.CMP
matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2,
3, 4, 5,
or 6. Unless otherwise specified, percent identity between two amino acid
sequences is to be determined using the GAP program using a Blosum 62 matrix,
a
GAP weight of 10 and a length weight of 3, and if such algorithm cannot
compute
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the desired percent identity, a suitable alternative disclosed herein should
be
selected.
In another embodiment, the percent identity between two amino acid
sequences is determined using the algorithm of E. Myers and W. Miller (CABIOS,
4:11-17 (1989)) which has been incorporated into the ALIGN program (version
2.0),
using a PAM120 weight residue table, a gap length penalty of 12 and a gap
penalty
of 4.
Another embodiment for determining the best overall alignment between two
amino acid sequences can be determined using the FASTDB computer program
based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6:237-245
(1990)). In a
sequence alignment the query and subject sequences are both amino acid
sequences.
The result of said global sequence alignment is presented in terms of percent
identity. In one embodiment, amino acid sequence identity is performed using
the
FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App.
Biosci., 6:237-245 (1990)). In a specific embodiment, parameters employed to
calculate percent identity and similarity of an amino acid alignment comprise:
Matrix=PAM 150, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20,
Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5 and Gap Size
Penalty=0.05.
In certain embodiments, ALKI ECD polypeptides comprise an extracellular
portion of a naturally occurring ALK1 protein such as a sequence of SEQ ID
NO:1,
and preferably a ligand binding portion of the ALK1 extracellular domain. In
certain embodiments, a soluble ALK1 polypeptide comprises an amino acid
sequence that is at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical to an amino acid sequence of amino acids 22-118 of the SEQ ID NO:1.
In
certain embodiments, a truncated extracellular ALKI polypeptide comprises at
least
30, 40 or 50 consecutive amino acids of an amino acid sequence of an
extracellular
portion of SEQ ID NO: 1.
In preferred embodiments, an ALK1 ECD polypeptide binds to one or more
of GDF5, GDF6, GDF7, BMP9 and BMPIO. Optionally the ALKI polypeptide
does not show substantial binding to TGF-131 or TGF-(33. Binding may be
assessed
using purified proteins in solution or in a surface plasmon resonance system,
such as
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a BiacoreTM system. Preferred soluble ALK1 polypeptides will exhibit an anti-
angiogenic activity. Bioassays for angiogenesis inhibitory activity include
the chick
chorioallantoic membrane (CAM) assay, the mouse corneal micropocket assay, an
assay for measuring the effect of administering isolated or synthesized
proteins on
implanted tumors. The CAM assay is described by O'Reilly, et at. in
"Angiogenic
Regulation of Metastatic Growth" Cell, vol. 79 (2), Oct. 1, 1994, pp. 315-328.
Briefly, 3 day old chicken embryos with intact yolks are separated from the
egg and
placed in a petri dish. After 3 days of incubation, a methylcellulose disc
containing
the protein to be tested is applied to the CAM of individual embryos. After 48
hours
of incubation, the embryos and CAMs are observed to determine whether
endothelial growth has been inhibited. The mouse corneal micropocket assay
involves implanting a growth factor-containing pellet, along with another
pellet
containing the suspected endothelial growth inhibitor, in the cornea of a
mouse and
observing the pattern of capillaries that are elaborated in the cornea. Other
assays
are described in the Examples.
ALK1 ECD polypeptides may be produced by removing the cytoplasmic tail
and the transmembrane region of an ALK1 polypeptide. Alternatively, the
transmembrane domain may be inactivated by deletion, or by substitution of the
normally hydrophobic amino acid residues which comprise a transmembrane
domain with hydrophilic ones. In either case, a substantially hydrophilic
hydropathy
profile is created which will reduce lipid affinity and improve aqueous
solubility.
Deletion of the transmembrane domain is preferred over substitution with
hydrophilic amino acid residues because it avoids introducing potentially
immunogenic epitopes.
ALK1 ECD polypeptides may additionally include any of various leader
sequences at the N-terminus. Such a sequence would allow the peptides to be
expressed and targeted to the secretion pathway in a eukaryotic system. See,
e.g.,
Ernst et al., U.S. Pat. No. 5,082,783 (1992). Alternatively, a native ALK1
signal
sequence may be used to effect extrusion from the cell. Possible leader
sequences
include native, tPa and honeybee mellitin leaders (SEQ ID Nos. 7-9,
respectively).
Processing of signal peptides may vary depending on the leader sequence
chosen,
the cell type used and culture conditions, among other variables, and
therefore actual
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N-terminal start sites for mature ALKI ECD polypeptides, including that of SEQ
ID
NO:5, may shift by 1-5 amino acids in either the N-terminal or C-terminal
direction.
In certain embodiments, the present disclosure contemplates specific
mutations of the ALKI polypeptides so as to alter the glycosylation of the
polypeptide. Such mutations may be selected so as to introduce or eliminate
one or
more glycosylation sites, such as O-linked or N-linked glycosylation sites.
Asparagine-linked glycosylation recognition sites generally comprise a
tripeptide
sequence, asparagine-X-threonine (or asparagines-X-serine) (where "X" is any
amino acid) which is specifically recognized by appropriate cellular
glycosylation
enzymes. The alteration may also be made by the addition of, or substitution
by,
one or more serine or threonine residues to the sequence of the wild-type ALKI
polypeptide (for O-linked glycosylation sites). A variety of amino acid
substitutions
or deletions at one or both of the first or third amino acid positions of a
glycosylation recognition site (and/or amino acid deletion at the second
position)
results in non-glycosylation at the modified tripeptide sequence. Another
means of
increasing the number of carbohydrate moieties on an ALKI polypeptide is by
chemical or enzymatic coupling of glycosides to the ALKI polypeptide.
Depending
on the coupling mode used, the sugar(s) may be attached to (a) arginine and
histidine; (b) free carboxyl groups; (c) free sulfhydryl groups such as those
of
cysteine; (d) free hydroxyl groups such as those of serine, threonine, or
hydroxyproline; (e) aromatic residues such as those of phenylalanine,
tyrosine, or
tryptophan; or (f) the amide group of glutamine. These methods are described
in
WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston (1981) CRC Crit.
Rev. Biochem., pp. 259-306, incorporated by reference herein. Removal of one
or
more carbohydrate moieties present on an ALKI polypeptide may be accomplished
chemically and/or enzymatically. Chemical deglycosylation may involve, for
example, exposure of the ALKI polypeptide to the compound
trifluoromethanesulfonic acid, or an equivalent compound. This treatment
results in
the cleavage of most or all sugars except the linking sugar (N-
acetylglucosamine or
N-acetylgalactosamine), while leaving the amino acid sequence intact. Chemical
deglycosylation is further described by Hakimuddin et al. (1987) Arch.
Biochem.
Biophys. 259:52 and by Edge et al. (1981) Anal. Biochem. 118:131. Enzymatic
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cleavage of carbohydrate moieties on ALKI polypeptides can be achieved by the
use of a variety of endo- and exo-glycosidases as described by Thotakura et
al.
(1987) Meth. Enzymol. 138:350. The sequence of an ALKI polypeptide may be
adjusted, as appropriate, depending on the type of expression system used, as
mammalian, yeast, insect and plant cells may all introduce differing
glycosylation
patterns that can be affected by the amino acid sequence of the peptide. In
general,
ALKI proteins for use in humans will be expressed in a mammalian cell line
that
provides proper glycosylation, such as HEK293 or CHO cell lines, although
other
mammalian expression cell lines, yeast cell lines with engineered
glycosylation
enzymes and insect cells are expected to be useful as well.
This disclosure further contemplates a method of generating mutants,
particularly sets of combinatorial mutants of an ALKI polypeptide, as well as
truncation mutants; pools of combinatorial mutants are especially useful for
identifying functional variant sequences. The purpose of screening such
combinatorial libraries may be to generate, for example, ALKI polypeptide
variants
which can act as either agonists or antagonist, or alternatively, which
possess novel
activities all together. A variety of screening assays are provided below, and
such
assays may be used to evaluate variants. For example, an ALKI polypeptide
variant
may be screened for ability to bind to an ALKI ligand, to prevent binding of
an
ALKI ligand to an ALKI polypeptide or to interfere with signaling caused by an
ALKI ligand. The activity of an ALKI polypeptide or its variants may also be
tested in a cell-based or in vivo assay, particularly any of the assays
disclosed in the
Examples.
Combinatorially-derived variants can be generated which have a selective or
generally increased potency relative to an ALKI ECD polypeptide comprising an
extracellular domain of a naturally occurring ALK I polypeptide. Likewise,
mutagenesis can give rise to variants which have serum half-lives dramatically
different than the corresponding a wild-type ALKI ECD polypeptide. For
example,
the altered protein can be rendered either more stable or less stable to
proteolytic
degradation or other processes which result in destruction of, or otherwise
elimination or inactivation of a native ALKI ECD polypeptide. Such variants,
and
the genes which encode them, can be utilized to alter ALKI ECD polypeptide
levels
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by modulating the half-life of the ALKI polypeptides. For instance, a short
half-life
can give rise to more transient biological effects and can allow tighter
control of
recombinant ALKI ECD polypeptide levels within the patient. In an Fc fusion
protein, mutations may be made in the linker (if any) and/or the Fc portion to
alter
the half-life of the protein.
A combinatorial library may be produced by way of a degenerate library of
genes encoding a library of polypeptides which each include at least a portion
of
potential ALKI polypeptide sequences. For instance, a mixture of synthetic
oligonucleotides can be enzymatically ligated into gene sequences such that
the
degenerate set of potential ALKI polypeptide nucleotide sequences are
expressible
as individual polypeptides, or alternatively, as a set of larger fusion
proteins (e.g.,
for phage display).
There are many ways by which the library of potential ALKI ECD variants
can be generated from a degenerate oligonucleotide sequence. Chemical
synthesis
of a degenerate gene sequence can be carried out in an automatic DNA
synthesizer,
and the synthetic genes then be ligated into an appropriate vector for
expression.
The synthesis of degenerate oligonucleotides is well known in the art (see for
example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al., (1981)
Recombinant
DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam:
Elsevier pp273-289; Itakura et al., (1984) Annu. Rev. Biochem. 53:323; Itakura
et
al., (1984) Science 198:1056; Ike et al., (1983) Nucleic Acid Res. 11:477).
Such
techniques have been employed in the directed evolution of other proteins
(see, for
example, Scott et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS
USA 89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et al.,
(1990) PNAS USA 87: 6378-6382; as well as U.S. Patent Nos: 5,223,409,
5,198,346, and 5,096,815).
