U.S. patent application number 16/039991 was filed with the patent office on 2018-11-15 for compositions comprising alkaline phosphatase and/or natriuretic peptide and methods of use thereof.
The applicant listed for this patent is Alexion Pharmaceuticals, Inc., Vanderbilt University. Invention is credited to Philippe CRINE, Florent ELEFTERIOU.
Application Number | 20180326019 16/039991 |
Document ID | / |
Family ID | 49670533 |
Filed Date | 2018-11-15 |
United States Patent
Application |
20180326019 |
Kind Code |
A1 |
CRINE; Philippe ; et
al. |
November 15, 2018 |
COMPOSITIONS COMPRISING ALKALINE PHOSPHATASE AND/OR NATRIURETIC
PEPTIDE AND METHODS OF USE THEREOF
Abstract
The present invention provides methods, compositions, and kits
for the treatment of neurocutaneous syndromes, such as
neurofibromatosis type I; disorders associated with overactivation
of FGFR3, such as achondroplasia; bone or cartilage disorders; or
vascular smooth muscle disorders; or for the elongation of bone. In
some embodiments, the present invention provides polypeptides
having an alkaline phosphatase peptide fused to an Fc domain of an
immunoglobulin or a natriuretic peptide fused to an Fc domain of an
immunoglobulin. Such polypeptides can be administered to subjects,
e.g., subcutaneously, to treat a neurocutaneous syndrome, a
disorder associated with overactivation of FGFR3, a bone or
cartilage disorder, or a vascular smooth muscle disorder, or to
elongate bone. The invention also features nucleic acid molecules
encoding such polypeptides and the use of the nucleic acid
molecules for treating neurocutaneous syndromes, disorders
associated with overactivation of FGFR3, bone or cartilage
disorders, or vascular smooth muscle disorders, or for elongating
bone.
Inventors: |
CRINE; Philippe; (Outremont,
CA) ; ELEFTERIOU; Florent; (Nashville, TN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alexion Pharmaceuticals, Inc.
Vanderbilt University |
New Haven
Nashville |
CT
TN |
US
US |
|
|
Family ID: |
49670533 |
Appl. No.: |
16/039991 |
Filed: |
July 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13899359 |
May 21, 2013 |
10052366 |
|
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16039991 |
|
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61649717 |
May 21, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/10 20130101;
C12N 9/16 20130101; C12Y 301/03001 20130101; C07K 2319/31 20130101;
C07K 7/08 20130101; C07K 14/58 20130101; A61K 38/465 20130101 |
International
Class: |
A61K 38/46 20060101
A61K038/46; C07K 14/58 20060101 C07K014/58; C12N 9/16 20060101
C12N009/16; A61K 38/10 20060101 A61K038/10; C07K 7/08 20060101
C07K007/08 |
Goverment Interests
STATEMENT AS TO FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under grant
numbers 5RO1AR055966-02 and W81XWH-09-01-0207) awarded by the NIH
and Department of Defense, respectively. The government has certain
rights in this invention.
Claims
1. A method of treating a neurocutaneous syndrome in a subject,
said method comprising administering to said subject a
therapeutically effective amount of a pharmaceutical composition
comprising: (a) a polypeptide comprising the structure A-sALP-B;
and (b) a pharmaceutically acceptable excipient, wherein sALP is
the extracellular domain of an alkaline phosphatase, A is absent or
is an amino acid sequence of at least one amino acid, and B is
absent or is an amino acid sequence of at least one amino acid,
thereby treating said syndrome in said subject.
2. The method of claim 1, wherein the amino acid sequence of said
polypeptide comprises the amino acid sequence of SEQ ID NOs: 1204
or 1221.
3. The method of claim 1, wherein the amino acid sequence of said
sALP comprises amino acid residues 23-508 of SEQ ID NO: 1215, amino
acid residues 18-498 of SEQ ID NO: 1216, amino acid residues 23-508
of SEQ ID NO: 1218, or amino acid residues 18-498 of SEQ ID NO:
1219, or the amino acid sequence of said sALP consists of amino
acid residues 23-512 of SEQ ID NO: 1215, amino acid residues 18-502
of SEQ ID NO: 1216, amino acid residues 23-512 of SEQ ID NO: 1218,
or amino acid residues 18-502 of SEQ ID NO: 1219.
4. The method of claim 1 or 3, wherein the amino acid sequence of
said sALP comprises an amino acid sequence having at least 85%
sequence identity to SEQ ID NO: 1205, or at least 95% sequence
identity to SEQ ID NO: 1205, or at least 99% sequence identity to
SEQ ID NO: 1205.
