U.S. patent application number 13/907798 was filed with the patent office on 2013-12-26 for alk1 receptor and ligand antagonists and uses thereof.
This patent application is currently assigned to Acceleron Pharma, Inc.. The applicant listed for this patent is Asya Grinberg, John Knopf, Ravindra Kumar, Robert S. Pearsall, Jasbir Seehra. Invention is credited to Asya Grinberg, John Knopf, Ravindra Kumar, Robert S. Pearsall, Jasbir Seehra.
Application Number | 20130344067 13/907798 |
Document ID | / |
Family ID | 39365087 |
Filed Date | 2013-12-26 |
United States Patent
Application |
20130344067 |
Kind Code |
A1 |
Grinberg; Asya ; et
al. |
December 26, 2013 |
ALK1 RECEPTOR AND LIGAND ANTAGONISTS AND USES THEREOF
Abstract
In certain aspects, the present disclosure relates to the
insight that a polypeptide comprising a ligand-binding portion of
the extracellular domain of activin-like kinase I (ALK1)
polypeptide may be used to inhibit angiogenesis in vivo,
particularly in mammals suffering angiogenesis-related disorders.
The disclosure also identifies ligands for ALK1 and demonstrates
that such ligands have pro-angiogenic activity, and antibodies that
inhibit receptor-ligand interaction.
Inventors: |
Grinberg; Asya; (Lexington,
MA) ; Knopf; John; (Carlisle, MA) ; Pearsall;
Robert S.; (North Reading, MA) ; Kumar; Ravindra;
(Acton, MA) ; Seehra; Jasbir; (Lexington,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Grinberg; Asya
Knopf; John
Pearsall; Robert S.
Kumar; Ravindra
Seehra; Jasbir |
Lexington
Carlisle
North Reading
Acton
Lexington |
MA
MA
MA
MA
MA |
US
US
US
US
US |
|
|
Assignee: |
Acceleron Pharma, Inc.
Cambridge
MA
|
Family ID: |
39365087 |
Appl. No.: |
13/907798 |
Filed: |
May 31, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11982738 |
Nov 2, 2007 |
8455428 |
|
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13907798 |
|
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60856592 |
Nov 2, 2006 |
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Current U.S.
Class: |
424/134.1 ;
424/139.1; 435/188; 530/387.3; 530/387.9; 536/23.2 |
Current CPC
Class: |
C07K 2317/52 20130101;
A61P 35/00 20180101; C07K 16/18 20130101; C07K 2319/30 20130101;
C07K 14/71 20130101; A61P 17/06 20180101; A61P 7/00 20180101; A61K
45/06 20130101; A61P 43/00 20180101; A61P 19/02 20180101; A61K
39/39558 20130101; A61P 27/02 20180101; A61P 35/04 20180101; A61P
9/00 20180101; A61P 29/00 20180101 |
Class at
Publication: |
424/134.1 ;
530/387.3; 530/387.9; 424/139.1; 536/23.2; 435/188 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 45/06 20060101 A61K045/06; C07K 16/18 20060101
C07K016/18 |
Claims
1. An ALK1-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 and BMP9 with a K.sub.D of less than
1.times.10.sup.-7 M and binds to TGF.beta.-1 with a K.sub.D of
greater than 1.times.10.sup.-6.
2. The ALK1-Fc fusion protein, wherein the Fc portion is an Fc
portion of a human IgG 1.
3. An ALK1-Fc fusion protein comprising the amino acid sequence of
SEQ ID NO: 3.
4. An ALK1-Fc fusion protein that is produced by expression of the
nucleic acid of SEQ ID NO:4 in a mammalian cell line.
5. The ALK1-Fc fusion protein of claim 4, wherein the mammalian
cell line is a Chinese Hamster Ovary (CHO) cell line.
6. A pharmaceutical preparation that is substantially pyrogen free,
comprising an ALK1-Fc fusion protein of claim 5.
7. 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.
8. The antibody of claim 7, wherein the antibody binds to the ALK1
polypeptide with a K.sub.D of less than 5.times.10.sup.-8 M.
9. The antibody of claim 7, wherein the antibody binds to the ALK1
polypeptide with a K.sub.D of less than 1.times.10.sup.-10 M.
10. The antibody of claim 8, wherein the antibody inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: GDF5, GDF6 and GDF7.
11. The antibody of claim 10, wherein the antibody inhibits binding
of BMP9 and BMP10 to ALK1.
12. A pharmaceutical preparation that is substantially pyrogen free
comprising the antibody of claim 7.
13. A method of inhibiting angiogenesis in a mammal, the method
comprising, administering to the mammal an effective amount of an
ALK1 ECD protein.
14. The method of claim 13, wherein the ALK-1 ECD protein is an
ALK1-Fc fusion protein.
15. The method of claim 14, wherein the ALK1-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
TGF.beta.-1 with a K.sub.D of greater than 1.times.10.sup.-6.
16. The method of claim 13, wherein the ALK1 ECD protein binds to
one or more ALK1 ligands selected from the group consisting of:
GDF5, GDF6, GDF7, BMP9 and BMP10.
17. The method of claim 13, wherein the ALK1 ECD polypeptide
comprises an amino acid sequence that is at least 90% identical to
the sequence of amino acids corresponding to amino acids 34-95 of
SEQ ID NO:1.
18. The method of claim 13, wherein the ALK1 ECD comprises an amino
acid sequence encoded by a nucleic acid that hybridized under
stringent hybridization conditions to nucleotides 100-285 of SEQ ID
NO:2 or a variant of nucleotides 100-285 of SEQ ID NO:2 that has
the same coding sequence.
19. The method of claim 14, wherein the ALK1-Fc fusion protein has
a sequence of SEQ ID NO:3.
20. The method of claim 13, wherein the ALK1 ECD fusion protein is
delivered intravenously or locally to the eye.
21. The method of claim 13, wherein the method further comprises
administering a second agent that inhibits angiogenesis.
22. The method of claim 13, wherein the angiogenesis to be
inhibited is angiogenesis occurring: in the eye of the mammal, in a
tumor or in a bone or joint.
23. A method of inhibiting angiogenesis in a mammal, the method
comprising, administering to the mammal an effective amount of 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.
24. The method of claim 23, wherein the antibody binds to the ALK1
polypeptide with a K.sub.D of less than 5.times.10.sup.-8 M.
25. The method of claim 23, wherein the antibody binds to the ALK1
polypeptide with a K.sub.D of less than 1.times.10.sup.-10 M.
26. The method of claim 23, wherein the antibody inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: GDF5, GDF6 and GDF7.
27. The method of claim 23, wherein the antibody inhibits binding
of ALK1 to an ALK1 ligand, wherein the ALK1 ligand is selected from
the group consisting of: BMP9 and BMP10.
28. The method of claim 23, wherein the antibody is delivered
intravenously.
29. The method of claim 23, wherein the method further comprises
administering a second agent that inhibits angiogenesis.
30. The method of claim 23, wherein the angiogenesis to be
inhibited is angiogenesis occurring: in the eye of the mammal, in a
tumor or in a bone or joint.
31. 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 and GDF7.
32. The antibody of claim 31, wherein the antibody binds to the
ALK1 ligand with a K.sub.D of less than 5.times.10.sup.-8 M.
33. The antibody of claim 31, wherein the antibody inhibits
angiogenesis stimulated by the ALK1 ligand.
34. A pharmaceutical preparation comprising the antibody of claim
31.
35. 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 BMP9 and BMP10.
36. The antibody of claim 35, wherein the antibody binds to the
ALK1 ligand with a K.sub.D of less than 1.times.10.sup.-10 M.
37. A pharmaceutical preparation comprising the antibody of claim
35.
38. A method of inhibiting angiogenesis in a mammal, the method
comprising, administering to the mammal an effective amount of 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.
39. The method of claim 38, wherein the antibody inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: GDF5, GDF6 and GDF7.
40. The method of claim 38, wherein the antibody inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: BMP9 and BMP10.
41. The method claim 38, wherein the antibody is delivered
intravenously.
42. The method of claim 38, wherein the method further comprises
administering a second agent that inhibits angiogenesis.
43. The method of claim 38, wherein the angiogenesis to be
inhibited is angiogenesis occurring: in the eye of the mammal, in a
tumor or in a bone or joint.
44. Use of an ALK1 ECD polypeptide or nucleic acids encoding the
same in the manufacture of a medicament for the inhibition of
angiogenesis in a mammal.
45. The use of claim 44, wherein the ALK1 ECD polypeptide is an
ALK-1 Fc fusion protein.
46. The use of claim 45, wherein the ALK-1 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
TGF.beta.-1 with a K.sub.D of greater than 1.times.10.sup.-6.
47. Use of an antibody that binds to ALK1 or nucleic acids encoding
the same in the manufacture of a medicament for the inhibition of
angiogenesis in a mammal, wherein the antibody 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.
48. Use of an antibody that binds to an ALK1 ligand and inhibits
the binding of the ALK1 ligand to ALK1, or nucleic acids encoding
the same, in the manufacture of a medicament for the inhibition of
angiogenesis in a mammal, wherein the antibody binds to an ALK1
ligand selected from the group consisting of GDF5, GDF6, GDF7, BMP9
and BMP10.
49. A method of inhibiting angiogenesis in a mammal, the method
comprising, administering to the mammal an effective amount of a
DAN protein.
50. The method of claim 49, wherein the DAN protein is a DAN-Fc
fusion protein.