Alternatively, other forms of mutagenesis can be utilized to generate a
combinatorial library. For example, ALKI polypeptide variants can be generated
and isolated from a library by screening using, for example, alanine scanning
mutagenesis and the like (Ruf et al., (1994) Biochemistry 33:1565-1572; Wang
et
al., (1994) J. Biol. Chem. 269:3095-3099; Balint et al., (1993) Gene 137:109-
118;
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Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et at., (1993)
J.
Biol. Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry 30:10832-10838;
and Cunningham et at., (1989) Science 244:1081-1085), by linker scanning
mutagenesis (Gustin et at., (1993) Virology 193:653-660; Brown et at., (1992)
Mol.
Cell Biol. 12:2644-2652; McKnight et at., (1982) Science 232:316); by
saturation
mutagenesis (Meyers et at., (1986) Science 232:613); by PCR mutagenesis (Leung
et at., (1989) Method Cell Mol Biol 1:11-19); or by random mutagenesis,
including
chemical mutagenesis, etc. (Miller et at., (1992) A Short Course in Bacterial
Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener et al., (1994)
Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis, particularly in
a
combinatorial setting, is an attractive method for identifying truncated
(bioactive)
forms of ALK 1 polypeptides.
A wide range of techniques are known in the art for screening gene products
of combinatorial libraries made by point mutations and truncations, and, for
that
matter, for screening cDNA libraries for gene products having a certain
property.
Such techniques will be generally adaptable for rapid screening of the gene
libraries
generated by the combinatorial mutagenesis of ALKI polypeptides. The most
widely used techniques for screening large gene libraries typically comprises
cloning the gene library into replicable expression vectors, transforming
appropriate
cells with the resulting library of vectors, and expressing the combinatorial
genes
under conditions in which detection of a desired activity facilitates
relatively easy
isolation of the vector encoding the gene whose product was detected.
Preferred
assays include ALKI ligand binding assays and ligand-mediated cell signaling
assays.
In certain embodiments, the ALKI ECD polypeptides of the disclosure may
further comprise post-translational modifications in addition to any that are
naturally
present in the ALKI polypeptides. Such modifications include, but are not
limited
to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation,
and
acylation. As a result, the modified ALKI ECD polypeptides may contain non-
amino acid elements, such as polyethylene glycols, lipids, poly- or mono-
saccharide,
and phosphates. Effects of such non-amino acid elements on the functionality
of an
ALKI ECD polypeptide may be tested as described herein for other ALKI ECD
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polypeptide variants. When an ALKI ECD polypeptide is produced in cells by
cleaving a nascent form of the ALKI polypeptide, post-translational processing
may
also be important for correct folding and/or function of the protein.
Different cells
(such as CHO, HeLa, MDCK, 293, W138, NIH-3T3 or HEK293) have specific
cellular machinery and characteristic mechanisms for such post-translational
activities and may be chosen to ensure the correct modification and processing
of the
ALKI polypeptides.
In certain aspects, functional variants or modified forms of the ALKI ECD
polypeptides include fusion proteins having at least a portion of the ALKI ECD
polypeptides and one or more fusion domains. Well known examples of such
fusion
domains include, but are not limited to, polyhistidine, Glu-Glu, glutathione S
transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin heavy
chain constant region (Fc), maltose binding protein (MBP), or human serum
albumin. A fusion domain may be selected so as to confer a desired property.
For
example, some fusion domains are particularly useful for isolation of the
fusion
proteins by affinity chromatography. For the purpose of affinity purification,
relevant matrices for affinity chromatography, such as glutathione-, amylase-,
and
nickel- or cobalt- conjugated resins are used. Many of such matrices are
available in
"kit" form, such as the Pharmacia GST purification system and the QIAexpressTM
system (Qiagen) useful with (HIS6) fusion partners. As another example, a
fusion
domain may be selected so as to facilitate detection of the ALKI ECD
polypeptides.
Examples of such detection domains include the various fluorescent proteins
(e.g.,
GFP) as well as "epitope tags," which are usually short peptide sequences for
which
a specific antibody is available. Well known epitope tags for which specific
monoclonal antibodies are readily available include FLAG, influenza virus
haemagglutinin (HA), and c-myc tags. In some cases, the fusion domains have a
protease cleavage site, such as for Factor Xa or Thrombin, which allows the
relevant
protease to partially digest the fusion proteins and thereby liberate the
recombinant
proteins therefrom. The liberated proteins can then be isolated from the
fusion
domain by subsequent chromatographic separation. In certain preferred
embodiments, an ALKI ECD polypeptide is fused with a domain that stabilizes
the
ALKI polypeptide in vivo (a "stabilizer" domain). By "stabilizing" is meant
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anything that increases serum half life, regardless of whether this is because
of
decreased destruction, decreased clearance by the kidney, or other
pharmacokinetic
effect. Fusions with the Fc portion of an immunoglobulin are known to confer
desirable pharmacokinetic properties on a wide range of proteins. Likewise,
fusions
to human serum albumin can confer desirable properties. Other types of fusion
domains that may be selected include multimerizing (e.g., dimerizing,
tetramerizing)
domains and functional domains.
As a specific example, the present disclosure provides a fusion protein
comprising a soluble extracellular domain of ALKI fused to an Fe domain (e.g.,
SEQ ID NO: 6).
THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKF
NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKAL
PVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
GQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYT
QKSLSLSPGK*
Optionally, the Fe domain has one or more mutations at residues such as
Asp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domain
having
one or more of these mutations (e.g., Asp-265 mutation) has reduced ability of
binding to the Fcy receptor relative to a wildtype Fc domain. In other cases,
the
mutant Fc domain having one or more of these mutations (e.g., Asn-434
mutation)
has increased ability of binding to the MHC class I-related Fc-receptor (FcRN)
relative to a wildtype Fc domain.
It is understood that different elements of the fusion proteins may be
arranged in any manner that is consistent with the desired functionality. For
example, an ALKI ECD polypeptide may be placed C-terminal to a heterologous
domain, or, alternatively, a heterologous domain may be placed C-terminal to
an
ALKI ECD polypeptide. The ALKI ECD polypeptide domain and the heterologous
domain need not be adjacent in a fusion protein, and additional domains or
amino
acid sequences may be included C- or N-terminal to either domain or between
the
domains.
As used herein, the term, "immunoglobulin Fc region" or simply "Fc" is
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understood to mean the carboxyl-terminal portion of an immunoglobulin chain
constant region, preferably an immunoglobulin heavy chain constant region, or
a
portion thereof. For example, an immunoglobulin Fc region may comprise 1) a
CHI
domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2 domain, 3)
a CHI domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, or 5) a
combination of two or more domains and an immunoglobulin hinge region. In a
preferred embodiment the immunoglobulin Fc region comprises at least an
immunoglobulin hinge region a CH2 domain and a CH3 domain, and preferably
lacks the CH1 domain.
In one embodiment, the class of immunoglobulin from which the heavy
chain constant region is derived is IgG (Igy) (y subclasses 1, 2, 3, or 4).
Other
classes of immunoglobulin, IgA (Iga), IgD (Ig5), IgE (IgE) and IgM (Igp), may
be
used. The choice of appropriate immunoglobulin heavy chain constant region is
discussed in detail in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of
particular immunoglobulin heavy chain constant region sequences from certain
immunoglobulin classes and subclasses to achieve a particular result is
considered to
be within the level of skill in the art. The portion of the DNA construct
encoding the
immunoglobulin Fc region preferably comprises at least a portion of a hinge
domain,
and preferably at least a portion of a CH3 domain of Fc gamma or the
homologous
domains in any of IgA, IgD, IgE, or IgM.
Furthermore, it is contemplated that substitution or deletion of amino acids
within the immunoglobulin heavy chain constant regions may be useful in the
practice of the methods and compositions disclosed herein. One example would
be
to introduce amino acid substitutions in the upper CH2 region to create an Fc
variant
with reduced affinity for Fc receptors (Cole et al. (1997) J. Immunol.
159:3613).
In certain embodiments, the present disclosure makes available isolated
and/or purified forms of the ALKI ECD polypeptides, which are isolated from,
or
otherwise substantially free of (e.g., at least 80%, 90%, 95%, 96%, 97%, 98%,
or
99% free of), other proteins and/or other ALK1 ECD polypeptide species. ALK1
polypeptides will generally be produced by expression from recombinant nucleic
acids.
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In certain embodiments, the disclosure includes nucleic acids encoding
soluble ALKI polypeptides comprising the coding sequence for an extracellular
portion of an ALKI proteins. In further embodiments, this disclosure also
pertains
to a host cell comprising such nucleic acids. The host cell may be any
prokaryotic
or eukaryotic cell. For example, a polypeptide of the present disclosure may
be
expressed in bacterial cells such as E. coli, insect cells (e.g., using a
baculovirus
expression system), yeast, or mammalian cells. Other suitable host cells are
known
to those skilled in the art. Accordingly, some embodiments of the present
disclosure
further pertain to methods of producing the ALKI ECD polypeptides. It has been
established that an ALKI -Fc fusion protein set forth in SEQ ID NO:3 and
expressed
in CHO cells has potent anti-angiogenic activity.
DAN polypeptides, including variants of wild type DAN, and fusion proteins
containing DAN proteins may be generated and characterized as described above
with respect to ALKI ECD proteins.
3. Nucleic Acids Encoding ALKI Polypeptides
In certain aspects, the disclosure provides isolated and/or recombinant
nucleic acids encoding any of the ALKI polypeptides (e.g., ALKI ECD
polypeptides), including fragments, functional variants and fusion proteins
disclosed
herein. For example, SEQ ID NO: 2 encodes the naturally occurring human ALKI
precursor polypeptide, while SEQ ID NO: 4 encodes the precursor of an ALKI
extracellular domain fused to an IgGI Fc domain. The subject nucleic acids may
be
single-stranded or double stranded. Such nucleic acids may be DNA or RNA
molecules. These nucleic acids may be used, for example, in methods for making
ALKI polypeptides or as direct therapeutic agents (e.g., in an antisense, RNAi
or
gene therapy approach).
In certain aspects, the subject nucleic acids encoding ALKI polypeptides are
further understood to include nucleic acids that are variants of SEQ ID NO: 2
or 4.
Variant nucleotide sequences include sequences that differ by one or more
nucleotide substitutions, additions or deletions, such as allelic variants.
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In certain embodiments, the disclosure provides isolated or recombinant
nucleic acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,
99%
or 100% identical to SEQ ID NO: 2 or 4. One of ordinary skill in the art will
appreciate that nucleic acid sequences complementary to SEQ ID NO: 2 or 4, and
variants of SEQ ID NO: 2 or 4 are also within the scope of this disclosure. In
further
embodiments, the nucleic acid sequences of the disclosure can be isolated,
recombinant, and/or fused with a heterologous nucleotide sequence, or in a DNA
library.