5. The method of claim 4, wherein the amino acid sequence of said
sALP comprises or consists of the amino acid sequence of SEQ ID NO:
1205.
6. The method of any one of claims 1-5, wherein A and/or B are
absent.
7. The method of any one of claims 1-6, wherein A or B comprises a
fragment crystallizable region (Fc).
8. The method of any one of claims 1-7, wherein said Fc comprises a
C.sub.H2 domain, a C.sub.H3 domain, and a hinge region, or wherein
said Fc is a constant domain of an immunoglobulin selected from the
group consisting of IgG-1, IgG-2, IgG-3, and IgG-4.
9. The method of claim 8, wherein the amino acid sequence of said
Fc comprises an amino acid sequence having at least 85% sequence
identity to SEQ ID NO: 401, or at least 95% sequence identity to
SEQ ID NO: 401, or at least 99% sequence identity to SEQ ID NO:
401.
10. The method of claim 9, wherein the amino acid sequence of said
Fc comprises or consists of the amino acid sequence of SEQ ID NO:
401.
11. The method of any one of claims 1-10, wherein A or B comprises
I.sub.n, and wherein I represents an aspartic acid or a glutamic
acid and n=10 to 16.
12. The method of any one of claims 1-10, wherein said polypeptide
does not comprise a polyaspartic acid or polyglutamic acid region
longer than three consecutive aspartic acid or glutamic acid
residues, or wherein said polypeptide does not comprise a
polyaspartic acid or polyglutamic acid region longer than two
consecutive aspartic acid or glutamic acid residues.
13. The method of any one of claims 1-10, wherein said polypeptide
does not comprise a bone-targeting moiety.
14. The method of any one of claims 1-10, wherein said polypeptide
comprises the structure C-sALP-D-Fc-E or the structure
C-Fc-D-sALP-E, C is absent or is an amino acid sequence of at least
one amino acid, D is absent or is an amino acid sequence of at
least one amino acid, and E is absent or is an amino acid sequence
of at least one amino acid.
15. The method of claim 14, wherein C and/or E are absent.
16. The method of any one of claims 1-10, wherein said polypeptide
comprises the structure C-sALP-D-Fc-G-I.sub.n-H or the structure
C-Fc-D-sALP-G-I.sub.n-H, C is absent or is an amino acid sequence
of at least one amino acid, D is absent or is an amino acid
sequence of at least one amino acid, G is absent or is an amino
acid sequence of at least one amino acid, H is absent or is an
amino acid sequence of at least one amino acid, I represents an
aspartic acid or a glutamic acid, and n=10 to 16.
17. The method of claim 16, wherein C and/or H are absent.
18. The method of claim 16 or 17, wherein G is two amino acid
residues.
19. The method of claim 18, wherein G is aspartic
acid-isoleucine.
20. The method of any one of claims 16-19, wherein I is aspartic
acid and n=10.
21. The method of any one of claims 14-20, wherein D is two amino
acid residues.
22. The method of claim 21, wherein D is leucine-lysine.
23. The method of any one of claims 1-22, wherein the amino acid
sequence of said polypeptide consists of the amino acid sequence of
SEQ ID NOs: 1201, 1204, 1220, or 1221.
24. The method of claim 23, wherein the amino acid sequence of said
polypeptide consists of the amino acid sequence of SEQ ID NO:
1204.
25. A method of treating a neurocutaneous syndrome in a subject,
said method comprising administering to said subject a
therapeutically effective amount of a pharmaceutical composition
comprising: (a) a polypeptide comprising the structure V-NP-W; and
(b) a pharmaceutically acceptable excipient, wherein NP is a
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), V is absent or is an amino acid sequence of at
least one amino acid, and W is absent or is an amino acid sequence
of at least one amino acid, thereby treating said syndrome in said
subject.
26. The method of claim 25, wherein the amino acid sequence of said
polypeptide comprises the amino acid sequence of SEQ ID NOs: 504,
512, 530, or 572.
27. The method of claim 25, wherein said NP comprises the
structure: [N-terminal extension]-[short segment]-[ring
domain]-[C-terminal extension], wherein said ring domain comprises
the amino acid sequence of SEQ ID NO: 6, amino acid residues 11-27
of SEQ ID NO: 30, or SEQ ID NO: 95, and each of said N-terminal
extension, short segment, and C-terminal extension is,
independently, absent or is an amino acid sequence of at least one
amino acid.
28. The method of claim 27, wherein said ring domain comprises
amino acid residues 6-22 of SEQ ID NO: 126.