51. The method of claim 49, wherein the DAN-Fc fusion protein
comprises a polypeptide having an amino acid sequence that is at
least 90% identical to the sequence of amino acids 21-125 of SEQ ID
NO:10, which polypeptide is fused to an Fc portion of an
immunoglobulin, and wherein the ALK1-Fc fusion protein binds to
TGF.beta.-1 with a K.sub.D of greater than 1.times.10.sup.-6.
52. The method of claim 49, wherein the DAN protein binds to one or
more ALK1 ligands selected from the group consisting of: GDF5, GDF6
and GDF7.
53. The method of claim 49, wherein the DAN protein comprises an
amino acid sequence that is at least 90% identical to the sequence
of amino acids corresponding to amino acids 17-180 of SEQ ID
NO:10.
54. The method of claim 49, wherein the DAN protein comprises an
amino acid sequence encoded by a nucleic acid that hybridizes under
stringent hybridization conditions to nucleotides 153-467 of SEQ ID
NO:11 or a variant of nucleotides 153-467 of SEQ ID NO:11 that has
the same coding sequence.
55. The method of claim 49, wherein the method further comprises
administering a second agent that inhibits angiogenesis.
56. The method of claim 49, wherein the angiogenesis to be
inhibited is angiogenesis occurring: in the eye of the mammal, in a
tumor or in a bone or joint.
57. A method 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: (a) an ALK1 ECD protein; (b) 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; (c) 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 (d)
a DAN polypeptide.
58. The method of claim 57, wherein the ALK-1 ECD protein is an
ALK1-Fc fusion protein.
59. The method of claim 57, wherein the ALK1-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
TGF.beta.-1 with a K.sub.D of greater than 1.times.10.sup.-6.
60. The method of claim 57, wherein the ALK1 ECD protein binds to
one or more ALK1 ligands selected from the group consisting of:
GDF5, GDF6, GDF7, BMP9 and BMP10.
61. The method of claim 58, wherein the ALK1-Fc fusion protein has
a sequence of SEQ ID NO:3.
62. The method of claim 57, wherein the ALK1 ECD polypeptide
comprises an amino acid sequence that is at least 90% identical to
the sequence of amino acids corresponding to amino acids 34-95 of
SEQ ID NO:1.
63. The method of claim 57, wherein the ALK1 ECD comprises an amino
acid sequence encoded by a nucleic acid that hybridized under
stringent hybridization conditions to nucleotides 100-285 of SEQ ID
NO:2 or a variant of nucleotides 100-285 of SEQ ID NO:2 that has
the same coding sequence.
64. The method of claim 57, wherein the antibody of (b) binds to
the ALK1 polypeptide with a K.sub.D of less than 5.times.10.sup.-8
M.
65. The method of claim 57, wherein the antibody of (b) binds to
the ALK1 polypeptide with a K.sub.D of less than 1.times.10.sup.-10
M.
66. The method of claim 57, wherein the antibody of (b) inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: GDF5, GDF6 and GDF7.
67. The method of claim 57, wherein the antibody of (b) inhibits
binding of ALK1 to an ALK1 ligand, wherein the ALK1 ligand is
selected from the group consisting of: BMP9 and BMP10.
68. The method of claim 57, wherein the antibody of (c) inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: GDF5, GDF6 and GDF7.
69. The method of claim 57, wherein the antibody of (c) inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: BMP9 and BMP10.
70. The method of claim 57, wherein the DAN protein is a DAN-Fc
fusion protein.
71. The method of claim 70, wherein the DAN-Fc fusion protein
comprises a polypeptide having an amino acid sequence that is at
least 90% identical to the sequence of amino acids 21-125 of SEQ ID
NO:10, which polypeptide is fused to an Fc portion of an
immunoglobulin, and wherein the ALK1-Fc fusion protein binds to
TGF.beta.-1 with a K.sub.D of greater than 1.times.10.sup.-6.
72. The method of claim 57, wherein the DAN protein binds to one or
more ALK1 ligands selected from the group consisting of: GDF5, GDF6
and GDF7.
73. The method of claim 57, wherein the DAN protein comprises an
amino acid sequence that is at least 90% identical to the sequence
of amino acids corresponding to amino acids 17-180 of SEQ ID
NO:10.
74. The method of claim 57, wherein the DAN protein comprises an
amino acid sequence encoded by a nucleic acid that hybridizes under
stringent hybridization conditions to nucleotides 153-467 of SEQ ID
NO:11 or a variant of nucleotides 153-467 of SEQ ID NO:11 that has
the same coding sequence.
75. The method of claim 57, wherein the agent is delivered
intravenously.
76. The method of claim 57, wherein the method further comprises
administering a second agent that inhibits angiogenesis.
77. A method for treating a tumor in a mammal, the method
comprising, administering to a mammal that has a tumor an effective
amount of an agent selected from the group consisting of: (a) an
ALK1 ECD protein; (b) 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; (c) 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 (d) a DAN
polypeptide.
78. The method of claim 77, wherein the ALK-1 ECD protein is an
ALK1-Fc fusion protein.
79. The method of claim 78, wherein the ALK1-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
TGF.beta.-1 with a K.sub.D of greater than 1.times.10.sup.-6.
80. The method of claim 77, wherein the ALK1 ECD protein binds to
one or more ALK1 ligands selected from the group consisting of:
GDF5, GDF6, GDF7, BMP9 and BMP10.
81. The method of claim 78, wherein the ALK1-Fc fusion protein has
a sequence of SEQ ID NO:3.
82. The method of claim 77, wherein the ALK1 ECD polypeptide
comprises an amino acid sequence that is at least 90% identical to
the sequence of amino acids corresponding to amino acids 34-95 of
SEQ ID NO:1.
83. The method of claim 77, wherein the ALK1 ECD comprises an amino
acid sequence encoded by a nucleic acid that hybridized under
stringent hybridization conditions to nucleotides 100-285 of SEQ ID
NO:2 or a variant of nucleotides 100-285 of SEQ ID NO:2 that has
the same coding sequence.
84. The method of claim 77, wherein the antibody of (b) binds to
the ALK1 polypeptide with a K.sub.D of less than 5.times.10.sup.-8
M.
85. The method of claim 77, wherein the antibody of (b) binds to
the ALK1 polypeptide with a K.sub.D of less than 1.times.10.sup.-10
M.
86. The method of claim 77, wherein the antibody of (b) inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: GDF5, GDF6 and GDF7.
87. The method of claim 77, wherein the antibody of (b) inhibits
binding of ALK1 to an ALK1 ligand, wherein the ALK1 ligand is
selected from the group consisting of: BMP9 and BMP10.
88. The method of claim 77, wherein the antibody of (c) inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: GDF5, GDF6 and GDF7.
89. The method of claim 77, wherein the antibody of (c) inhibits
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: BMP9 and BMP10.
90. The method of claim 77, wherein the DAN protein is a DAN-Fc
fusion protein.
91. The method of claim 90, wherein the DAN-Fc fusion protein
comprises a polypeptide having an amino acid sequence that is at
least 90% identical to the sequence of amino acids 21-125 of SEQ ID
NO:10, which polypeptide is fused to an Fc portion of an
immunoglobulin, and wherein the ALK1-Fc fusion protein binds to
TGF.beta.-1 with a K.sub.D of greater than 1.times.10.sup.-6.
92. The method of claim 77, wherein the DAN protein binds to one or
more ALK1 ligands selected from the group consisting of: GDF5, GDF6
and GDF7.
93. The method of claim 77, wherein the DAN protein comprises an
amino acid sequence that is at least 90% identical to the sequence
of amino acids corresponding to amino acids 17-180 of SEQ ID
NO:10.
94. The method of claim 77, wherein the DAN protein comprises an
amino acid sequence encoded by a nucleic acid that hybridizes under
stringent hybridization conditions to nucleotides 153-467 of SEQ ID
NO:11 or a variant of nucleotides 153-467 of SEQ ID NO:11 that has
the same coding sequence.
95. The method of claim 77, wherein the agent is delivered
intravenously.
96. The method of claim 77, wherein the method further comprises
administering a second agent that inhibits angiogenesis.
97. The method of claim 77, wherein the method further comprises
administering a second agent that inhibits angiogenesis.
98. The method of claim 77, wherein the tumor is associated with
bone.
99. The method of claim 77, wherein the tumor is a myeloma or a
tumor that has metastasized to the bone.
100. The method of claim 77, wherein the tumor is resistant to
anti-VEGF therapy.
101. An ophthalmic pharmaceutical formulation comprising an agent
selected from the group consisting of: (a) an ALK1 ECD protein; (b)
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; (c)
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 (d) a DAN polypeptide.
102. A method of treating an angiogenesis related disease of the
eye comprising administering systemically or to said eye a
pharmaceutical formulation comprising: an effective amount of an
agent selected from the group consisting of: (a) an ALK1 ECD
protein; (b) 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; (c) 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 (d) a DAN polypeptide.
Description
RELATED APPLICATION
[0001] This application is a Continuation of U.S. application Ser.
No. 11/982,738, filed on Nov. 2, 2007, which claims the benefit of
the filing date under 35 U.S.C. .sctn.119(e) of U.S. provisional
application 60/856,592, filed Nov. 2, 2006, the entire contents of
each of which are incorporated herein by reference.
BACKGROUND
[0002] 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.
[0003] 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.
[0004] 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 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.
[0005] Agents that inhibit angiogenesis have proven to be effective
in treating a variety of disorders. Avastin.TM. (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. Macugen.TM., 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-1H-2-benzopyran-3-yl) propionic acid
(NM-3) has completed phase I 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 December; 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 January; 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.
[0006] 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.