In other embodiments, nucleic acids of the disclosure also include nucleotide
sequences that hybridize under highly stringent conditions to the nucleotide
sequence designated in SEQ ID NO: 2 or 4, complement sequence of SEQ ID NO: 2
or 4, or fragments thereof. As discussed above, one of ordinary skill in the
art will
understand readily that appropriate stringency conditions which promote DNA
hybridization can be varied. One of ordinary skill in the art will understand
readily
that appropriate stringency conditions which promote DNA hybridization can be
varied. For example, one could perform the hybridization at 6.0 x sodium
chloride/sodium citrate (SSC) at about 45 C, followed by a wash of 2.0 x SSC
at 50
C. For example, the salt concentration in the wash step can be selected from a
low
stringency of about 2.0 x SSC at 50 C to a high stringency of about 0.2 x SSC
at 50
C. In addition, the temperature in the wash step can be increased from low
stringency conditions at room temperature, about 22 C, to high stringency
conditions at about 65 C. Both temperature and salt may be varied, or
temperature
or salt concentration may be held constant while the other variable is
changed. In
one embodiment, the disclosure provides nucleic acids which hybridize under
low
stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x
SSC at room temperature.
Isolated nucleic acids which differ from the nucleic acids as set forth in SEQ
ID NOs: 2 or 4 due to degeneracy in the genetic code are also within the scope
of the
disclosure. For example, a number of amino acids are designated by more than
one
triplet. Codons that specify the same amino acid, or synonyms (for example,
CAU
and CAC are synonyms for histidine) may result in "silent" mutations which do
not
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affect the amino acid sequence of the protein. However, it is expected that
DNA
sequence polymorphisms that do lead to changes in the amino acid sequences of
the
subject proteins will exist among mammalian cells. One skilled in the art will
appreciate that these variations in one or more nucleotides (up to about 3-5%
of the
nucleotides) of the nucleic acids encoding a particular protein may exist
among
individuals of a given species due to natural allelic variation. Any and all
such
nucleotide variations and resulting amino acid polymorphisms are within the
scope
of this disclosure.
In certain embodiments, the recombinant nucleic acids of the disclosure may
be operably linked to one or more regulatory nucleotide sequences in an
expression
construct. Regulatory nucleotide sequences will generally be appropriate to
the host
cell used for expression. Numerous types of appropriate expression vectors and
suitable regulatory sequences are known in the art for a variety of host
cells.
Typically, said one or more regulatory nucleotide sequences may include, but
are
not limited to, promoter sequences, leader or signal sequences, ribosomal
binding
sites, transcriptional start and termination sequences, translational start
and
termination sequences, and enhancer or activator sequences. Constitutive or
inducible promoters as known in the art are contemplated by the disclosure.
The
promoters may be either naturally occurring promoters, or hybrid promoters
that
combine elements of more than one promoter. An expression construct may be
present in a cell on an episome, such as a plasmid, or the expression
construct may
be inserted in a chromosome. In a preferred embodiment, the expression vector
contains a selectable marker gene to allow the selection of transformed host
cells.
Selectable marker genes are well known in the art and will vary with the host
cell
used.
In certain aspects disclosed herein, the subject nucleic acid is provided in
an
expression vector comprising a nucleotide sequence encoding an ALK1
polypeptide
and operably linked to at least one regulatory sequence. Regulatory sequences
are
art-recognized and are selected to direct expression of the ALKI polypeptide.
Accordingly, the term regulatory sequence includes promoters, enhancers, and
other
expression control elements. Exemplary regulatory sequences are described in
Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press,
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San Diego, CA (1990). For instance, any of a wide variety of expression
control
sequences that control the expression of a DNA sequence when operatively
linked to
it may be used in these vectors to express DNA sequences encoding an ALK1
polypeptide. Such useful expression control sequences, include, for example,
the
early and late promoters of SV40, tet promoter, adenovirus or cytomegalovirus
immediate early promoter, RSV promoters, the lac system, the trp system, the
TAC
or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase,
the major operator and promoter regions of phage lambda , the control regions
for fd
coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic
enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the
yeast
a-mating factors, the polyhedron promoter of the baculovirus system and other
sequences known to control the expression of genes of prokaryotic or
eukaryotic
cells or their viruses, and various combinations thereof. It should be
understood that
the design of the expression vector may depend on such factors as the choice
of the
host cell to be transformed and/or the type of protein desired to be
expressed.
Moreover, the vector's copy number, the ability to control that copy number
and the
expression of any other protein encoded by the vector, such as antibiotic
markers,
should also be considered.
A recombinant nucleic acid included in the disclosure can be produced by
ligating the cloned gene, or a portion thereof, into a vector suitable for
expression in
either prokaryotic cells, eukaryotic cells (yeast, avian, insect or
mammalian), or
both. Expression vehicles for production of a recombinant ALK1 polypeptide
include plasmids and other vectors. For instance, suitable vectors include
plasmids
of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived
plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in
prokaryotic cells, such as E. coli.
Some mammalian expression vectors contain both prokaryotic sequences to
facilitate the propagation of the vector in bacteria, and one or more
eukaryotic
transcription units that are expressed in eukaryotic cells. The pcDNAI/amp,
pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG,
pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression
vectors suitable for transfection of eukaryotic cells. Some of these vectors
are
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modified with sequences from bacterial plasmids, such as pBR322, to facilitate
replication and drug resistance selection in both prokaryotic and eukaryotic
cells.
Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-
1), or
Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient
expression of proteins in eukaryotic cells. Examples of other viral (including
retroviral) expression systems can be found below in the description of gene
therapy
delivery systems. The various methods employed in the preparation of the
plasmids
and in transformation of host organisms are well known in the art. For other
suitable
expression systems for both prokaryotic and eukaryotic cells, as well as
general
recombinant procedures, see Molecular Cloning A Laboratory Manual, 3rd Ed.,
ed.
by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 2001).
In some instances, it may be desirable to express the recombinant polypeptides
by
the use of a baculovirus expression system. Examples of such baculovirus
expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and
pVL941), pAcUW-derived vectors (such as pAcUWI), and pBlueBac-derived
vectors (such as the 13-gal containing pBlueBac III).
In a preferred embodiment, a vector will be designed for production of the
subject ALKI polypeptides in CHO cells, such as a Pcmv-Script vector
(Stratagene,
La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.) and pCI-neo
vectors
(Promega, Madison, Wisc.). As will be apparent, the subject gene constructs
can be
used to cause expression of the subject ALKI polypeptides in cells propagated
in
culture, e.g., to produce proteins, including fusion proteins or variant
proteins, for
purification.
This disclosure also pertains to a host cell transfected with a recombinant
gene including a coding sequence (e.g., SEQ ID NO: 2 or 4) for one or more of
the
subject ALKI polypeptides. The host cell may be any prokaryotic or eukaryotic
cell. For example, an ALKI polypeptide disclosed herein may be expressed in
bacterial cells such as E. coli, insect cells (e.g., using a baculovirus
expression
system), yeast, or mammalian cells. Other suitable host cells are known to
those
skilled in the art.
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Accordingly, the present disclosure further pertains to methods of producing
the subject ALKI polypeptides, including ALKI ECD polypeptides. For example, a
host cell transfected with an expression vector encoding an ALKI polypeptide
can
be cultured under appropriate conditions to allow expression of the ALKI
polypeptide to occur. The ALKI polypeptide may be secreted and isolated from a
mixture of cells and medium containing the ALKI polypeptide. Alternatively,
the
ALKI polypeptide may be retained cytoplasmically or in a membrane fraction and
the cells harvested, lysed and the protein isolated. A cell culture includes
host cells,
media and other byproducts. Suitable media for cell culture are well known in
the
art. The subject ALKI polypeptides can be isolated from cell culture medium,
host
cells, or both, using techniques known in the art for purifying proteins,
including
ion-exchange chromatography, gel filtration chromatography, ultrafiltration,
electrophoresis, immunoaffinity purification with antibodies specific for
particular
epitopes of the ALKI polypeptides and affinity purification with an agent that
binds
to a domain fused to the ALKI polypeptide (e.g., a protein A column may be
used to
purify an ALKI-Fc fusion). In a preferred embodiment, the ALKI polypeptide is
a
fusion protein containing a domain which facilitates its purification. In a
preferred
embodiment, purification is achieved by a series of column chromatography
steps,
including, for example, three or more of the following, in any order: protein
A
chromatography, Q sepharose chromatography, phenylsepharose chromatography,
size exclusion chromatography, and cation exchange chromatography. The
purification could be completed with viral filtration and buffer exchange.
In another embodiment, a fusion gene coding for a purification leader
sequence, such as a poly-(His)/enterokinase cleavage site sequence at the N-
terminus of the desired portion of the recombinant ALKI polypeptide, can allow
purification of the expressed fusion protein by affinity chromatography using
a Ni2+
metal resin. The purification leader sequence can then be subsequently removed
by
treatment with enterokinase to provide the purified ALKI polypeptide (e.g.,
see
Hochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al., PNAS
USA
88:8972).
Techniques for making fusion genes are well known. Essentially, the joining
of various DNA fragments coding for different polypeptide sequences is
performed
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in accordance with conventional techniques, employing blunt-ended or stagger-
ended termini for ligation, restriction enzyme digestion to provide for
appropriate
termini, filling-in of cohesive ends as appropriate, alkaline phosphatase
treatment to
avoid undesirable joining, and enzymatic ligation. In another embodiment, the
fusion gene can be synthesized by conventional techniques including automated
DNA synthesizers. Alternatively, PCR amplification of gene fragments can be
carried out using anchor primers which give rise to complementary overhangs
between two consecutive gene fragments which can subsequently be annealed to
generate a chimeric gene sequence (see, for example, Current Protocols in
Molecular Biology, eds. Ausubel et al., John Wiley & Sons: 1992).
Examples of categories of nucleic acid compounds that are antagonists of
ALK1, BMP9, BMP10, GDF5, GDF6 or GDF7 include antisense nucleic acids,
RNAi constructs and catalytic nucleic acid constructs. A nucleic acid compound
may be single or double stranded. A double stranded compound may also include
regions of overhang or non-complementarity, where one or the other of the
strands is
single stranded. A single stranded compound may include regions of self-
complementarity, meaning that the compound forms a so-called "hairpin" or
"stem-
loop" structure, with a region of double helical structure. A nucleic acid
compound
may comprise a nucleotide sequence that is complementary to a region
consisting of
no more than 1000, no more than 500, no more than 250, no more than 100 or no
more than 50, 35, 30, 25, 22, 20 or 18 nucleotides of the full-length ALKI
nucleic
acid sequence or ligand nucleic acid sequence. The region of complementarity
will
preferably be at least 8 nucleotides, and optionally at least 10 or at least
15
nucleotides, and optionally between 15 and 25 nucleotides. A region of
complementarity may fall within an intron, a coding sequence or a noncoding
sequence of the target transcript, such as the coding sequence portion.