29. The method of claim 28, wherein the amino acid at position 17
of SEQ ID NO: 126 is Phe, Leu, Ile, Thr, Val, Ala, Ser, Glu, Arg,
Tyr, Cys, Pro, or Asp.
30. The method of claim 27, wherein said ring domain comprises the
amino acid sequence of SEQ ID NO: 12.
31. The method of any one of claims 27-30, wherein said short
segment and said ring domain together comprise the amino acid
sequence of any one of SEQ ID NOs: 4, 13-30, 119-122, 126, or
156-161.
32. The method of any one of claims 27-31, wherein the amino acid
sequence of said short segment consists of amino acid residues 1-5,
2-5, 3-5, 4-5, or 5 of SEQ ID NO: 4, amino acid residues 1-10 of
SEQ ID NO: 17, amino acid residues 1-5 of SEQ ID NO: 19, amino acid
residues 1-3 of SEQ ID NO: 20, amino acid residues 1-5 of SEQ ID
NO: 21, or amino acid residues 1-6 of SEQ ID NO: 29.
33. The method of any one of claims 27-32, wherein the amino acid
sequence of said N-terminal extension comprises amino acid residues
1-31 or 17-31 of SEQ ID NO: 11.
34. The method of any one of claims 27-32, wherein the amino acid
sequence of said N-terminal extension comprises KGANKK (SEQ ID NO:
314) or KGANQK (SEQ ID NO: 315).
35. The method of claim 27, wherein said N-terminal extension,
short segment, and ring domain together comprise the amino acid
sequence of SEQ ID NO: 11.
36. The method of any one of claims 27-35, wherein said C-terminal
extension comprises the amino acid sequence of SEQ ID NOs: 117 or
118 or comprises amino acid residues 23-37 selected from any one of
SEQ ID NOs: 101-116.
37. The method of claim 27, wherein the amino acid sequence of said
NP consists of SEQ ID NOs: 4 or 11, or the amino acid sequence of
any one of SEQ ID NOs: 31-94, or a fragment thereof comprising at
least a ring domain, or the amino acid sequence of any one of SEQ
ID NOs: 13-29, 100-116, 119-125, 127-233, or 1001-1155.
38. The method of any one of claims 27-37, wherein V and/or W are
absent.
39. The method of any one of claims 27-38, wherein V or W comprises
a fragment crystallizable region (Fc).
40. The method of claim 39, wherein said Fc comprises a C.sub.H2
domain, a C.sub.H3 domain, and a hinge region, or wherein said Fc
is a constant domain of an immunoglobulin selected from the group
consisting of IgG-1, IgG-2, IgG-3, and IgG-4.
41. The method of claim 40, wherein the amino acid sequence of said
Fc comprises an amino acid sequence having at least 85% sequence
identity to SEQ ID NO: 401, or at least 95% sequence identity to
SEQ ID NO: 401, or at least 99% sequence identity to SEQ ID NO:
401.
42. The method of claim 41, wherein the amino acid sequence of said
Fc comprises or consists of the amino acid sequence of SEQ ID NO:
401.
43. The method of any one of claims 27-42, wherein V or W comprises
a glycine-rich region.
44. The method of claim 43, wherein the amino acid sequence of V or
W consists of one or more glycines and one or more serines.
45. The method of claim 43 or 44, wherein the amino acid sequence
of V or W comprises [(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, and wherein each of m, n, and
p is, independently, between 0 and 20.
46. The method of claim 45, wherein m is between 1 and 6; n is
between 1 and 10; and p is between 0 and 4.
47. The method of claim 46, wherein m is 4 and n is 1-6.
48. The method of claim 46, wherein combinations of m, n, and p are
selected from a single row of Table 2, or wherein the amino acid
sequence of V or W comprises the amino acid sequence of any one of
SEQ ID NOs: 301-391.
49. The method of any one of claims 27-48, wherein V or W does not
comprise a bone-targeting moiety.
50. The method of any one of claims 27-48, wherein V or W comprises
a bone-targeting moiety.
51. The method of claim 50, wherein said bone-targeting moiety
comprises six consecutive acidic residues.
52. The method of claim 51, wherein said bone-targeting moiety
comprises ten consecutive acidic residues.
53. The method of claim 51 or 52, wherein said acidic residues are
aspartic acid or glutamic acid.
54. The method of claim 53, wherein said bone-targeting moiety
comprises E.sub.6, E.sub.10, D.sub.6, or D.sub.10.
55. The method of any one of claims 27-54, wherein V or W comprises
a cathepsin cleavage sequence.
56. The method of claim 55, wherein said cathepsin cleavage
sequence comprises a cathepsin K cleavage sequence.