SUMMARY
[0007] In part, the present disclosure presents a characterization
of an activin-like kinase I (ALK1)-mediated regulatory system and
the role of this system in angiogenesis in vivo. 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, ALK1 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. This disclosure demonstrates that
signaling mediated by ALK1 and the ligands described above is
involved in angiogenesis in vivo, and that inhibition of this
regulatory system has a potent anti-angiogenic effect. Thus, in
certain aspects, the disclosure provides antagonists of the ALK1
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 ALK1 ligands for
the treatment of cancers, particularly multiple myeloma, rheumatoid
arthritis, and disorders associated with pathological angiogenesis
in the eye.
[0008] In certain aspects, the disclosure provides polypeptides
comprising a ligand binding portion of the extracellular domain of
ALK1 ("ALK1 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 ALK1 and inhibiting the ability of these ligands to
interact with ALK1 as well as other receptors. In certain
embodiments, an ALK1 ECD polypeptide comprises an amino acid
sequence that is at least 70%, 80%, 90%, 95%, 97%, 99% or 100%
identical to the sequence of amino acids 22-118 of the human ALK1
sequence of SEQ ID NO:1. An ALK1 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 ALK1 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 ALK1 ECD
polypeptide in systemic administration (e.g., intravenous,
intraarterial and intra-peritoneal administration). As demonstrated
herein, a systemically administered ALK1-Fc polypeptide has a
potent anti-angiogenic effect in the eye and also provides positive
effects in murine models of rheumatoid arthritis and multiple
myeloma. In certain embodiments, an ALK1-Fc fusion protein
comprises a polypeptide having an amino acid sequence that is at
least 70%, 80%, 90%, 95%, 97%, 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 and BMP9 with a K.sub.D of less than
1.times.10.sup.-7 M and binds to TGF.beta.-1 with a K.sub.D of
greater than 1.times.10.sup.-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 IgG1. In a preferred embodiment, the ALK1-Fc
fusion protein comprises amino acids 22-118 of SEQ ID NO:1.
Optionally, the ALK1-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. ALK1-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).
[0009] In certain aspects, the disclosure provides methods for
inhibiting angiogenesis in a mammal by administering any of the
ALK1 ECD polypeptides described generally or specifically herein.
In one embodiment, a method comprises administering to the mammal
an effective amount of an ALK1-Fc fusion protein, wherein the ALK1
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 TGF.beta.-1 with a K.sub.D of greater than
1.times.10.sup.-6. Optionally, the ALK1-Fc fusion protein binds to
one or more ALK1 ligands selected from the group consisting of:
GDF5, GDF6, GDF7, BMP9 and BMP10. Optionally, the ALK1-Fc fusion
protein has a sequence of SEQ ID NO:3. The ALK1 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 ALK1-Fc
protein to the mammal at a location distal to the eye, e.g. by
systemic administration.
[0010] In certain aspects, the disclosure provides antibodies that
bind to ALK1, particularly an epitope situated in the extracellular
domain, amino acids 22-118 of SEQ ID NO:1, and inhibit the binding
of ALK1 to at least one ALK1 ligand selected from the group
consisting of: GDF5, GDF6, GDF7, BMP9 and BMP10. Based on the
affinity of these ligands for ALK1, an antibody may bind with a
K.sub.D of less than 5.times.10.sup.-8 M, and optionally between
5.times.10.sup.-8 and 1.times.10.sup.-10. An antibody with affinity
within this range would be expected to inhibit signaling by one or
more of GDF5, 6 and 7 while having less effect on signaling by BMP9
and 10. Such an antibody preferably inhibits angiogenesis
stimulated by at least one ALK1 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 ALK1 activity directly, which should be
contrasted to the activity of an ALK1-Fc fusion protein, which is
expected to inhibit the activity of ALK1 ligands. An anti-ALK1
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 BMPR1a, BMPR1b and BMPRII complexes. However,
an anti-ALK1 antibody is expected to interfere with the ability of
low affinity ligands for ALK1 (e.g., TGF-.beta., which is generally
recognized as triggering significant signaling events through ALK-1
even though binding is relatively weak) to signal through ALK1,
even though an ALK1 ECD may not bind to or inhibit such low
affinity ligands. An antibody may bind to the ALK1 polypeptide with
a K.sub.D of less than 1.times.10.sup.-10 M. An antibody with
affinity within this range would be expected to inhibit signaling
by BMP9 or 10. Such an antibody preferably inhibits binding of BMP9
and BMP10 to ALK1. Notably, based on the data disclosed herein, an
antibody that binds relatively poorly to ALK1 may inhibit TGF.beta.
binding to ALK1 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
ALK1-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).
[0011] 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 ALK1
polypeptide, described herein either generally or specifically. An
antibody useful for this purpose may bind to the extracellular
domain of ALK1 (e.g., bind to a polypeptide consisting of amino
acids 22-118 of SEQ ID NO:1) or another portion of ALK1. 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 ALK1 ligand
selected from the group consisting of: GDF5, GDF6, GDF7, BMP9 and
BMP10. The antibody may bind to the ALK1 polypeptide with a K.sub.D
of less than 5.times.10.sup.-8 M, and optionally between
5.times.10.sup.-8 and 1.times.10.sup.-10. The antibody may inhibit
angiogenesis stimulated by at least one ALK1 ligand selected from
the group consisting of: GDF5, GDF6 and GDF7. An antibody that
selectively inhibits signaling mediated by GDF5, 6 or 7 relative to
signaling by BMP9 or 10 may be used as a selective inhibitor of
angiogenesis that occurs in tissues where GDF5, 6 or 7 are
localized: primarily bone or joints. The antibody may bind to the
ALK1 polypeptide with a K.sub.D of less than 1.times.10.sup.-10 M.
The antibody may inhibit the binding of ALK1 to an ALK1 ligand,
wherein the ALK1 ligand is selected from the group consisting of:
BMP9 and BMP10. The anti-ALK1 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-ALK1 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.
[0012] In certain aspects, the disclosure provides antibodies that
bind to an ALK1 ligand disclosed herein and inhibit the binding of
the ALK1 ligand to ALK1. While not wishing to be bound to any
particular mechanism, it is expected that antibodies that bind to
ALK1 ligands will have effects that are similar in nature to ALK1
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 ALK1 ligand with
a K.sub.D of less than 5.times.10.sup.-8 M. The antibody may be
selected for inhibition of angiogenesis stimulated by the ALK1
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).
[0013] In certain aspects, the disclosure provides antibodies that
bind to an ALK1 ligand and inhibit the binding of the ALK1 ligand
to ALK1, wherein the ALK1 ligand is selected from the group
consisting of BMP9 and BMP10. The antibody may bind to the ALK1
ligand with a K.sub.D of less than 1.times.10.sup.-10 M. 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).
[0014] 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 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. The antibody may
inhibit angiogenesis stimulated by at least one ALK1 ligand
selected from the group consisting of: GDF5, GDF6 and GDF7.
[0015] 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 ALK1 ligand and inhibits the ligand to
ALK1 is an antibody that binds at or near the type I receptor
binding site of the ligand.
[0016] In certain aspects, the disclosure provides methods for
inhibiting angiogenesis in a mammal by administering other
inhibitors of the ALK1 signaling system disclosed herein. Such
inhibitors may include nucleic acids (e.g., antisense or RNAi
constructs) that decrease the production of ALK1, 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 ALK1 regulatory system disclosed herein, either by
disrupting the ALK1-ligand interaction or by inhibiting the
signaling that occurs after binding.
[0017] In a further embodiment, the disclosure describes the role
of DAN as a regulator of the ALK1 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 BMP10. DAN may therefore be used as a selective
agent for inhibiting angiogenesis in the bone or joints, where the
GDF5 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 ALK1
ligands selected from the group consisting of: GDF5, GDF6 and GDF7,
while having relatively poor binding to BMP9 or BMP10. A DAN
protein may comprise an amino acid sequence that is at least 70%,
80%, 90%, 95%, 97%, 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:11 or a variant of nucleotides 153-467 of SEQ
ID NO:11 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:11 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.
[0018] 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.
[0019] 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 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. A method may
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 also be one that utilizes multiple
pro-angiogenic factors, such as a tumor that is resistant to
anti-VEGF therapy.
[0020] In certain aspects the disclosure provides ophthalmic
formulations. Such formulations may comprise 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.
[0021] 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 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.
[0022] 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.
[0023] The disclosure also provides an ophthalmic pharmaceutical
formulation comprising an ALK1-Fc fusion protein 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 and BMP9 with a K.sub.D of less than
1.times.10.sup.-7 M and binds to TGF.beta.-1 with a K.sub.D of
greater than 1.times.10.sup.-6. In one embodiment, the fusion
protein has the sequence of SEQ ID NO: 3. In one embodiment, the Fc
portion is from human IgG1. 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.
[0024] 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 ALK1 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 K.sub.D of less than 5.times.10.sup.-8 M. In
another embodiment, the antibody binds to the ALK1 polypeptide with
a K.sub.D of less than 1.times.10.sup.-10 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.
[0025] In certain aspects, the disclosure provides for an
ophthalmic pharmaceutical formulation comprising an antibody that
binds to an ALK1 ligand disclosed herein and inhibits the binding
of the ALK1 ligand to ALK1. 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 K.sub.D
of less than 5.times.10.sup.-8 M. The antibody may be selected for
inhibition of angiogenesis stimulated by the ALK1 ligand. A CAM
assay is an appropriate assay system for selection of desirable
antibodies. Such antibodies are preferably recombinant 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.