Generally, a
nucleic acid compound will have a length of about 8 to about 500 nucleotides
or
base pairs in length, and optionally the length will be about 14 to about 50
nucleotides. A nucleic acid may be a DNA (particularly for use as an
antisense),
RNA or RNA:DNA hybrid. Any one strand may include a mixture of DNA and
RNA, as well as modified forms that cannot readily be classified as either DNA
or
RNA. Likewise, a double stranded compound may be DNA:DNA, DNA:RNA or
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RNA:RNA, and any one strand may also include a mixture of DNA and RNA, as
well as modified forms that cannot readily be classified as either DNA or RNA.
A
nucleic acid compound may include any of a variety of modifications, including
one
or modifications to the backbone (the sugar-phosphate portion in a natural
nucleic
acid, including internucleotide linkages) or the base portion (the purine or
pyrimidine portion of a natural nucleic acid). An antisense nucleic acid
compound
will preferably have a length of about 15 to about 30 nucleotides and will
often
contain one or more modifications to improve characteristics such as stability
in the
serum, in a cell or in a place where the compound is likely to be delivered,
such as
the stomach in the case of orally delivered compounds and the lung for inhaled
compounds. In the case of an RNAi construct, the strand complementary to the
target transcript will generally be RNA or modifications thereof. The other
strand
may be RNA, DNA or any other variation. The duplex portion of double stranded
or
single stranded "hairpin" RNAi construct will preferably have a length of 18
to 40
nucleotides in length and optionally about 21 to 23 nucleotides in length, so
long as
it serves as a Dicer substrate. Catalytic or enzymatic nucleic acids may be
ribozymes or DNA enzymes and may also contain modified forms. Nucleic acid
compounds may inhibit expression of the target by about 50%, 75%, 90% or more
when contacted with cells under physiological conditions and at a
concentration
where a nonsense or sense control has little or no effect. Preferred
concentrations
for testing the effect of nucleic acid compounds are 1, 5 and 10 micromolar.
Nucleic
acid compounds may also be tested for effects on, for example, angiogenesis.
Nucleic acids encoding DAN polypeptides, including variants of wild type
DAN, and those encoding fusion proteins containing DAN proteins may be
generated and characterized as described above with respect to nucleic acids
encoding ALK1 ECD proteins.
4. Antibodies
Another aspect of the disclosure pertains to an antibody reactive with an
extracellular portion of an ALKI polypeptide, preferably antibodies that are
specifically reactive with ALKI polypeptide. In a preferred embodiment, such
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antibody may interfere with ALK1 binding to a ligand such as GDF5, GDF6, GDF7
BMP-9 or BMP-10 - it will be understood that an antibody against a ligand of
ALK1 should bind to the mature, processed form of the relevant protein. The
disclosure also provides antibodies that bind to GDF5, GDF6, GDF7, BMP9 and/or
BMP10 and inhibit ALKI binding to such ligands. Preferred antibodies will
exhibit
an anti-angiogenic activity in a bioassay, such as a CAM assay or corneal
micropocket assay (see above). A preferred anti-BMP9 antibody is described in
Example 10, below. In certain embodiments, an antibody that inhibits both BMP9
and BMP10 may be desirable; such an antibody may inhibit both ligands in an
ALK-
1 binding assay, in an angiogenesis assay (e.g. HUVEC tube forming assay, CAM
assay, Matrigel assay, or other such assays described herein).
The term "antibody" as used herein is intended to include whole antibodies,
e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments or
domains of
immunoglobulins which are reactive with a selected antigen. Antibodies can be
fragmented using conventional techniques and the fragments screened for
utility
and/or interaction with a specific epitope of interest. Thus, the term
includes
segments of proteolytically-cleaved or recombinantly-prepared portions of an
antibody molecule that are capable of selectively reacting with a certain
protein.
Non-limiting examples of such proteolytic and/or recombinant fragments include
Fab, F(ab')2, Fab', Fv, and single chain antibodies (scFv) containing a V[L]
and/or
V[H] domain joined by a peptide linker. The scFv's may be covalently or non-
covalently linked to form antibodies having two or more binding sites. The
term
antibody also includes polyclonal, monoclonal, or other purified preparations
of
antibodies and recombinant antibodies. The term "recombinant antibody", means
an
antibody, or antigen binding domain of an immunoglobulin, expressed from a
nucleic acid that has been constructed using the techniques of molecular
biology,
such as a humanized antibody or a fully human antibody developed from a single
chain antibody. Single domain and single chain antibodies are also included
within
the term "recombinant antibody".
Antibodies may be generated by any of the various methods known in the
art, including administration of antigen to an animal, administration of
antigen to an
animal that carries human immunoglobulin genes, or screening with an antigen
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against a library of antibodies (often single chain antibodies or antibody
domains).
Once antigen binding activity is detected, the relevant portions of the
protein may be
grafted into other antibody frameworks, including full-length IgG frameworks.
For
example, by using immunogens derived from an ALKI polypeptide or an ALKI
ligand (e.g., BMP9 or BMP10, or an immunogen common to both BMP9 and
BMP10), anti-protein/anti-peptide antisera or monoclonal antibodies can be
made by
standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by
Harlow and Lane (Cold Spring Harbor Press: 1988)). As shown in Figure 19, BMP9
and BMP10 have considerable amino acid identity, and therefore, each protein
may
be used as an immunogen to generate antibodies that can cross-react with both
BMP9 and BMPI O. Fragments of highly similar sequence may also be used as
immunogens. A mammal, such as a mouse, a hamster or rabbit can be immunized
with an immunogenic form of the peptide (e.g., a ALKI polypeptide or an
antigenic
fragment which is capable of eliciting an antibody response, or a fusion
protein).
Techniques for conferring immunogenicity on a protein or peptide include
conjugation to carriers or other techniques well known in the art. An
immunogenic
portion (preferably an extracellular portion) of an ALKI polypeptide or an
ALKI
ligand such as BMP9 or BMP10 can be administered in the presence of adjuvant.
The progress of immunization can be monitored by detection of antibody titers
in
plasma or serum. Standard ELISA or other immunoassays can be used with the
immunogen as antigen to assess the levels of antibodies.
Following immunization of an animal with an antigenic preparation of an
ALKI polypeptide or ligand polypeptide (e.g., BMP9 or BMP10), anti-ALKI or
anti-ligand antisera can be obtained and, if desired, polyclonal antibodies
can be
isolated from the serum. To produce monoclonal antibodies, antibody-producing
cells (lymphocytes) can be harvested from an immunized animal and fused by
standard somatic cell fusion procedures with immortalizing cells such as
myeloma
cells to yield hybridoma cells. Such techniques are well known in the art, and
include, for example, the hybridoma technique (originally developed by Kohler
and
Milstein, (1975) Nature, 256: 495-497), the human B cell hybridoma technique
(Kozbar et al., (1983) Immunology Today, 4: 72), and the EBV-hybridoma
technique to produce human monoclonal antibodies (Cole et al., (1985)
Monoclonal
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Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96). Hybridoma cells
can
be screened immunochemically for production of antibodies specifically
reactive
with a mammalian ALKI polypeptide of the present disclosure or ligands such as
BMP9 or BMP10 and monoclonal antibodies isolated from a culture comprising
such hybridoma cells. Antibodies with specificity for both BMP9 and BMP10 may
be selected from hybridomas that are obtained from animals inoculated with
either
BMP9 or BMP10 alone.
The term antibody as used herein is intended to include fragments thereof
which are also specifically reactive with one of the subject ALKI polypeptides
or
ALKI ligand polypeptides or a combination of target antigens (e.g., BMP9 and
BMP10). Antibodies can be fragmented using conventional techniques and the
fragments screened for utility in the same manner as described above for whole
antibodies. For example, F(ab)2 fragments can be generated by treating
antibody
with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide
bridges to produce Fab fragments. The antibody of the present disclosure is
further
intended to include bispecific, single-chain, and chimeric and humanized
molecules
having affinity for an ALKI polypeptide conferred by at least one CDR region
of
the antibody. In preferred embodiments, the antibody further comprises a label
attached thereto and is able to be detected, (e.g., the label can be a
radioisotope,
fluorescent compound, enzyme or enzyme co-factor).
In certain preferred embodiments, an antibody of the disclosure is a
recombinant antibody, particularly a humanized monoclonal antibody or a fully
human recombinant antibody.
The adjective "specifically reactive with" as used in reference to an antibody
is intended to mean, as is generally understood in the art, that the antibody
is
sufficiently selective between the antigen of interest (e.g. an ALKI
polypeptide or
an ALKI ligand) and other antigens that are not of interest that the antibody
is useful
for, at minimum, detecting the presence of the antigen of interest in a
particular type
of biological sample. In certain methods employing the antibody, a higher
degree of
specificity in binding may be desirable. For example, an antibody for use in
detecting a low abundance protein of interest in the presence of one or more
very
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high abundance protein that are not of interest may perform better if it has a
higher
degree of selectivity between the antigen of interest and other cross-
reactants.
Monoclonal antibodies generally have a greater tendency (as compared to
polyclonal
antibodies) to discriminate effectively between the desired antigens and cross-
reacting polypeptides. In addition, an antibody that is effective at
selectively
identifying an antigen of interest in one type of biological sample (e.g. a
stool
sample) may not be as effective for selectively identifying the same antigen
in a
different type of biological sample (e.g. a blood sample). Likewise, an
antibody that
is effective at identifying an antigen of interest in a purified protein
preparation that
is devoid of other biological contaminants may not be as effective at
identifying an
antigen of interest in a crude biological sample, such as a blood or urine
sample.
Accordingly, in preferred embodiments, the application provides antibodies
that
have demonstrated specificity for an antigen of interest in a sample type that
is likely
to be the sample type of choice for use of the antibody.
One characteristic that influences the specificity of an antibody: antigen
interaction is the affinity of the antibody for the antigen. Although the
desired
specificity may be reached with a range of different affinities, generally
preferred
antibodies will have an affinity (a dissociation constant) of about 10-6, 10-
7, 10-1, 10"9
or less. Given the apparently low binding affinity of TGF(3 for ALKI, it is
expected
that many anti-ALKI antibodies will inhibit TGF 3 binding. However, the
GDF5,6,7
group of ligands bind with a KD of approximately 5x 10"8 M and the BMP9,10
ligands bind with a KD of approximately 1x10"10 M. Thus, antibodies of
appropriate
affinity may be selected to interfere with the signaling activities of these
ligands.