57. The method of claim 55 or 56, wherein said cathepsin cleavage
sequence is HGPQG (SEQ ID NO: 374) or HKLRG (SEQ ID NO: 375).
58. The method of any one of claims 27-57, wherein said polypeptide
comprises the structure V-NP-W, NP is a natriuretic peptide that is
an agonist of natriuretic peptide receptor B (NPR-B), each of V and
W is, independently, absent or is an amino acid sequence of at
least one amino acid, and said NP comprises the amino acid sequence
of any one of SEQ ID NOs: 17-29, 31-40, 42-94, 101-116, 119-122,
128-161, or 163-233, or V or W comprises the amino acid sequence of
any one of SEQ ID NOs: 304-313, 322-333, or 337-391.
59. The method of any one of claims 27-57, wherein said polypeptide
comprises the structure V-NP or NP-W, NP is a natriuretic peptide
that is an agonist of natriuretic peptide receptor B (NPR-B), and
each of V and W comprises, independently, the amino acid sequence
of any one of SEQ ID NOs: 304-313, 322-333, or 337-391.
60. The method of any one of claims 27-57, wherein said polypeptide
comprises the structure X-Fc-Y-NP-Z or the structure X-NP-Y-Fc-Z,
NP is a natriuretic peptide that is an agonist of natriuretic
peptide receptor B (NPR-B), and each of X, Y, and Z is,
independently, absent or is an amino acid sequence of at least one
amino acid.
61. The method of claim 60, wherein Y comprises a glycine-rich
region.
62. The method of claim 61, wherein the amino acid sequence of Y
consists of one or more glycines and one or more serines.
63. The method of claim 62, wherein the amino acid sequence of Y
comprises [(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, and wherein each of m, n, and
p is, independently, between 0 and 20.
64. The method of any one of claims 60-63, wherein X is absent, Z
is absent, or X and Z are both absent.
65. The method of any one of claims 60-64, wherein X, Y, or Z
comprises a bone-targeting moiety.
66. The method of claim 65, wherein said bone-targeting moiety
comprises six or ten consecutive acidic residues.
67. The method of claim 66, wherein said acidic residues are
aspartic acid or glutamic acid.
68. The method of any one of claims 65-67, wherein said
bone-targeting moiety comprises E.sub.6, E.sub.10, D.sub.6, or
D.sub.10.
69. The method of any one of claims 60-68, wherein X, Y, or Z
comprises a cathepsin cleavage sequence.
70. The method of claim 69, wherein said cathepsin cleavage
sequence comprises a cathepsin K cleavage sequence.
71. The method of any one of claims 25-70, wherein the amino acid
sequence of said polypeptide comprises the amino acid sequence of
any one of SEQ ID NOs: 501-608.
72. The method of claim 71, wherein the amino acid sequence of said
polypeptide comprises the amino acid sequence of any one of SEQ ID
NOs: 502, 504, 506, 512, 514, 516, 530, 560, 562, 564, 572, 574,
576, 584, 586, 588, 596, 598, 600, or 608.
73. The method of claim 72, wherein the amino acid sequence of said
polypeptide comprises or consists of the amino acid sequence of SEQ
ID NOs: 504, 512, 530, or 572.
74. The method of any one of claims 1-73, wherein said polypeptide
is in dimeric form.
75. The method of any one of claims 1-74, wherein said polypeptide
is glycosylated or pegylated.
76. The method of any one of claims 1-75, wherein said
pharmaceutical composition is administered in a dosage between
about 0.2 mg/kg to about 20 mg/kg of said polypeptide.
77. The method of any one of claims 1-75, wherein said
pharmaceutical composition is administered in a dosage between
about 0.5 mg/kg to about 500 mg/kg of said polypeptide.
78. The method of any one of claims 1-75, wherein said
pharmaceutical composition is administered in a dosage between
about 10 .mu.g/kg to about 1,000 .mu.g/kg of said polypeptide.
79. The method of any one of claims 1-78, wherein said
pharmaceutical composition is administered subcutaneously.
80. The method of any one of claims 1-79, wherein said
pharmaceutical composition is administered one time, two times, or
three times per week.
81. The method of any one of claims 1-80, wherein said subject is
human.
82. The method of any one of claims 1-81, wherein said
neurocutaneous syndrome is neurofibromatosis type I.
83. A composition comprising a first polypeptide and a second
polypeptide, wherein a) said first polypeptide comprises the
structure A-sALP-B, wherein i) sALP is the extracellular domain of
an alkaline phosphatase, ii) A is absent or is an amino acid
sequence of at least one amino acid, and iii) B is absent or is an
amino acid sequence of at least one amino acid; and b) said second
polypeptide comprises the structure V-NP-W, wherein i) NP is a
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), ii) V is absent or is an amino acid sequence of
at least one amino acid, and iii) W is absent or is an amino acid
sequence of at least one amino acid.