[0026] 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
ALK1-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 and BMP9 with a K.sub.D of less than
1.times.10.sup.-7 M and binds to TGF.beta.-1 with a K.sub.D of
greater than 1.times.10.sup.-6. In one embodiment, the fusion
protein has the sequence of SEQ ID NO: 3. In one embodiment, the Fc
portion is from human IgG1. 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.
[0027] 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 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. 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 K.sub.D of less
than 5.times.10.sup.-8 M. In another embodiment, the antibody binds
to the ALK1 polypeptide with a K.sub.D of less than
1.times.10.sup.-10 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.
[0028] In one embodiment of the disclosed methods, the angiogenesis
related disease of the eye 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
[0029] FIG. 1 shows the amino acid sequence for the human Activin
Like Kinase 1, ALK1 (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.
[0030] FIG. 2 shows the nucleic acid sequence of a human ALK1 cDNA
(SEQ ID NO:2). The coding sequence is underlined. The portion
encoding the extracellular domain is double underlined.
[0031] FIG. 3 shows an example of a fusion of the extracellular
domain of human ALK1 to an Fc domain (SEQ ID NO:3). The hALK1-Fc
protein includes amino acids 22-120 of the human ALK1 protein,
fused at the C-terminus to a linker (underlined) and an IgG1 Fc
region.
[0032] FIG. 4 shows the nucleic acid sequence for expression of the
hALK1-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.
[0033] FIG. 5 shows the anti-angiogenic effect of murine ALK1-Fc
("RAP") and human ALK1-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.
[0034] FIG. 6 shows the angiogenic effect of GDF7 in a chick
chorioallantoic membrane (CAM) assay. The GDF7 effect is comparable
to that of VEGF.
[0035] FIG. 7 shows the anti-angiogenic effect of the human ALK1-Fc
fusion in the CAM assay. hALK1-Fc inhibits angiogenesis stimulated
by VEGF, FGF and GDF7.
[0036] FIG. 8 shows comparative anti-angiogenic effects of murine
ALK1-Fc (mALK1-Fc), hALK1-Fc, a commercially available anti-ALK1
monoclonal antibody (Anti-ALK1 mAb) and a commercially available,
neutralizing anti-VEGF monoclonal antibody. The anti-angiogenic
effect of the ALK1-Fc constructs is comparable to the effects of
the anti-VEGF antibody.
[0037] FIG. 9 shows the anti-angiogenic effects of hALK1-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.
[0038] FIG. 10 shows the effects of mALK1-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/1 arthritic
mice. RAP-041 is mALK1-Fc. Avastin.TM. is the anti-VEGF antibody
bevacizumab.
DETAILED DESCRIPTION
1. Overview
[0039] ALK1 is a type I cell-surface receptor for the TGF-0
superfamily of ligands and is also known as ACVRL1 and ACVRLK1.
ALK1 has been implicated as a receptor for TGF-.beta.1, TGF-.beta.3
and BMP-9 (Marchuk et al., Hum Mol. Genet. 2003; Brown et al., J
Biol. Chem. 2005 Jul. 1; 280(26):25111-8).
[0040] 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, 2626-2631; Urness et al., Nat.
Genet. 2000, 26, 328-331).
[0041] In humans, loss-of-function mutations in ALK1 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.
[0042] 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 ALK1
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.
[0043] The disclosure relates to the discovery that polypeptides
comprising a portion of the extracellular domain of ALK1 ("ALK1 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 ALK1 ECD polypeptide can exert an anti-angiogenic effect
even in the case where the ALK1 ECD polypeptide does not exhibit
meaningful binding to TGF-.beta.1. Moreover, ALK1 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 ALK1 regulatory system, in
which ALK1 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. Further, the disclosure demonstrates that signaling
mediated by ALK1 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 ALK1 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 ALK1 itself
are expected to have different effects from an ALK1 ECD
polypeptide. A pan-neutralizing antibody against ALK1 (one that
inhibits the binding of all strong and weak ligands) would be
expected to inhibit the signaling of such ligands through ALK1 but
would not be expected to inhibit the ability of such ligands to
signal through other receptors (e.g., BMPR1a, BMPR1b, BMPRII in the
case of GDF5-7 and BMP9-10 and TBRI and TBRII in the case of
TGF.beta.). On the other hand, an ALK1 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-.beta.. So, while a pan-neutralizing antibody
against ALK1 would block BMP9 and TGF-.beta. signaling through ALK1
it would not block BMP9 and TGF-.beta. signaling through another
receptor, and while an ALK1 ECD polypeptide may inhibit BMP9
signaling through all receptors (even receptors other than ALK1) it
would not be expected to inhibit TGF-.beta. signaling through any
receptor, even ALK1.
[0044] 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.sub.--878248; human BMP9, Q9UK05; human BMP10, O95393; human
DAN, BAA92265. ALK1 sequences are set forth in FIGS. 1-5.
TABLE-US-00001 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
[0045] 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).
TABLE-US-00002 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 cagctacagc
gtccccaaca ccttcccaca gtccacagag tccctggttc actgtgactc ctgcatgcca
gcccagtcca tgtgggagat tgtgacgctg gagtgcccgg gccacgagga ggtgcccagg
gtggacaagc tggtggagaa gatcctgcac tgtagctgcc aggcctgcgg caaggagcct
agtcacgagg ggctgagcgt ctatgtgcag ggcgaggacg ggccgggatc ccagcccggc
acccaccctc acccccatcc ccacccccat cctggcgggc agacccctga gcccgaggac
ccccctgggg ccccccacac agaggaagag ggggctgagg actgaggccc ccccaactct
tcctcccctc tcatccccct gtggaatgtt gggtctcact ctctggggaa gtcaggggag
aagctgaagc ccccctttgg cactggatgg acttggcttc agactcggac ttgaatgctg
cccggttgcc atggagatct gaaggggcgg ggttagagcc aagctgcaca atttaatata
ttcaagagtg gggggaggaa gcagaggtct tcagggctct ttttttgggg ggggggtggt
ctcttcctgt ctggcttcta gagatgtgcc tgtgggaggg ggaggaagtt ggctgagcca
ttgagtgctg ggggaggcca tccaagatgg catgaatcgg gctaaggtcc ctgggggtgc
agatggtact gctgaggtcc cgggcttagt gtgagcatct tgccagcctc aggcttgagg
gagggctggg ctagaaagac cactggcaga aacaggaggc tccggcccca caggtttccc
caaggcctct caccccactt cccatctcca gggaagcgtc gccccagtgg 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
ggggacctgg ggggagggac agggcaggcg ggcccatgaa gaaagcccct cgttgcccag
cactgtctgc gtctgctctt ctgtgcccag ggtggctgcc agcccactgc ctcctgcctg
gggtggcctg gccctcctgg ctgttgcgac gcgggcttct ggagcttgtc accattggac
agtctccctg atggaccctc agtcttctca tgaataaatt ccttcaacgc caaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa
[0046] 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.
[0047] 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
[0048] Naturally occurring ALK1 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 ALK1.
[0049] 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 ALK1 polypeptide, and optionally at
least 60%, at least 70%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 97%, 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-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, 5.times.SSC, 2% w/v blocking agent, 0.1%
N-lauroylsarcosine, 0.3% SDS at 65 C..degree. overnight and washing
in, for example, SxSSC at about 65 C..degree.). 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, 5.times.SSC, 2% w/v blocking agent, 0.1%
N-lauroylsarcosine, 0.3% SDS at 65 C..degree. overnight and washing
in, for example, 5.times.SSC at about 65 C..degree.). The term
"ALK1 ECD polypeptide" accordingly encompasses isolated
extracellular portions of ALK1 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:1 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 ALK1 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.
[0050] As described above, the disclosure provides ALK1 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 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.
[0051] 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).
[0052] 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 the desired percent identity, a suitable alternative
disclosed herein should be selected.
[0053] 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.
[0054] 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=1, Joining Penalty=20,
Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5 and Gap
Size Penalty=0.05.
[0055] In certain embodiments, ALK1 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%, 97% 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 ALK1 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.
[0056] In preferred embodiments, an ALK1 ECD polypeptide binds to
one or more of GDF5, GDF6, GDF7, BMP9 and BMP10. Optionally the
ALK1 polypeptide does not show substantial binding to TGF-131 or
TGF-133. Binding may be assessed using purified proteins in
solution or in a surface plasmon resonance system, such as a
Biacore.TM. 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 al. 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.
[0057] 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.
[0058] ALK1 ECD polypeptides may additionally include any number of
well-known 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
N-terminal start sites for mature ALK1 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.
[0059] In certain embodiments, the present disclosure contemplates
specific mutations of the ALK1 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 ALK1
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 ALK1
polypeptide is by chemical or enzymatic coupling of glycosides to
the ALK1 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 ALK1 polypeptide
may be accomplished chemically and/or enzymatically. Chemical
deglycosylation may involve, for example, exposure of the ALK1
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 cleavage of carbohydrate
moieties on ALK1 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 ALK1
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,
ALK1 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.
[0060] This disclosure further contemplates a method of generating
mutants, particularly sets of combinatorial mutants of an ALK1
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, ALK1 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 ALK1 polypeptide variant may be screened
for ability to bind to an ALK1 ligand, to prevent binding of an
ALK1 ligand to an ALK1 polypeptide or to interfere with signaling
caused by an ALK1 ligand. The activity of an ALK1 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.
[0061] Combinatorially-derived variants can be generated which have
a selective or generally increased potency relative to an ALK1 ECD
polypeptide comprising an extracellular domain of a naturally
occurring ALK1 polypeptide. Likewise, mutagenesis can give rise to
variants which have serum half-lives dramatically different than
the corresponding a wild-type ALK1 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 ALK1 ECD polypeptide. Such variants, and the genes which
encode them, can be utilized to alter ALK1 ECD polypeptide levels
by modulating the half-life of the ALK1 polypeptides. For instance,
a short half-life can give rise to more transient biological
effects and can allow tighter control of recombinant ALK1 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.