In addition, the techniques used to screen antibodies in order to identify a
desirable antibody may influence the. properties of the antibody obtained. For
example, an antibody to be used for certain therapeutic purposes will
preferably be
able to target a particular cell type. Accordingly, to obtain antibodies of
this type, it
may be desirable to screen for antibodies that bind to cells that express the
antigen of
interest (e.g. by fluorescence activated cell sorting). Likewise, if an
antibody is to
be used for binding an antigen in solution, it may be desirable to test
solution
binding. A variety of different techniques are available for testing antibody:
antigen
interactions to identify particularly desirable antibodies. Such techniques
include
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ELISAs, surface plasmon resonance binding assays (e.g. the Biacore binding
assay,
Bia-core AB, Uppsala, Sweden), sandwich assays (e.g. the paramagnetic bead
system of IGEN International, Inc., Gaithersburg, Maryland), western blots,
immunoprecipitation assays and immunohistochemistry.
In a preferred embodiment, an antibody disclosed herein is an antibody that
binds to the mature portion of human BMP9, the amino acid sequence of which is
shown below:
RS AGAGSHCQKT SLRVNFEDIG WDSWIIAPKE YEAYECKGGC FFPLADDVTP TKHAIVQTLV
HLKFPTKVGK ACCVPTKLSP ISVLYKDDMG VPTLKYHYEG MSVAECGCR (SEQ ID NO:
12)
In an additional embodiment, an antibody disclosed herein is an antibody
that binds to the mature portion of human BMP10, the amino acid sequence of
which is shown below:
NAKG NYCKRTPLYI DFKEIGWDSW IIAPPGYEAY ECRGVCNYPL
AEHLTPTKHA IIQALVHLKN SQKASKACCV PTKLEPISIL YLDKGVVTYK FKYEGMAVSE
CGCR
(SEQ ID NO: 13)
Additionally, non-antibody proteins that bind to BMP9 or BMPIO may be
generated by selection from libraries. A wide variety of technologies are
available
for selecting random peptides, as well as framework based proteins, that bind
to a
particular ligand. In general, an approach to identifying a useful non-
antibody
protein will involve screening or selecting from a library those proteins that
bind to
BMP9 and/or BMP10 or inhibit a BMP9 or BMP10 activity, such as receptor (e.g.,
ALK1) binding or cellular signaling (e.g, SMAD 1/5 signaling).
5. Alterations in antibodies and Fc-fusion proteins
The application further provides antibodies, ALK1-Fc fusion proteins and
DAN-Fc fusion proteins with engineered or variant Fc regions. Such antibodies
and
Fc fusion proteins may be useful, for example, in modulating effector
functions,
such as, antigen-dependent cytotoxicity (ADCC) and complement-dependent
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cytotoxicity (CDC). Additionally, the modifications may improve the stability
of
the antibodies and Fc fusion proteins. Amino acid sequence variants of the
antibodies and Fc fusion proteins are prepared by introducing appropriate
nucleotide
changes into the DNA, or by peptide synthesis. Such variants include, for
example,
deletions from, and/or insertions into and/or substitutions of, residues
within the
amino acid sequences of the antibodies and Fc fusion proteins disclosed
herein. Any
combination of deletion, insertion, and substitution is made to arrive at the
final
construct, provided that the final construct possesses the desired
characteristics. The
amino acid changes also may alter post-translational processes of the
antibodies and
Fc fusion proteins, such as changing the number or position of glycosylation
sites.
Antibodies and Fc fusion proteins with reduced effector function may be
produced by introducing changes in the amino acid sequence, including, but are
not
limited to, the Ala-Ala mutation described by Bluestone et al. (see WO
94/28027
and WO 98/4753 1; also see Xu et al. 2000 Cell Immunol 200; 16-26). Thus in
certain embodiments, antibodies and Fc fusion proteins of the disclosure with
mutations within the constant region including the Ala-Ala mutation may be
used to
reduce or abolish effector function. According to these embodiments,
antibodies
and Fc fusion proteins may comprise a mutation to an alanine at position 234
or a
mutation to an alanine at position 235, or a combination thereof. In one
embodiment, the antibody or Fc fusion protein comprises an IgG4 framework,
wherein the Ala-Ala mutation would describe a mutation(s) from phenylalanine
to
alanine at position 234 and/or a mutation from leucine to alanine at position
235. In
another embodiment, the antibody or Fc fusion protein comprises an IgGI
framework, wherein the Ala-Ala mutation would describe a mutation(s) from
leucine to alanine at position 234 and/or a mutation from leucine to alanine
at
position 235. The antibody or Fc fusion protein may alternatively or
additionally
carry other mutations, including the point mutation K322A in the CH2 domain
(Hezareh et al. 2001 J Virol. 75: 12161-8).
In particular embodiments, the antibody or Fc fusion protein may be
modified to either enhance or inhibit complement dependent cytotoxicity (CDC).
Modulated CDC activity may be achieved by introducing one or more amino acid
substitutions, insertions, or deletions in an Fc region (see, e.g., U.S. Pat.
No.
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6,194,551). Alternatively or additionally, cysteine residue(s) may be
introduced in
the Fc region, thereby allowing interchain disulfide bond formation in this
region.
The homodimeric antibody thus generated may have improved or reduced
internalization capability and/or increased or decreased complement-mediated
cell
killing. See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J.
Immunol. 148:2918-2922 (1992), W099/51642, Duncan & Winter Nature 322: 738-
40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and W094/29351.
6. Methods and compositions for modulating angiogenesis and treating
certain disorders
The disclosure provides methods of inhibiting angiogenesis in a mammal by
administering to a subject an effective amount of a an ALKI ECD polypeptide,
such
as an ALKI-Fc fusion protein, a DAN protein, such as a DAN-Fc fusion protein,
or
an antibody disclosed herein, such as an antibody against GDF5, GDF6, GDF7,
BMP9, BMP10, or the ECD of ALKI, or nucleic acid antagonists (e.g., antisense
or
siRNA) of any of the foregoing hereafter collectively referred to as
"therapeutic
agents". The data presented indicate specifically that the anti-angiogenic
therapeutic
agents disclosed herein may be used to inhibit angiogenesis in the eye of a
mammal.
It is expected that these therapeutic agents will also be useful in inhibiting
angiogenesis in bones and joints, and in tumors, particularly tumors
associated with
bones and joints.
Angiogenesis associated diseases include, but are not limited to,
angiogenesis-dependent cancer, including, for example, solid tumors, blood
born
tumors such as leukemias, and tumor metastases; benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic
granulomas; rheumatoid arthritis; psoriasis; rubeosis; Osler-Webber Syndrome;
myocardial angiogenesis; plaque neovascularization; telangiectasia;
hemophiliac
joints; and angiofibroma.
In particular, polypeptide therapeutic agents of the present disclosure are
useful for treating or preventing a cancer (tumor), and particularly such
cancers as
are known to rely on angiogenic processes to support growth. Unlike most anti-
angiogenic agents, ALKI ECD polypeptides affect angiogenesis that is
stimulated
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by multiple factors. This is highly relevant in cancers, where a cancer will
frequently acquire multiple factors that support tumor angiogenesis. Thus, the
therapeutic agents disclosed herein will be particularly effective in treating
tumors
that are resistant to treatment with a drug that targets a single angiogenic
factor (e.g.,
bevacizumab, which targets VEGF). As demonstrated herein, an ALK1-Fc fusion
protein is effective in reducing the pathological effects of melanoma, lung
cancer
and multiple myeloma. Multiple myeloma is widely recognized as a cancer that
includes a significant angiogenic component. Accordingly, it is expected that
ALKI-Fc fusion proteins and other therapeutic agents disclosed herein will be
useful
in treating multiple myeloma and other tumors associated with the bone. As
demonstrated herein, therapeutic agents disclosed herein may be used to
ameliorate
the bone damage associated with multiple myeloma, and therefore may be used to
ameliorate bone damage associated with bone metastases of other tumors, such
as
breast or prostate tumors. As noted herein, the GDF5-7 ligands are highly
expressed
in bone, and, while not wishing to be limited to any particular mechanism,
interference with these ligands may disrupt processes that are required for
tumor
development in bone.
In certain embodiments of such methods, one or more polypeptide
therapeutic agents can be administered, together (simultaneously) or at
different
times (sequentially). In addition, polypeptide therapeutic agents can be
administered
with another type of compounds for treating cancer or for inhibiting
angiogenesis.
In certain embodiments, the subject methods of the disclosure can be used
alone. Alternatively, the subject methods may be used in combination with
other
conventional anti-cancer therapeutic approaches directed to treatment or
prevention
of proliferative disorders (e.g., tumor). For example, such methods can be
used in
prophylactic cancer prevention, prevention of cancer recurrence and metastases
after
surgery, and as an adjuvant of other conventional cancer therapy. The present
disclosure recognizes that the effectiveness of conventional cancer therapies
(e.g.,
chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can
be
enhanced through the use of a subject polypeptide therapeutic agent.
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A wide array of conventional compounds have been shown to have anti-
neoplastic activities. These compounds have been used as pharmaceutical agents
in
chemotherapy to shrink solid tumors, prevent metastases and further growth, or
decrease the number of malignant cells in leukemic or bone marrow
malignancies.
Although chemotherapy has been effective in treating various types of
malignancies,
many anti-neoplastic compounds induce undesirable side effects. It has been
shown
that when two or more different treatments are combined, the treatments may
work
synergistically and allow reduction of dosage of each of the treatments,
thereby
reducing the detrimental side effects exerted by each compound at higher
dosages.
In other instances, malignancies that are refractory to a treatment may
respond to a
combination therapy of two or more different treatments.
When a polypeptide therapeutic agent disclosed herein is administered in
combination with another conventional anti-neoplastic agent, either
concomitantly
or sequentially, such therapeutic agent may enhance the therapeutic effect of
the
anti-neoplastic agent or overcome cellular resistance to such anti-neoplastic
agent.
This allows decrease of dosage of an anti-neoplastic agent, thereby reducing
the
undesirable side effects, or restores the effectiveness of an anti-neoplastic
agent in
resistant cells.
According to the present disclosure, the antiangiogenic agents described
herein may be used in combination with other compositions and procedures for
the
treatment of diseases. For example, a tumor may be treated conventionally with
surgery, radiation or chemotherapy combined with the ALKI or ALKI ligand
antagonist and then the antagonist may be subsequently administered to the
patient
to extend the dormancy of micrometastases and to stabilize any residual
primary
tumor.
Angiogenesis-inhibiting agents can also be given prophylactically to
individuals known to be at high risk for developing new or re-current cancers.
Accordingly, an aspect of the disclosure encompasses methods for prophylactic
prevention of cancer in a subject, comprising administrating to the subject an
effective amount of an ALKI or ALKI ligand antagonist and/or a derivative
thereof,
or another angiogenesis-inhibiting agent of the present disclosure.
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As demonstrated herein, ALK1-Fc is effective for diminishing the phenotype
of a murine model of rheumatoid arthritis. Accordingly, therapeutic agents
disclosed herein may be used for the treatment of rheumatoid arthritis and
other type
of bone or joint inflammation.
Certain normal physiological processes are also associated with
angiogenesis, for example, ovulation, menstruation, and placentation. The
angiogenesis inhibiting proteins of the present disclosure are useful in the
treatment
of disease of excessive or abnormal stimulation of endothelial cells. These
diseases
include, but are not limited to, intestinal adhesions, atherosclerosis,
scleroderma, and
hypertrophic scars, i.e., keloids. They are also useful in the treatment of
diseases that
have angiogenesis as a pathologic consequence such as cat scratch disease
(Rochele
minalia quintosa) and ulcers (Helicobacter pylori).
General angiogenesis inhibiting proteins can be used as a birth control agent
by reducing or preventing uterine vascularization required for embryo
implantation.
Thus, the present disclosure provides an effective birth control method when
an
amount of the inhibitory protein sufficient to prevent embryo implantation is
administered to a female. In one aspect of the birth control method, an amount
of the
inhibiting protein sufficient to block embryo implantation is administered
before or
after intercourse and fertilization have occurred, thus providing an effective
method
of birth control, possibly a "morning after" method. While not wanting to be
bound
by this statement, it is believed that inhibition of vascularization of the
uterine
endometrium interferes with implantation of the blastocyst. Similar inhibition
of
vascularization of the mucosa of the uterine tube interferes with implantation
of the
blastocyst, preventing occurrence of a tubal pregnancy. Administration methods
may
include, but are not limited to, pills, injections (intravenous, subcutaneous,
intramuscular), suppositories, vaginal sponges, vaginal tampons, and
intrauterine
devices. It is also believed that administration of angiogenesis inhibiting
agents of
the present disclosure will interfere with normal enhanced vascularization of
the
placenta, and also with the development of vessels within a successfully
implanted
blastocyst and developing embryo and fetus.
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In the eye, angiogenesis is associated with, for example, diabetic
retinopathy,
retinopathy of prematurity, macular degeneration, corneal graft rejection,
neovascular glaucoma, and retrolental fibroplasias. The therapeutic agents
disclosed
herein may be administered intra-ocularly or by other local administration to
the eye.
Furthermore, as shown in the Examples, ALK1-Fc may be administered
systemically and yet have the desired effect on ocular angiogenesis.
Other diseases associated with angiogenesis in the eye include, but are not
limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens
overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis
sicca,
sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections,
lipid
degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex
infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma,
Mooren
ulcer, Terrien's marginal degeneration, mariginal keratolysis, rheumatoid
arthritis,
systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis,
Steven's
Johnson disease, periphigoid radial keratotomy, and corneal graft rejection.
sickle cell anemia, sarcoid, pseudoxanthoma elasticum, Pagets disease, vein
occlusion, artery occlusion, carotid obstructive disease, chronic
uveitis/vitritis,
mycobacterial infections, Lyme's disease, systemic lupus erythematosis,
retinopathy
of prematurity, Eales disease, Bechets disease, infections causing a retinitis
or
choroiditis, presumed ocular histoplasmosis, Bests disease, myopia, optic
pits,
Stargarts disease, pars planitis, chronic retinal detachment, hyperviscosity
syndromes, toxoplasmosis, trauma and post-laser complications. Other diseases
include, but are not limited to, diseases associated with rubeosis
(neovasculariation
of the angle) and diseases caused by the abnormal proliferation of
fibrovascular or
fibrous tissue including all forms of proliferative vitreoretinopathy.
Conditions of the eye can be treated or prevented by, e.g., systemic, topical,
intraocular injection of a therapeutic agent, or by insertion of a sustained
release
device that releases a therapeutic agent. A therapeutic agent may be delivered
in a
pharmaceutically acceptable ophthalmic vehicle, such that the compound is
maintained in contact with the ocular surface for a sufficient time period to
allow the
compound to penetrate the corneal and internal regions of the eye, as for
example
the anterior chamber, posterior chamber, vitreous body, aqueous humor,
vitreous
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humor, cornea, iris/ciliary, lens, choroid/retina and sclera. The
pharmaceutically-
acceptable ophthalmic vehicle may, for example, be an ointment, vegetable oil
or an
encapsulating material. Alternatively, the therapeutic agents of the
disclosure may
be injected directly into the vitreous and aqueous humour. In a further
alternative,
the compounds may be administered systemically, such as by intravenous
infusion
or injection, for treatment of the eye.
One or more therapeutic agents can be administered. The methods of the
disclosure also include co-administration with other medicaments that are used
to
treat conditions of the eye. When administering more than one agent or a
combination of agents and medicaments, administration can occur simultaneously
or
sequentially in time. The therapeutic agents and/or medicaments may be
administered by different routes of administration or by the same route of
administration. In one embodiment, a therapeutic agent and a medicament are
administered together in an ophthalmic pharmaceutical formulation.
In one embodiment, a therapeutic agent is used to treat a disease associated
with angiogenesis in the eye by concurrent administration with other
medicaments
that act to block angiogenesis by pharmacological mechanisms. Medicaments that
can be concurrently administered with a therapeutic agent of the disclosure
include,
but are not limited to, pegaptanib (MacugenTM), ranibizumab (LucentisTM),
squalamine lactate (EvizonTM), heparinase, and glucocorticoids (e.g.
Triamcinolone). In one embodiment, a method is provided to treat a disease
associated with angiogenesis is treated by administering an ophthalmic
pharmaceutical formulation containing at least one therapeutic agent disclosed
herein and at least one of the following medicaments: pegaptanib (MacugenTM),
ranibizumab (LucentisTM), squalamine lactate (EvizonTM), heparinase, and
glucocorticoids (e.g. Triamcinolone).
7. Formulations and Effective Doses
The therapeutic agents described herein may be formulated into
pharmaceutical compositions. Pharmaceutical compositions for use in accordance
with the present disclosure may be formulated in conventional manner using one
or
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more physiologically acceptable carriers or excipients. Such formulations will
generally be substantially pyrogen free, in compliance with most regulatory
requirements.
In certain embodiments, the therapeutic method of the disclosure includes
administering the composition systemically, or locally as an implant or
device.
When administered, the therapeutic composition for use in this disclosure is
in a
pyrogen-free, physiologically acceptable form. Therapeutically useful agents
other
than the ALK1 signaling antagonists which may also optionally be included in
the
composition as described above, may be administered simultaneously or
sequentially with the subject compounds (e.g., ALKI ECD polypeptides or any of
the antibodies disclosed herein) in the methods disclosed herein.
Typically, protein therapeutic agents disclosed herein will be administered
parentally, and particularly intravenously or subcutaneously. Pharmaceutical
compositions suitable for parenteral administration may comprise one or more
ALK1 ECD polypeptides or other antibodies in combination with one or more
pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions, suspensions or emulsions, or sterile powders which may be
reconstituted into sterile injectable solutions or dispersions just prior to
use, which
may contain antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or suspending or
thickening agents. Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the disclosure include
water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene
glycol, and
the like), and suitable mixtures thereof, vegetable oils, such as olive oil,
and
injectable organic esters, such as ethyl oleate. Proper fluidity can be
maintained, for
example, by the use of coating materials, such as lecithin, by the maintenance
of the
required particle size in the case of dispersions, and by the use of
surfactants.
In one embodiment, the antibodies and ALK1 ECD proteins disclosed herein
are administered in an ophthalmic pharmaceutical formulation. In some
embodiments, the ophthalmic pharmaceutical formulation is a sterile aqueous
solution, preferable of suitable concentration for injection, or a salve or
ointment.
Such salves or ointments typically comprise one or more antibodies or ALKI ECD
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proteins disclosed herein dissolved or suspended in a sterile pharmaceutically
acceptable salve or ointment base, such as a mineral oil-white petrolatum
base. In
salve or ointment compositions, anhydrous lanolin may also be included in the
formulation. Thimerosal or chlorobutanol are also preferably added to such
ointment
compositions as antimicrobial agents. In one embodiment, the sterile aqueous
solution is as described in U.S. Pat. No. 6,071,958.
The disclosure provides formulations that may be varied to include acids and
bases to adjust the pH; and buffering agents to keep the pH within a narrow
range.
Additional medicaments may be added to the formulation. These include, but are
not
limited to, pegaptanib, heparinase, ranibizumab, or glucocorticoids. The
ophthalmic
pharmaceutical formulation according to the disclosure is prepared by aseptic
manipulation, or sterilization is performed at a suitable stage of
preparation.
The compositions and formulations may, if desired, be presented in a pack or
dispenser device which may contain one or more unit dosage forms containing
the
active ingredient. The pack may for example comprise metal or plastic foil,
such as
a blister pack. The pack or dispenser device may be accompanied by
instructions for
administration.
EXAMPLES:
Example 1: Expression ofALK1-Fc fusion proteins
Applicants constructed a soluble ALKI fusion protein that has the
extracellular domain of human ALKI fused to a human Fe or mouse ALKI fused to
a murine Fc domain with a minimal linker in between. The constructs are
referred
to as hALK I-Fc and mALK I-Fc, respectively.
hALKI-Fc is shown as purified from CHO cell lines in Figure 3 (SEQ ID
NO: 3). Notably, while the conventional C-terminus of the extracellular domain
of
human ALKI protein is amino acid 118 of SEQ ID NO:1, we have determined that
it is desirable to avoid having a domain that ends at a glutamine residue.
Accordingly, the portion of SEQ ID NO:3 that derives from human ALKI
incorporates two residues c-terminal to Q118, a leucine and an alanine. The
disclosure therefore provides ALKI ECD polypeptides (including Fc fusion
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proteins) having a c-terminus of the ALK1 derived sequence that is anywhere
from 1
to 5 amino acids upstream (113-117 relative to SEQ ID NO:1) or downstream (119-
123) ofQ118.
The hALK1-Fc and mALKI-Fc proteins were expressed in CHO cell lines.
Three different leader sequences were considered:
(i) Honey bee mellitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO:
7)
(ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP
(SEQ ID NO: 8)
(iii) Native: MTLGSPRKGLLMLLMALVTQG (SEQ ID NO: 9).
The selected form employs the TPA leader and has the unprocessed amino
acid sequence shown in Figure 4 (SEQ ID NO:5).
This polypeptide is encoded by the nucleic acid sequence shown in Figure 4
(SEQ ID NO:4).
Purification can be achieved by a series of column chromatography steps,
including, for example, three or more of the following, in any order: protein
A
chromatography, Q sepharose chromatography, phenylsepharose chromatography,
size exclusion chromatography, and cation exchange chromatography. The
purification can be completed with viral filtration and buffer exchange. The
hALK1-Fc protein was purified to a purity of >98% as determined by size
exclusion
chromatography and >95% as determined by SDS PAGE.