[0062] 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 ALK1 polypeptide
sequences. For instance, a mixture of synthetic oligonucleotides
can be enzymatically ligated into gene sequences such that the
degenerate set of potential ALK1 polypeptide nucleotide sequences
are expressible as individual polypeptides, or alternatively, as a
set of larger fusion proteins (e.g., for phage display).
[0063] There are many ways by which the library of potential ALK1
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, S A (1983) Tetrahedron 39:3; Itakura
et al., (1981) Recombinant DNA, Proc. 3rd Cleveland Sympos.
Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp 273-289;
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. Pat. Nos. 5,223,409,
5,198,346, and 5,096,815).
[0064] Alternatively, other forms of mutagenesis can be utilized to
generate a combinatorial library. For example, ALK1 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;
Grodberg et al., (1993) Eur. J. Biochem. 218:597-601; Nagashima et
al., (1993) J. Biol. Chem. 268:2888-2892; Lowman et al., (1991)
Biochemistry 30:10832-10838; and Cunningham et al., (1989) Science
244:1081-1085), by linker scanning mutagenesis (Gustin et al.,
(1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol.
12:2644-2652; McKnight et al., (1982) Science 232:316); by
saturation mutagenesis (Meyers et al., (1986) Science 232:613); by
PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol
1:11-19); or by random mutagenesis, including chemical mutagenesis,
etc. (Miller et al., (1992) A Short Course in Bacterial Genetics,
CSHL Press, Cold Spring Harbor, N.Y.; 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 ALK1
polypeptides.
[0065] 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 ALK1
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 ALK1
ligand binding assays and ligand-mediated cell signaling
assays.
[0066] In certain embodiments, the ALK1 ECD polypeptides of the
disclosure may further comprise post-translational modifications in
addition to any that are naturally present in the ALK1
polypeptides. Such modifications include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. As a result, the modified ALK1 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 ALK1 ECD polypeptide may be tested as described
herein for other ALK1 ECD polypeptide variants. When an ALK1 ECD
polypeptide is produced in cells by cleaving a nascent form of the
ALK1 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, WI38, 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 ALK1
polypeptides.
[0067] In certain aspects, functional variants or modified forms of
the ALK1 ECD polypeptides include fusion proteins having at least a
portion of the ALK1 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 QIAexpress.TM.
system (Qiagen) useful with (HIS.sub.6) fusion partners. As another
example, a fusion domain may be selected so as to facilitate
detection of the ALK1 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
ALK1 ECD polypeptide is fused with a domain that stabilizes the
ALK1 polypeptide in vivo (a "stabilizer" domain). By "stabilizing"
is meant 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.
[0068] As a specific example, the present disclosure provides a
fusion protein comprising a soluble extracellular domain of ALK1
fused to an Fc domain (e.g., SEQ ID NO: 6).
TABLE-US-00003 THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT
CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*
[0069] Optionally, the Fc 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 Fey
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.
[0070] 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 ALK1 ECD polypeptide may be
placed C-terminal to a heterologous domain, or, alternatively, a
heterologous domain may be placed C-terminal to an ALK1 ECD
polypeptide. The ALK1 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.
[0071] As used herein, the term, "immunoglobulin Fc region" or
simply "Fc" is 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 CH1
domain, a CH2 domain, and a CH3 domain, 2) a CH1 domain and a CH2
domain, 3) a CH1 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.
[0072] In one embodiment, the class of immunoglobulin from which
the heavy chain constant region is derived is IgG (Ig.gamma.)
(.gamma. subclasses 1, 2, 3, or 4). Other classes of
immunoglobulin, IgA (Ig.alpha.), IgD (Ig.delta.), IgE (Ig.epsilon.)
and IgM (Ig.mu.), 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 CH.sub.3
domain of Fc .gamma. or the homologous domains in any of IgA, IgD,
IgE, or IgM.
[0073] 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).
[0074] In certain embodiments, the present disclosure makes
available isolated and/or purified forms of the ALK1 ECD
polypeptides, which are isolated from, or otherwise substantially
free of (e.g., at least 80%, 90%, 95%, 97% 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.
[0075] In certain embodiments, the disclosure includes nucleic
acids encoding soluble ALK1 polypeptides comprising the coding
sequence for an extracellular portion of an ALK1 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 ALK1 ECD
polypeptides. It has been established that an ALK1-Fc fusion
protein set forth in SEQ ID NO:3 and expressed in CHO cells has
potent anti-angiogenic activity.
[0076] 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 ALK1 ECD
proteins.
3. Nucleic Acids Encoding ALK1 Polypeptides
[0077] In certain aspects, the disclosure provides isolated and/or
recombinant nucleic acids encoding any of the ALK1 polypeptides
(e.g., ALK1 ECD polypeptides), including fragments, functional
variants and fusion proteins disclosed herein. For example, SEQ ID
NO: 2 encodes the naturally occurring human ALK1 precursor
polypeptide, while SEQ ID NO: 4 encodes the precursor of an ALK1
extracellular domain fused to an IgG1 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 ALK1 polypeptides or as
direct therapeutic agents (e.g., in an antisense, RNAi or gene
therapy approach).
[0078] In certain aspects, the subject nucleic acids encoding ALK1
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.
[0079] In certain embodiments, the disclosure provides isolated or
recombinant nucleic acid sequences that are at least 80%, 85%, 90%,
95%, 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.
[0080] 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.times. sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by a
wash of 2.0.times.SSC at 50.degree. C. For example, the salt
concentration in the wash step can be selected from a low
stringency of about 2.0.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 50.degree. C. In addition, the
temperature in the wash step can be increased from low stringency
conditions at room temperature, about 22.degree. C., to high
stringency conditions at about 65.degree. 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.times.SSC at room temperature
followed by a wash at 2.times.SSC at room temperature.
[0081] 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 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.
[0082] 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.
[0083] 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 ALK1
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, San
Diego, Calif. (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., PhoS, the
promoters of the yeast .alpha.-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.
[0084] 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.
[0085] 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 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-Ban 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 pAcUW1), and pBlueBac-derived vectors (such as the
.beta.-gal containing pBlueBac III).
[0086] In a preferred embodiment, a vector will be designed for
production of the subject ALK1 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, Wis.). As will be apparent, the subject gene constructs
can be used to cause expression of the subject ALK1 polypeptides in
cells propagated in culture, e.g., to produce proteins, including
fusion proteins or variant proteins, for purification.
[0087] 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 ALK1 polypeptides. The
host cell may be any prokaryotic or eukaryotic cell. For example,
an ALK1 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.
[0088] Accordingly, the present disclosure further pertains to
methods of producing the subject ALK1 polypeptides, including ALK1
ECD polypeptides. For example, a host cell transfected with an
expression vector encoding an ALK1 polypeptide can be cultured
under appropriate conditions to allow expression of the ALK1
polypeptide to occur. The ALK1 polypeptide may be secreted and
isolated from a mixture of cells and medium containing the ALK1
polypeptide. Alternatively, the ALK1 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 ALK1 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 ALK1
polypeptides and affinity purification with an agent that binds to
a domain fused to the ALK1 polypeptide (e.g., a protein A column
may be used to purify an ALK1-Fc fusion). In a preferred
embodiment, the ALK1 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.
[0089] 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 ALK1 polypeptide, can allow purification of the
expressed fusion protein by affinity chromatography using a
Ni.sup.2+ metal resin. The purification leader sequence can then be
subsequently removed by treatment with enterokinase to provide the
purified ALK1 polypeptide (e.g., see Hochuli et al., (1987) J.
Chromatography 411:177; and Janknecht et al., PNAS USA
88:8972).
[0090] Techniques for making fusion genes are well known.
Essentially, the joining of various DNA fragments coding for
different polypeptide sequences is performed 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).
[0091] 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 ALK1 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 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.
[0092] 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
[0093] Another aspect of the disclosure pertains to an antibody
reactive with an extracellular portion of an ALK1 polypeptide,
preferably antibodies that are specifically reactive with ALK1
polypeptide. In a preferred embodiment, such 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 ALK1 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).
[0094] 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".
[0095] 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 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 ALK1 polypeptide or an ALK1
ligand, 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)). A mammal, such as a mouse, a hamster or rabbit can be
immunized with an immunogenic form of the peptide (e.g., a ALK1
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
ALK1 polypeptide 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.
[0096] Following immunization of an animal with an antigenic
preparation of an ALK1 polypeptide, anti-ALK1 antisera can be
obtained and, if desired, polyclonal anti-ALK1 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 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 ALK1 polypeptide of the present
disclosure and monoclonal antibodies isolated from a culture
comprising such hybridoma cells.
[0097] The term antibody as used herein is intended to include
fragments thereof which are also specifically reactive with one of
the subject ALK1 polypeptides. 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).sub.2 fragments can be generated by treating
antibody with pepsin. The resulting F(ab).sub.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 ALK1 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).
[0098] In certain preferred embodiments, an antibody of the
disclosure is a recombinant antibody, particularly a humanized
monoclonal antibody or a fully human recombinant antibody.
[0099] 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 ALK1 polypeptide or an
ALK1 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 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.