In the course of protein production and purification, we observed that
hALK1-Fc tends to be expressed in a mixture of dimers and higher order
aggregates
which, while appearing pure under denaturing, reducing conditions (e.g.,
reducing
SDS-PAGE), are problematic for administration to a patient. The aggregates may
be
immunogenic or poorly bioavailable, and because of their heterogeneity, these
aggregates make it difficult to characterize the pharmaceutical preparation at
a level
that is desirable for drug development. Thus, various approaches were tested
to
reduce the amount of aggregate in final preparations.
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In one approach, a number of different cell culture media were tested. IS
CHO-CD (Cat. No. 91119, Irvine Scientific, Santa Ana, CA) showed a remarkable
reduction in the production of aggregated products, while maintaining high
level
production of the hALKI-Fc. Additionally, elution of the material from a
hydrophobic interaction column (e.g., phenylsepharose) at a pH of 8.0 resulted
in
further resolution of the aggregated product. The resulting material is
comprised of
greater than 99% dimers. A comparison to an ALKI-Fc fusion protein sold by R&D
Systems (cat. no. 370-AL, Minneapolis, MN) shows that this protein, produced
in
NSO cells, is 84% dimers, with the remaining protein appearing as high
molecular
weight species by size exclusion chromatography. A comparison of the sizing
column profile for the preparations is shown in Figure 11. Having identified
aggregate formation as a significant problem in ALKI-Fc production, it is
expected
that other approaches may be developed, including approaches that involve
additional purification steps (although such approaches may result in lower
yield of
purified protein).
Example 2: Identification of ALKI-Fc Ligands
ALKI is a type I receptor for ligands of the TGF(3 family. Multiple
members of the TGF(3 family were tested for binding to a human ALKI-Fc fusion
protein, using a BiacoreTM system. TGF(3 itself, GDF8, GDF11, BMP2 and BMP4
all failed to show substantial binding to the hALKI-Fc protein, while BMP2 and
BMP4 showed only limited binding. In contrast, GDF5 and GDF7 displayed
significant binding, with KD values of approximately 5 x 10-8 M in both cases.
Based on the structural similarity of GDF5 and GDF7 to GDF6, it is expected
that
GDF6 will bind the fusion protein with similar affinity. The highest binding
affinity
to hALKI-Fc was observed for BMP9, with KD values ranging from 1 x 10"10 to 2
x
10"9, and BMP10, with a KD of approximately 3 x 10-9.
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Example 3: Characterization of ALK 1-Fc and anti-ALK 1 Antibody Effects on
Endothelial Cells
Using a luciferase reporter construct under the control of four sequential
consensus SBE sites (SBE4-luc), which are responsive to Smad1/5/8-mediated
signaling, we measured BMP-9 mediated activity in the presence and absence of
hALKI-Fc drug or neutralizing ALKI specific monoclonal antibody in HMVEC
cells. HMVEC cells were stimulated with rhBMP-9 (50ng/ml), which induced
Smad1/5/8-mediated transcriptional activation, evidenced here by the increase
in
SBE4-luc modulated transcriptional upregulation. When added, the hALKI-Fc
compound (10 g/ml) or antibody (10 g/ml) diminished this transcriptional
response, each by nearly 60%, indicating that the presence of ALKI-Fc
significantly
reduces BMP9 signaling, and moreover, that the BMP9 signaling is related to
ALKI
activity.
Activation of SMAD phosphorylation is commonly used to assay activation
of upstream activin receptors. ALKI is known to modulate phosphorylation of
SMAD proteins 1,5 and 8 upon activation by its ligand. Here, we added rhBMP-9
(50ng/ml) to initiate SMAD phosphorylation in HUVEC cells, a human endothelial
cell line which innately expresses ALKI receptor, over a timecourse of 30
minutes.
Phosphorylation of SMAD 1/5/8 was seen 5 minutes after treatment of cells with
ligand and phosphorylation was maintained for the entirety of the 30 minute
period.
In the presence of relatively low concentrations of hALKI-Fc (250ng/ml), SMAD
1/5/8 phosphorylation was reduced, confirming that this agent inhibits
Smadl/5/8
activation in endothelial cells.
In order to evaluate the angiogenic effect of ALKI-Fc in an in vitro system,
we assayed the effectiveness of the compound in reducing tube formation of
endothelial cells on a Matrigel substrate. This technique is commonly used to
assess
neovascularization, giving both rapid and highly reproducible results.
Endothelial
Cell Growth Supplement (ECGS) is used to induce the formation of microvessels
from endothelial cells on Matrigel, and the efficacy of anti-angiogenic
compounds
are then gauged as a reduction of cord formation in the presence of both the
drug
and ECGS over an 18 hour timecourse. As expected, addition of ECGS (200ng/ml)
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induced significant cord formation, as compared to the negative control (no
treatment added), which indicates basal levels of endothelial cell cord
formation
produced on Matrigel substrate (Fig 5). Upon addition of either hALKI-Fc (100
ng/ml) or mALK1-Fc (100ng/mI), cord formation was visibly reduced. Final
quantification of vessel length in all samples revealed that every
concentration of
hALK1-fc or mALKI-Fc reduced neovascularization to basal levels. Additionally,
hALK 1-Fc and mALK I-Fc in the presence of the strongly pro-angiogenic factor
ECGS maintained strong inhibition of neovascularization demonstrating even
more
potent anti-angiogenic activity than the negative control endostatin
(100ng/ml).
Example 4: CAM Assays
VEGF and FGF are well-known to stimulate angiogenesis. A CAM (chick
chorioallantoic membrane) assay system was used to assess the angiogenic
effects of
GDF7. As shown in Figure 6, GDF7 stimulates angiogenesis with a potency that
is
similar to that of VEGF. Similar results were observed with GDF5 and GDF6.
ALK1-Fc fusions were tested for anti-angiogenic activity in the CAM assay.
These fusion proteins showed a potent anti-angiogenic effect on angiogenesis
stimulated by VEGF, FGF and GDF7. See Figure 7. BMP9 and PDGF showed a
relatively poor capability to induce angiogenesis in this assay, but such
angiogenesic
effect of these factors was nonetheless inhibited by ALKI.
ALKI-Fc proteins and a commercially available, anti-angiogenic anti-VEGF
monoclonal antibody were compared in the CAM assay. The ALKI-Fc proteins had
similar potency as compared to anti-VEGF. The anti-VEGF antibody bevacizumab
is currently used in the treatment of cancer and macular degeneration in
humans. See
Figure 8.
Interestingly, an anti-ALK1 antibody (R&D Systems) failed to significantly
inhibit angiogenesis in this assay system. We expect that this may reflect the
difference in the ALKI sequence in different species.
Example 5: Mouse Corneal Micropocket Assay
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The mouse corneal micropocket assay was used to assess the effects of
ALKI-Fc on angiogenesis in the mouse eye. hALKI-Fc, administered
intraperitoneally, significantly inhibited ocular angiogenesis. As shown in
Figure 9,
hALK1-Fc inhibited ocular angiogenesis to the same degree as anti-VEGF. hALKI-
Fc and anti-VEGF were used at identical weight/weight dosages. Similar data
were
obtained when a Matrigel plug impregnated with VEGF was implanted in a non-
ocular location.
These data demonstrate that high affinity ligands for ALKI promote
angiogenesis and that an ALK1-Fc fusion protein has potent anti-angiogenic
activity. The ligands for ALKI fall into two categories, with the GDF5,6,7
grouping
having an intermediate affinity for ALKI and the BMP9,10 grouping having a
high
affinity for ALK 1.
GDF5, GDF6, and GDF7 are primarily localized to bone and joints, while
BMP9 is circulated in the blood. Thus, there appears to be a pro-angiogenic
system
of the bones and joints that includes ALK1, GDF5, GDF6, and GDF7 and a
systemic angiogenic system that includes ALKI and BMP9 (and possibly BMP10).
Example 6: Murine Model of Rheumatoid Arthritis
The murine collagen-induced arthritis model is a well-accepted model of
rheumatoid arthritis. In this study, groups of 10 mice were treated with
vehicle, anti-
VEGF (bevacizumab - as a negative control, because bevacizumab does not
inhibit
murine VEGF), or doses of mALK 1-Fc ("RAP-041 ") at 1 mg/kg, 10 mg/kg or 25
mg/kg. Following the collagen boost on day 21 arthritic scores (see Figure 10)
and
paw swelling steadily increased in all groups, peaking around day 38. Mice
treated
with mALK I -Fc ("RAP-041 ") showed reduced scores for both characteristics,
particularly at the highest dose (25mg/kg), although the reduction did not
achieve
statistical significance. Nonetheless, a dose-related trend is apparent.
By study termination at day 42 the incidence of arthritis had reached 10/10 in
the vehicle control treated mice, 9/10 in the bevacizumab treated mice, 8/10
in the
mALKI-Fc at Img/kg treated group and 9/10 in the mALKI-Fc 10mg/kg treated
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group. In the mALKI-Fc 25mg/kg treated group disease incidence was lower at
6/10.
Example 7: Ligand Binding Characteristics of DAN
DAN is a member of a family of secreted cystine knot proteins that inhibit
BMP activity. DAN is known to bind to and antagonize GDF5. We determined that
DAN also binds tightly to GDF7, but not to BMP9. Thus, we conclude that DAN
inhibits the suite of bone and joint localized ligands for ALK1, and DAN is
expected
to be a potent antagonist of bone and joint related angiogenesis. Thus DAN may
be
useful in treating cancers of the bone, e.g., multiple myeloma and bone
metastases,
as well as rheumatoid arthritis and osteoarthritis.
Taken together, the findings disclosed in these Examples provide numerous
reagents, described herein, for inhibiting angiogenesis in vivo, and
particularly
ocular angiogenesis. These findings also indicate that agents targeted to
GDF5,
GDF6, and GDF7 can be used to selectively inhibit bone and joint angiogenesis.
These findings further indicate that such agents can be used to treat cancers
and
rheumatoid arthritis.
Example 8: ALK 1-Fc Reduces Tumor Angiogenesis in a CAM Assay
Tumors, as with any tissue, have a basic nutrient and oxygen requirement.
Although small tumors are capable of acquiring adequate amounts via diffusion
from neighboring blood vessels, as the tumor increases in size, it must secure
nutrients by recruiting and maintaining existing capillaries. In order to test
the
capacity of ALKI-Fc proteins to limit tumor growth through vessel inhibition,
we
tested varying concentrations of mALKI-Fc in a melanoma explant CAM assay. As
with CAM assays described above, small windows were made in the surface of
each
egg through which 5x105 B 16 melanoma cells were implanted. Eggs were then
treated daily with 0.02 mg/ml mALK1-Fc, 0.2 mg/ml mALKI-Fc, or left untreated
for a period of a week. At the end of the experiment, tumors were carefully
removed, weighed and digital images were captured. Tumors originating from
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CAMs treated with mALK1-Fc showed a significant decrease in size as compared
to
untreated CAM tumors. Quantification of tumor weight demonstrated that weight
of
tumors treated daily with either 0.02 mg/ml or 0.2 mg/ml mALKI-Fc showed a
reduction of 65% and 85% compared to the untreated CAMs. In conclusion,
neovascularization and tumor growth was significantly suppressed upon addition
of
ALK1-Fc in a dose-responsive manner, indicating that ALK1-Fc is a powerful
anti-
angiogenic agent.
Example 9: Lung Cancer Experimental Model
To further confirm the effects of ALK 1-Fc on tumor progression, a mouse
model of lung cancer was tested. Fluorescently labeled murine Lewis lung
cancer
cells (LL/2-luc) were administered to albino Black 6 mice through the tail
vein. On
the same day, the mice began treatment with either PBS control (n=7) or
10mg/kg
mALKI-Fc (n=7) administered intraperitoneally. In-life fluorescent imaging
showed substantial development of tumors localized to the lungs in the control
mice,
to the point that the mice became moribund and had to be sacrificed by day 22
post-
implantation. By contrast, the ALK1-Fc treated mice showed a substantially
delayed growth of lung tumors and exhibited 100% survival as of day 22. See
Figure 12.
These data demonstrate that ALK 1-Fc has substantial effect on tumor growth
in a mouse model of lung cancer and provides a survival benefit.
Example 10. BMP9 and Anti-BMP9, Effects on Angiogenesis
A CAM (chick chorioallantoic membrane) assay system was used to assess
the angiogenic effects of recombinant human BMP9 (rhB9) and anti-BMP9
monoclonal antibody (mabB9) (R&D Systems, Minneapolis, MN, Cat. No.
MAB3209). This antibody is known to neutralize BMP9/ALK1 signaling. See, e.g.,
Scharpfenecker et al., JCell Sci. 2007 Mar 15;120(Pt 6):964-72; David et al.
Blood
Mar 1;109(5):1953-61; 2007; David et al., Circ Res. 2008 Apr 25;102(8):914-22.
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Neither BMP9 nor anti-BMP9 had a substantial effect on angiogenesis in the
absence of exogenous VEGF, probably because the lack of angiogenesis in the
absence of exogenous VEGF decreases the sensitivity of the assay. See Figure
13,
right hand columns. In the absence of VEGF, both proteins were used at the
50ng
dosed Ix/day on days 1 and 3 in the 5-day cycle. However, in the presence of
VEGF, both BMP9 and its antibody had a substantial anti-angiogenic effect. See
Figure 13. These data are consistent with data from Scharpfenecker et al.,
with
respect to BMP9 and VEGF in combination, and are also consistent with the
conclusions of Scharpfenecker et al. and David et al. with respect to the anti-
angiogenic effects of BMP9 itself. However, the effects of the anti-BMP9
antibody
are in remarkable contrast to the published literature. Based on these data,
we
hypothesize that optimal or physiological levels of BMP9 may be needed for
proper
angiogenesis, and that either an excess or deficiency in BMP9 will inhibit
angiogenesis.
Intriguingly, the effects of the anti-BMP9 antibody are consistent with data
presented here showing that ALKI-Fc (which is an alternative BMP9 antagonist)
also inhibits angiogenesis. Thus, these data demonstrate that ALKI-Fc and anti-
BMP9 each have anti-angiogenic effects, and that anti-BMP9 antibody is likely
to be
useful in the treatment of angiogenic disorders, such as tumors, rheumatoid
arthritis
and ocular disorders, in much the same way that ALKI-Fc is shown to be.
Given the anti-angiogenic activity of the MAB3209, we propose that this
murine monoclonal antibody could be humanized to provide a therapeutic agent
for
use in humans. The antibody may be humanized by a variety of art-recognized
techniques, including chimerization, CDR-grafting, resurfacing, back
mutations,
superhumanization, human string content optimization, and empirical methods,
such
as FR library generation and selection, FR shuffling and humaneering. See,
e.g,
Almagro and Fransson, Frontiers in Biosciences, 13: 1619-1633, 2008.
Example 11. Effects of ALKI-Fc Fusion Protein on Breast Cancer Tumor Models
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mALKI-Fc was effective in delaying the growth of breast cancer tumor cell
lines derived from both estrogen receptor positive (ER+) and estrogen receptor
negative tumor cells (ER-).
The MDA-MB-231 breast cancer cell line (derived from ER- cells) was
stably transfected with the luciferase gene to allow for the in vivo detection
of tumor
growth and potential metastasis. In this study, 1 x 106 MDA-MB-231-Luc cells
were implanted orthotopically in the mammary fat pad of athymic nude mice
(Harlan). Tumor progression was followed by bioluminescent detection using an
IVIS Spectrum imaging system (Caliper Life Sciences). An increase in the
luminescence (number of photons detected) corresponds to an increase in tumor
burden.
Thirty female nude mice were injected with 1 x 106 tumor cells into the
mammary fat pad. Three days after tumor implantation the mice were treated
with
either vehicle control or mALKI-Fc (30 mg/kg) twice per week by subcutaneous
(SC) injection. Treatment was continued and tumor progression was monitored by
bioluminescent imaging for 10 weeks. mALKI-Fc treatment at 30 mg/kg slowed
tumor progression as determined by bioluminescent detection when compared to
vehicle treated controls (Figure 14). Treatment with mALKI-Fc delayed, but did
not reverse tumor growth in this model. This may be expected of an
antiangiogenic
compound in that tumors may be able to survive to a certain size before
requiring
new blood vessel formation to support continued growth. In a similar
experiment,
hALKI-Fc produced similar, if slightly lesser, effects at dose levels as low
as 3
mg/kg.
The estrogen-receptor-positive (ER+), luciferase expressing cell line, MCF-
7, was also tested in an orthotopic implantation model. In this model, female
nude
mice are implanted subcutaneously with a 60 day slow release pellet of 17(3-
estradiol. Two days following pellet implantation, 5 x 106 MCF-7 tumor cells
were
implanted into the mammary fat pad. Mice were treated twice per week with
hALKI-Fc at 3, 10 and 30 mg/kg, or vehicle control, by the IP route. Tumor
progression was followed by bioluminescent imaging on a weekly basis with an
IVIS-Spectrum imager (Caliper Life Sciences). In vehicle treated mice tumors
progressed rapidly until study day 26 (Figure 15). After day 26, there were
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fluctuations in tumor luminescence until the conclusion of the study at day 60
(when
the estradiol pellets were depleted). These fluctuations are due to a common
feature
of this model in that the rapid tumor growth can exceed the angiogenic
response of
the host animals leading to tumor necrosis and a concomitant drop-off in
luminescent signal. The remaining cells continue to grow leading to an
increased
signal. Mice treated with 10 or 30 mg/kg of hALKI-Fc were able to maintain
tumor
size at a constant level during the study, compared to vehicle-treated
controls,
indicating a potent effect of this molecule on tumor growth.
Example 12. Inhibition of BMP10 Signaling by hALKI-Fc in a Cell-based Assay
Effects of hALK-Fc on BMP10 signaling were determined in a cell-based
assay, in which human glioblastoma T98G cells were transfected with three
plasmids: 1) an expression construct encoding full-length ALK1; 2) a firefly-
luciferase reporter construct (see Example 3) responsive to Smadl/5/8-mediated
signaling, and 3) a Renilla-luciferase control construct. Treatment of
transfected
cells with recombinant human BMPIO (1 ng/ml) strongly stimulated firefly
luciferase activity relative to Renilla luciferase activity (Figure 16).
Omission of the
ALK1 expression construct reduced BMP10-stimulated activity by approximately
two-thirds (data not shown), thus implicating ALK1 as a major mediator of the
BMP10 signal. Treatment of fully transfected cells with hALKI-Fc (65 ng/ml)
and
BMPIO (1 ng/ml) reduced the transcriptional response compared to BMP10 alone
by more than 80% (Figure 16). Together, these results indicate that ALK1 is a
major mediator of BMP10 signaling and that ALK1-Fc can markedly inhibit such
signaling.
Example 13. BMP9/10 Antagonists Inhibit VEGF- and BMP I 0-induced
Angiogenesis in a CAM Assay
A chick chorioallantoic membrane (CAM) assay system was used to
investigate the effect of BMP9/10 antagonists on angiogenesis. In the first
experiment, VEGF and BMPIO were used separately to promote angiogenesis.
Compared to VEGF alone, combined treatment with VEGF (50 ng daily) and
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BMP9/10 antagonist (82 pmol daily) reduced vessel count by more than half
(Figure
17). Compared to BMPIO alone, combined treatement with BMPIO (50 ng daily)
and BMP9/10 anagonist (82 pmol daily) also reduced vessel count by more than
half
(Figure 17). Similar inhibitory effects on VEGF- or BMP10-induced angiogenesis
were observed with hALK1-Fc (41 pmol daily). In a second CAM experiment, three
doses of a different BMP9/10 antagonist were evaluated for their ability to
inhibit
angiogenesis induced by a higher dose (150 ng) of BMP10. A clear dose-
dependent
inhibition of BMP10-stimulated angiogenesis was observed, with the highest
dose of
BMP9/10 antagonist (164 pmol daily, 4x) resulting in a 49% reduction in vessel
count compared to BMP10 alone (Figure 18). These data demonstrate that, in
contrast to BMP9, BMP10 stimulates angiogenesis, and that antagonists of BMP10
can inhibit angiogenesis. This result is in contrast to published literature
suggesting
that BMP10 should, like BMP9, inhibit angiogenesis. These in vivo data
demonstrate that BMP9 and/or BMP10 antagonists and ALKI-Fc can significantly
inhibit angiogenesis induced by either VEGF or BMP10.
INCORPORATION BY REFERENCE
All publications and patents mentioned herein are hereby incorporated by
reference in their entirety as if each individual publication or patent was
specifically
and individually indicated to be incorporated by reference. In case of
conflict, the
present application, including any definitions herein, will control.
EQUIVALENTS
While specific embodiments of the subject inventions are explicitly disclosed
herein, the above specification is illustrative and not restrictive. Many
variations of
the inventions will become apparent to those skilled in the art upon review of
this
specification and the claims below. The full scope of the inventions should be
CA 02724525 2010-11-15
WO 2009/139891 PCT/US2009/003016
determined by reference to the claims, along with their full scope of
equivalents, and
the specification, along with such variations.
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