[0100] 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.sup.-6,
10.sup.-7, 10.sup.-8, 10.sup.-9 or less. Given the apparently low
binding affinity of TGF.beta. for ALK1, it is expected that many
anti-ALK1 antibodies will inhibit TGF.beta. binding. However, the
GDF5, 6, 7 group of ligands bind with a K.sub.D of approximately
5.times.10.sup.-8 M and the BMP9, 10 ligands bind with a K.sub.D of
approximately 1.times.10.sup.-10 M. Thus, antibodies of appropriate
affinity may be selected to interfere with the signaling activities
of these ligands.
[0101] 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 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,
Md.), western blots, immunoprecipitation assays and
immunohistochemistry.
5. Alterations in Antibodies and Fc-Fusion Proteins
[0102] 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
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.
[0103] 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 98/47531;
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 IgG1
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).
[0104] 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. 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), WO99/51642, Duncan & Winter
Nature 322: 738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No.
5,624,821; and WO94/29351.
6. Methods and Compositions for Modulating Angiogenesis and Certain
Disorders
[0105] The disclosure provides methods of inhibiting angiogenesis
in a mammal by administering to a subject an effective amount of a
an ALK1 ECD polypeptide, such as an ALK1-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 ALK1, 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.
[0106] 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.
[0107] 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,
ALK1 ECD polypeptides affect angiogenesis that is stimulated 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 multiple myeloma. Multiple myeloma is widely recognized
as a cancer that includes a significant angiogenic component.
Accordingly, it is expected that ALK1-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.
[0108] 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.
[0109] 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.
[0110] 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.
[0111] 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.
[0112] 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 ALK1 or ALK1 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.
[0113] 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 ALK1 or ALK1 ligand antagonist and/or a
derivative thereof, or another angiogenesis-inhibiting agent of the
present disclosure.
[0114] 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.
[0115] 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).
[0116] 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.
[0117] 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.
[0118] 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 graph 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.
[0119] 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 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.
[0120] 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.
[0121] 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
(Macugen.TM.), ranibizumab (Lucentis.TM.), squalamine lactate
(Evizon.TM.), 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 (Macugen.TM.), ranibizumab
(Lucentis.TM.), squalamine lactate (Evizon.TM.), heparinase, and
glucocorticoids (e.g. Triamcinolone).
7. Formulations and Effective Doses
[0122] 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 more physiologically acceptable
carriers or excipients. Such formulations will generally be
substantially pyrogen free, in compliance with most regulatory
requirements.
[0123] 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., ALK1 ECD polypeptides or any of the antibodies
disclosed herein) in the methods disclosed herein.
[0124] 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.
[0125] 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 ALK1 ECD
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.
[0126] 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.
[0127] 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 of ALK1-Fc Fusion Proteins
[0128] Applicants constructed a soluble ALK1 fusion protein that
has the extracellular domain of human ALK1 fused to a human Fc or
mouse ALK1 fused to a murine Fc domain with a minimal linker in
between. The constructs are referred to as hALK1-Fc and mALK1-Fc,
respectively.
[0129] hALK1-Fc is shown as purified from CHO cell lines in FIG. 3
(SEQ ID NO: 3). Notably, while the conventional C-terminus of the
extracellular domain of human ALK1 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 ALK1 incorporates two
residues c-terminal to Q118, a leucine and an alanine. The
disclosure therefore provides ALK1 ECD polypeptides (including Fc
fusion 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) of Q118.
[0130] The hALK1-Fc and mALK1-Fc proteins were expressed in CHO
cell lines. Three different leader sequences were considered:
TABLE-US-00004 (i) Honey bee mellitin (HBML): (SEQ ID NO: 7)
MKFLVNVALVFMVVYISYIYA (ii) Tissue Plasminogen Activator (TPA): (SEQ
ID NO: 8) MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 9)
MTLGSPRKGLLMLLMALVTQG.
[0131] The selected form employs the TPA leader and has the
unprocessed amino acid sequence shown in FIG. 4 (SEQ ID NO:5).
[0132] This polypeptide is encoded by the nucleic acid sequence
shown in FIG. 4 (SEQ ID NO:4).
[0133] 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.
Example 2
Identification of ALK1-Fc Ligands
[0134] ALK1 is a type 1 receptors for members of the TGF.beta.
family. A variety of members of the TGF.beta. family were tested
for binding to a human ALK1-Fc fusion protein, using a Biacore.TM.
system. TGF.beta. itself, GDF5, GDF11, BMP2 and BMP4 all failed to
show substantial binding to the hALK1-Fc protein. BMP2 and BMP4
showed limited binding. GDF5, GDF7 and BMP9 showed binding with
K.sub.D values of approximately 5.times.10.sup.-8 M,
5.times.10.sup.-8 M and 1.times.10.sup.-10 M, respectively. Based
on the similarity of GDF5 and GDF7 to GDF6, it is expected that
GDF6 will bind with similar affinity. BMP10 is closely related to
BMP9 and is also expected to bind with similar affinity.
Example 3
Characterization of ALK1-Fc and Anti-ALK1 Antibody Effects on
Endothelial Cells
[0135] 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 hALK1-Fc drug or
neutralizing ALK1 specific monoclonal antibody in HMVEC cells.
HMVEC cells were stimulated with rhBMP-9 (50 ng/ml), which induced
Smad1/5/8-mediated transcriptional activation, evidenced here by
the increase in SBE4-luc modulated transcriptional upregulation.
When added, the hALK1-Fc compound (10 .mu.g/ml) or antibody (10
.mu.g/ml) diminished this transcriptional response, each by nearly
60%, indicating that the presence of ALK1-Fc significantly reduces
BMP9 signaling, and moreover, that the BMP9 signaling is related to
ALK1 activity.
[0136] Activation of SMAD phosphorylation is commonly used to assay
activation of upstream activin receptors. ALK1 is known to modulate
phosphorylation of SMAD proteins 1, 5 and 8 upon activation by its
ligand. Here, we added rhBMP-9 (50 ng/ml) to initiate SMAD
phosphorylation in HUVEC cells, a human endothelial cell line which
innately expresses ALK1 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 hALK1-Fc (250 ng/ml), SMAD 1/5/8 phosphorylation
was reduced, confirming that this agent inhibits Smad1/5/8
activation in endothelial cells.
[0137] In order to evaluate the angiogenic effect of ALK1-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 (200 ng/ml) 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 hALK1-Fc (100 ng/ml) or
mALK1-Fc (100 ng/ml), cord formation was visibly reduced. Final
quantification of vessel length in all samples revealed that every
concentration of hALK1-fc or mALK1-Fc reduced neovascularization to
basal levels. Additionally, hALK1-Fc and mALK1-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 (100
ng/ml).
Example 4
CAM Assays
[0138] 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 FIG. 6, GDF7 stimulates
angiogenesis with a potency that is similar to that of VEGF.
Similar results were observed with GDF5 and GDF6.
[0139] 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 FIG. 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 ALK1.
[0140] ALK1-Fc proteins and a commercially available,
anti-angiogenic anti-VEGF monoclonal antibody were compared in the
CAM assay. The ALK1-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
FIG. 8.
[0141] 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 ALK1 sequence
in different species.
Example 5
Mouse Corneal Micropocket Assay
[0142] The mouse corneal micropocket assay was used to assess the
effects of ALK1-Fc on angiogenesis in the mouse eye. hALK1-Fc,
administered intraperitoneally, significantly inhibited ocular
angiogenesis. As shown in FIG. 9, hALK1-Fc inhibited ocular
angiogenesis to the same degree as anti-VEGF. hALK1-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.
[0143] These data demonstrate that high affinity ligands for ALK1
promote angiogenesis and that an ALK1-Fc fusion protein has potent
anti-angiogenic activity. The ligands for ALK1 fall into two
categories, with the GDF5, 6, 7 grouping having an intermediate
affinity for ALK1 and the BMP9, 10 grouping having a high affinity
for ALK1.
[0144] GDF5, 6 and 7 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, 6 and 7 and a systemic angiogenic system that includes ALK1
and BMP9 (and possibly BMP10).
Example 6
Murine Model of Rheumatoid Arthritis
[0145] 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 mALK1-Fc ("RAP-041") at 1 mg/kg, 10 mg/kg or 25
mg/kg. Following the collagen boost on day 21 arthritic scores (see
FIG. 10) and paw swelling steadily increased in all groups, peaking
around day 38. Mice treated with mALK1-Fc ("RAP-041") showed
reduced scores for both characteristics, particularly at the
highest dose (25 mg/kg), although the reduction did not achieve
statistical significance. Nonetheless, a dose-related trend is
apparent.
[0146] 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 mALK1-Fc at 1 mg/kg treated
group and 9/10 in the mALK1-Fc 10 mg/kg treated group. In the
mALK1-Fc 25 mg/kg treated group disease incidence was lower at
6/10.
Example 7
Murine Model of Multiple Myeloma
[0147] Multiple myeloma is a cancer primarily of the bone that is
associated with substantial bone loss. The 5T2MM model of myeloma
in mice is based on the use of tumor cells (5T2MM cells) from a
type of spontaneous tumor that develops in aged mice and causes
effects in mice that are similar to those seen in human multiple
myeloma patients. See, e.g., Vanderkerken et al., Methods Mol. Med.
2005; 113:191-205. mALK1-Fc was tested for effects in this
model.
[0148] 5T2MM cells injected into C57BL/KaLwRij mice promotes an
increase in osteoclast surface, the formation of osteolytic lesions
and caused a decrease in bone area. Bone disease is associated with
a decrease in osteoblast number, osteoblast surface and a reduction
in mineralization.
[0149] Mice bearing 5T2MM cells were treated with mALK1-Fc
(RAP-041) (10 mg/kg, i.p. twice weekly), or a vehicle, from the
time of 5T2MM injection, for a total of 12 weeks. MicroCT analysis
of the proximal tibia and lumbar vertebrae demonstrated a
statistically significant reduction in cancellous bone volume and
trabecular number in 5T2MM-bearing mice compared to naive mice
(bone volume reduced by 40% relative to controls; trabecular number
reduced by 40%). RAP-041 completely prevented 5T2MM-induced
decreases in bone volume and trabecular number when compared to
vehicle treated mice (treated mice had a bone volume of 120%
relative to untumored controls and trabecular number of 115%
relative to untumored controls). Additionally, the tumor treated
mice developed lytic bone lesions that were detected by microCT.
mALK1-Fc treatment reduced the number of lytic bone lesions by 50%
relative to vehicle treated mice.
[0150] Based on the anti-angiogenic effects of ALK1-Fc, we infer
that the protective effect for bone provided by this agent is a
consequence of diminished tumor growth.
[0151] Therefore, ALK1-Fc may be used to treat multiple myeloma and
to decrease the effects of bone disease resulting from this tumor
type.
Example 8
Ligand Binding Characteristics of DAN
[0152] 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.
[0153] 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, 6 and 7 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.
INCORPORATION BY REFERENCE
[0154] 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
[0155] 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 determined by reference to the claims, along
with their full scope of equivalents, and the specification, along
with such variations.
Sequence CWU 1
1
111503PRThomo sapiens 1Met Thr Leu Gly Ser Pro Arg Lys Gly Leu Leu
Met Leu Leu Met Ala 1 5 10 15 Leu Val Thr Gln Gly Asp Pro Val Lys
Pro Ser Arg Gly Pro Leu Val 20 25 30 Thr Cys Thr Cys Glu Ser Pro
His Cys Lys Gly Pro Thr Cys Arg Gly 35 40 45 Ala Trp Cys Thr Val
Val Leu Val Arg Glu Glu Gly Arg His Pro Gln 50 55 60 Glu His Arg
Gly Cys Gly Asn Leu His Arg Glu Leu Cys Arg Gly Arg 65 70 75 80 Pro
Thr Glu Phe Val Asn His Tyr Cys Cys Asp Ser His Leu Cys Asn 85 90
95 His Asn Val Ser Leu Val Leu Glu Ala Thr Gln Pro Pro Ser Glu Gln
100 105 110 Pro Gly Thr Asp Gly Gln Leu Ala Leu Ile Leu Gly Pro Val
Leu Ala 115 120 125 Leu Leu Ala Leu Val Ala Leu Gly Val Leu Gly Leu
Trp His Val Arg 130 135 140 Arg Arg Gln Glu Lys Gln Arg Gly Leu His
Ser Glu Leu Gly Glu Ser 145 150 155 160 Ser Leu Ile Leu Lys Ala Ser
Glu Gln Gly Asp Ser Met Leu Gly Asp 165 170 175 Leu Leu Asp Ser Asp
Cys Thr Thr Gly Ser Gly Ser Gly Leu Pro Phe 180 185 190 Leu Val Gln
Arg Thr Val Ala Arg Gln Val Ala Leu Val Glu Cys Val 195 200 205 Gly
Lys Gly Arg Tyr Gly Glu Val Trp Arg Gly Leu Trp His Gly Glu 210 215
220 Ser Val Ala Val Lys Ile Phe Ser Ser Arg Asp Glu Gln Ser Trp Phe
225 230 235 240 Arg Glu Thr Glu Ile Tyr Asn Thr Val Leu Leu Arg His
Asp Asn Ile 245 250 255 Leu Gly Phe Ile Ala Ser Asp Met Thr Ser Arg
Asn Ser Ser Thr Gln 260 265 270 Leu Trp Leu Ile Thr His Tyr His Glu
His Gly Ser Leu Tyr Asp Phe 275 280 285 Leu Gln Arg Gln Thr Leu Glu
Pro His Leu Ala Leu Arg Leu Ala Val 290 295 300 Ser Ala Ala Cys Gly
Leu Ala His Leu His Val Glu Ile Phe Gly Thr 305 310 315 320 Gln Gly
Lys Pro Ala Ile Ala His Arg Asp Phe Lys Ser Arg Asn Val 325 330 335
Leu Val Lys Ser Asn Leu Gln Cys Cys Ile Ala Asp Leu Gly Leu Ala 340
345 350 Val Met His Ser Gln Gly Ser Asp Tyr Leu Asp Ile Gly Asn Asn
Pro 355 360 365 Arg Val Gly Thr Lys Arg Tyr Met Ala Pro Glu Val Leu
Asp Glu Gln 370 375 380 Ile Arg Thr Asp Cys Phe Glu Ser Tyr Lys Trp
Thr Asp Ile Trp Ala 385 390 395 400 Phe Gly Leu Val Leu Trp Glu Ile
Ala Arg Arg Thr Ile Val Asn Gly 405 410 415 Ile Val Glu Asp Tyr Arg
Pro Pro Phe Tyr Asp Val Val Pro Asn Asp 420 425 430 Pro Ser Phe Glu
Asp Met Lys Lys Val Val Cys Val Asp Gln Gln Thr 435 440 445 Pro Thr
Ile Pro Asn Arg Leu Ala Ala Asp Pro Val Leu Ser Gly Leu 450 455 460
Ala Gln Met Met Arg Glu Cys Trp Tyr Pro Asn Pro Ser Ala Arg Leu 465
470 475 480 Thr Ala Leu Arg Ile Lys Lys Thr Leu Gln Lys Ile Ser Asn
Ser Pro 485 490 495 Glu Lys Pro Lys Val Ile Gln 500 21512DNAhomo
sapiens 2atgaccttgg gctcccccag gaaaggcctt ctgatgctgc tgatggcctt
ggtgacccag 60ggagaccctg tgaagccgtc tcggggcccg ctggtgacct gcacgtgtga
gagcccacat 120tgcaaggggc ctacctgccg gggggcctgg tgcacagtag
tgctggtgcg ggaggagggg 180aggcaccccc aggaacatcg gggctgcggg
aacttgcaca gggagctctg cagggggcgc 240cccaccgagt tcgtcaacca
ctactgctgc gacagccacc tctgcaacca caacgtgtcc 300ctggtgctgg
aggccaccca acctccttcg gagcagccgg gaacagatgg ccagctggcc
360ctgatcctgg gccccgtgct ggccttgctg gccctggtgg ccctgggtgt
cctgggcctg 420tggcatgtcc gacggaggca ggagaagcag cgtggcctgc
acagcgagct gggagagtcc 480agtctcatcc tgaaagcatc tgagcagggc
gacagcatgt tgggggacct cctggacagt 540gactgcacca cagggagtgg
ctcagggctc cccttcctgg tgcagaggac agtggcacgg 600caggttgcct
tggtggagtg tgtgggaaaa ggccgctatg gcgaagtgtg gcggggcttg
660tggcacggtg agagtgtggc cgtcaagatc ttctcctcga gggatgaaca
gtcctggttc 720cgggagactg agatctataa cacagtgttg ctcagacacg
acaacatcct aggcttcatc 780gcctcagaca tgacctcccg caactcgagc
acgcagctgt ggctcatcac gcactaccac 840gagcacggct ccctctacga
ctttctgcag agacagacgc tggagcccca tctggctctg 900aggctagctg
tgtccgcggc atgcggcctg gcgcacctgc acgtggagat cttcggtaca
960cagggcaaac cagccattgc ccaccgcgac ttcaagagcc gcaatgtgct
ggtcaagagc 1020aacctgcagt gttgcatcgc cgacctgggc ctggctgtga
tgcactcaca gggcagcgat 1080tacctggaca tcggcaacaa cccgagagtg
ggcaccaagc ggtacatggc acccgaggtg 1140ctggacgagc agatccgcac
ggactgcttt gagtcctaca agtggactga catctgggcc 1200tttggcctgg
tgctgtggga gattgcccgc cggaccatcg tgaatggcat cgtggaggac
1260tatagaccac ccttctatga tgtggtgccc aatgacccca gctttgagga
catgaagaag 1320gtggtgtgtg tggatcagca gacccccacc atccctaacc
ggctggctgc agacccggtc 1380ctctcaggcc tagctcagat gatgcgggag
tgctggtacc caaacccctc tgcccgactc 1440accgcgctgc ggatcaagaa
gacactacaa aaaattagca acagtccaga gaagcctaaa 1500gtgattcaat tg
15123328PRTartificial sequenceSynthetic Peptide 3Asp Pro Val Lys
Pro Ser Arg Gly Pro Leu Val Thr Cys Thr Cys Glu 1 5 10 15 Ser Pro
His Cys Lys Gly Pro Thr Cys Arg Gly Ala Trp Cys Thr Val 20 25 30
Val Leu Val Arg Glu Glu Gly Arg His Pro Gln Glu His Arg Gly Cys 35
40 45 Gly Asn Leu His Arg Glu Leu Cys Arg Gly Arg Pro Thr Glu Phe
Val 50 55 60 Asn His Tyr Cys Cys Asp Ser His Leu Cys Asn His Asn
Val Ser Leu 65 70 75 80 Val Leu Glu Ala Thr Gln Pro Pro Ser Glu Gln
Pro Gly Thr Asp Gly 85 90 95 Gln Leu Ala Thr Gly Gly Gly Thr His
Thr Cys Pro Pro Cys Pro Ala 100 105 110 Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro 115 120 125 Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 130 135 140 Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 145 150 155 160
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 165
170 175 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln 180 185 190 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala 195 200 205 Leu Pro Val Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro 210 215 220 Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr 225 230 235 240 Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 245 250 255 Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 260 265 270 Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 275 280 285
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 290
295 300 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys 305 310 315 320 Ser Leu Ser Leu Ser Pro Gly Lys 325
41075DNAartificial sequenceSynthetic Oligonucleotide 4gctagcacca
tggatgcaat gaagagaggg ctctgctgtg tgctgctgct gtgtggagca 60gtcttcgttt
cgcccggcgc cgaccctgtg aagccgtctc ggggcccgct ggtgacctgc
120acgtgtgaga gcccacattg caaggggcct acctgccggg gggcctggtg
cacagtagtg 180ctggtgcggg aggaggggag gcacccccag gaacatcggg
gctgcgggaa cttgcacagg 240gagctctgca ggggccgccc caccgagttc
gtcaaccact actgctgcga cagccacctc 300tgcaaccaca acgtgtccct
ggtgctggag gccacccaac ctccttcgga gcagccggga 360acagatggcc
agctggccac cggtggtgga actcacacat gcccaccgtg cccagcacct
420gaagccctgg gggcaccgtc agtcttcctc ttccccccaa aacccaagga
caccctcatg 480atctcccgga cccctgaggt cacatgcgtg gtggtggacg
tgagccacga agaccctgag 540gtcaagttca actggtacgt ggacggcgtg
gaggtgcata atgccaagac aaagccgcgg 600gaggagcagt acaacagcac
gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 660tggctgaatg
gcaaggagta caagtgcaag gtctccaaca aagccctccc agtccccatc
720gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta
caccctgccc 780ccatcccggg aggagatgac caagaaccag gtcagcctga
cctgcctggt caaaggcttc 840tatcccagcg acatcgccgt ggagtgggag
agcaatgggc agccggagaa caactacaag 900accacgcctc ccgtgctgga
ctccgacggc cccttcttcc tctacagcaa gctcaccgtg 960gacaagagca
ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg
1020cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatgagg aattc
10755352PRTartificial sequenceSynthetic Peptide 5Met Asp Ala Met
Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val
Phe Val Ser Pro Gly Ala Asp Pro Val Lys Pro Ser Arg Gly 20 25 30
Pro Leu Val Thr Cys Thr Cys Glu Ser Pro His Cys Lys Gly Pro Thr 35
40 45 Cys Arg Gly Ala Trp Cys Thr Val Val Leu Val Arg Glu Glu Gly
Arg 50 55 60 His Pro Gln Glu His Arg Gly Cys Gly Asn Leu His Arg
Glu Leu Cys 65 70 75 80 Arg Gly Arg Pro Thr Glu Phe Val Asn His Tyr
Cys Cys Asp Ser His 85 90 95 Leu Cys Asn His Asn Val Ser Leu Val
Leu Glu Ala Thr Gln Pro Pro 100 105 110 Ser Glu Gln Pro Gly Thr Asp
Gly Gln Leu Ala Thr Gly Gly Gly Thr 115 120 125 His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Ala Leu Gly Ala Pro Ser 130 135 140 Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 165
170 175 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Val Pro Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290
295 300 Ser Asp Gly Pro Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser 305 310 315 320 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 340 345 350 6228PRTartificial
sequenceSynthetic Peptide 6Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro 1 5 10 15 Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser 20 25 30 Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Ala Val Ser His Glu 35 40 45 Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 50 55 60 Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 65 70 75 80
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 85
90 95 Glu Tyr Lys Cys Lys Ala Val Ser Asn Lys Ala Leu Pro Val Pro
Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser
Asp Gly Pro Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205
His Glu Ala Leu His Asn Ala His Tyr Thr Gln Lys Ser Leu Ser Leu 210
215 220 Ser Pro Gly Lys 225 721PRTartificial sequenceSynthetic
Peptide 7Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val
Tyr Ile 1 5 10 15 Ser Tyr Ile Tyr Ala 20 822PRTartificial
sequenceSynthetic Peptide 8Met Asp Ala Met Lys Arg Gly Leu Cys Cys
Val Leu Leu Leu Cys Gly 1 5 10 15 Ala Val Phe Val Ser Pro 20
921PRTartificial sequenceSynthetic Peptide 9Met Thr Leu Gly Ser Pro
Arg Lys Gly Leu Leu Met Leu Leu Met Ala 1 5 10 15 Leu Val Thr Gln
Gly 20 10180PRThomo sapiens 10Met Leu Arg Val Leu Val Gly Ala Val
Leu Pro Ala Met Leu Leu Ala 1 5 10 15 Ala Pro Pro Pro Ile Asn Lys
Leu Ala Leu Phe Pro Asp Lys Ser Ala 20 25 30 Trp Cys Glu Ala Lys
Asn Ile Thr Gln Ile Val Gly His Ser Gly Cys 35 40 45 Glu Ala Lys
Ser Ile Gln Asn Arg Ala Cys Leu Gly Gln Cys Phe Ser 50 55 60 Tyr
Ser Val Pro Asn Thr Phe Pro Gln Ser Thr Glu Ser Leu Val His 65 70
75 80 Cys Asp Ser Cys Met Pro Ala Gln Ser Met Trp Glu Ile Val Thr
Leu 85 90 95 Glu Cys Pro Gly His Glu Glu Val Pro Arg Val Asp Lys
Leu Val Glu 100 105 110 Lys Ile Leu His Cys Ser Cys Gln Ala Cys Gly
Lys Glu Pro Ser His 115 120 125 Glu Gly Leu Ser Val Tyr Val Gln Gly
Glu Asp Gly Pro Gly Ser Gln 130 135 140 Pro Gly Thr His Pro His Pro
His Pro His Pro His Pro Gly Gly Gln 145 150 155 160 Thr Pro Glu Pro
Glu Asp Pro Pro Gly Ala Pro His Thr Glu Glu Glu 165 170 175 Gly Ala
Glu Asp 180 112003DNAhomo sapiens 11gccgagcctc ctggggcgcc
cgggcccgcg acccccgcac ccagctccgc aggaccggcg 60ggcgcgcgcg ggctctggag
gccacgggca tgatgcttcg ggtcctggtg ggggctgtcc 120tccctgccat
gctactggct gccccaccac ccatcaacaa gctggcactg ttcccagata
180agagtgcctg gtgcgaagcc aagaacatca cccagatcgt gggccacagc
ggctgtgagg 240ccaagtccat ccagaacagg gcgtgcctag gacagtgctt
cagctacagc gtccccaaca 300ccttcccaca gtccacagag tccctggttc
actgtgactc ctgcatgcca gcccagtcca 360tgtgggagat tgtgacgctg
gagtgcccgg gccacgagga ggtgcccagg gtggacaagc 420tggtggagaa
gatcctgcac tgtagctgcc aggcctgcgg caaggagcct agtcacgagg
480ggctgagcgt ctatgtgcag ggcgaggacg ggccgggatc ccagcccggc
acccaccctc 540acccccatcc ccacccccat cctggcgggc agacccctga
gcccgaggac ccccctgggg 600ccccccacac agaggaagag ggggctgagg
actgaggccc ccccaactct tcctcccctc 660tcatccccct gtggaatgtt
gggtctcact ctctggggaa gtcaggggag aagctgaagc 720ccccctttgg
cactggatgg acttggcttc agactcggac ttgaatgctg cccggttgcc
780atggagatct gaaggggcgg ggttagagcc aagctgcaca atttaatata
ttcaagagtg 840gggggaggaa gcagaggtct tcagggctct ttttttgggg
ggggggtggt ctcttcctgt 900ctggcttcta gagatgtgcc tgtgggaggg
ggaggaagtt ggctgagcca ttgagtgctg 960ggggaggcca tccaagatgg
catgaatcgg gctaaggtcc ctgggggtgc agatggtact 1020gctgaggtcc
cgggcttagt gtgagcatct tgccagcctc aggcttgagg gagggctggg
1080ctagaaagac cactggcaga aacaggaggc tccggcccca caggtttccc
caaggcctct 1140caccccactt cccatctcca gggaagcgtc gccccagtgg
cactgaagtg gccctccctc 1200agcggagggg tttgggagtc aggcctgggc
aggaccctgc
tgactcgtgg cgcgggagct 1260gggagccagg ctctccgggc ctttctctgg
cttccttggc ttgcctggtg ggggaagggg 1320aggaggggaa gaaggaaagg
gaagagtctt ccaaggccag aaggaggggg acaacccccc 1380aagaccatcc
ctgaagacga gcatccccct cctctccctg ttagaaatgt tagtgccccg
1440cactgtgccc caagttctag gccccccaga aagctgtcag agccggccgc
cttctcccct 1500ctcccaggga tgctctttgt aaatatcgga tgggtgtggg
agtgaggggt tacctccctc 1560gccccaaggt tccagaggcc ctaggcggga
tgggctcgct gaacctcgag gaactccagg 1620acgaggagga catgggactt
gcgtggacag tcagggttca cttgggctct ctctagctcc 1680ccaattctgc
ctgcctcctc cctcccagct gcactttaac cctagaaggt ggggacctgg
1740ggggagggac agggcaggcg ggcccatgaa gaaagcccct cgttgcccag
cactgtctgc 1800gtctgctctt ctgtgcccag ggtggctgcc agcccactgc
ctcctgcctg gggtggcctg 1860gccctcctgg ctgttgcgac gcgggcttct
ggagcttgtc accattggac agtctccctg 1920atggaccctc agtcttctca
tgaataaatt ccttcaacgc caaaaaaaaa aaaaaaaaaa 1980aaaaaaaaaa
aaaaaaaaaa aaa 2003
* * * * *
References