84. The composition of claim 83, wherein the amino acid sequence of
said first polypeptide comprises the amino acid sequence of SEQ ID
NOs: 1204 or 1221 and the amino acid sequence of said second
polypeptide comprises the amino acid sequence of SEQ ID NOs: 504,
512, 530, or 572.
85. The composition of claim 83, wherein the amino acid sequence of
said sALP of said first polypeptide comprises amino acid residues
23-508 of SEQ ID NO: 1215, amino acid residues 18-498 of SEQ ID NO:
1216, amino acid residues 23-508 of SEQ ID NO: 1218, or amino acid
residues 18-498 of SEQ ID NO: 1219, or the amino acid sequence of
said sALP of said first polypeptide consists of amino acid residues
23-512 of SEQ ID NO: 1215, amino acid residues 18-502 of SEQ ID NO:
1216, amino acid residues 23-512 of SEQ ID NO: 1218, or amino acid
residues 18-502 of SEQ ID NO: 1219, or the amino acid sequence of
said sALP of said first polypeptide comprises an amino acid
sequence having at least 85% sequence identity to SEQ ID NO: 1205,
or at least 95% sequence identity to SEQ ID NO: 1205, or at least
99% sequence identity to SEQ ID NO: 1205.
86. The composition of claim 83 or 85, wherein A and/or B of said
first polypeptide are absent.
87. The composition of any one of claims 83-86, wherein A or B of
said first polypeptide comprises a fragment crystallizable region
(Fc).
88. The composition of any one of claims 83-87, wherein A or B of
said first polypeptide comprises I.sub.n, and wherein I represents
an aspartic acid or a glutamic acid and n=10 to 16.
89. The composition of any one of claims 83-88, wherein said first
polypeptide comprises the structure C-sALP-D-Fc-G-I.sub.n-H, C is
absent or is an amino acid sequence of at least one amino acid, D
is absent or is an amino acid sequence of at least one amino acid,
G is absent or is an amino acid sequence of at least one amino
acid, H is absent or is an amino acid sequence of at least one
amino acid, I represents an aspartic acid or a glutamic acid, and
n=10 to 16.
90. The composition of any one of claims 83-89, wherein the amino
acid sequence of said first polypeptide comprises or consists of
the amino acid sequence of SEQ ID NO: 1204.
91. The composition of any one of claims 83-90, wherein said NP of
said second polypeptide comprises the structure: [N-terminal
extension]-[short segment]-[ring domain]-[C-terminal extension],
wherein said ring domain comprises the amino acid sequence of SEQ
ID NO: 6, amino acid residues 11-27 of SEQ ID NO: 30, or SEQ ID NO:
95, and each of said N-terminal extension, short segment, and
C-terminal extension is, independently, absent or is an amino acid
sequence of at least one amino acid.
92. The composition of claim 91, wherein said ring domain comprises
amino acid residues 6-22 of SEQ ID NO: 126.
93. The composition of claim 92, wherein the amino acid at position
17 of SEQ ID NO: 126 is Phe, Leu, Ile, Thr, Val, Ala, Ser, Glu,
Arg, Tyr, Cys, Pro, or Asp.
94. The composition of any one of claims 91-93, wherein the amino
acid sequence of said N-terminal extension comprises amino acid
residues 1-31 or 17-31 of SEQ ID NO: 11, KGANKK (SEQ ID NO: 314),
or KGANQK (SEQ ID NO: 315).
95. The composition of any one of claims 91-94, wherein said
C-terminal extension comprises the amino acid sequence of SEQ ID
NOs: 117 or 118 or comprises amino acid residues 23-37 selected
from any one of SEQ ID NOs: 101-116.
96. The composition of claim 91, wherein the amino acid sequence of
said NP consists of SEQ ID NOs: 4 or 11, or the amino acid sequence
of any one of SEQ ID NOs: 31-94, or a fragment thereof comprising
at least a ring domain, or the amino acid sequence of any one of
SEQ ID NOs: 13-29, 100-116, 119-125, 127-233, or 1001-1155.
97. The composition of any one of claims 83-96, wherein V and/or W
of said second polypeptide are absent.
98. The composition of any one of claims 83-97, wherein V or W of
said second polypeptide comprises a fragment crystallizable region
(Fc).
99. The composition of any one of claims 83-98, wherein V or W of
said second polypeptide comprises a glycine-rich region.
100. The composition of any one of claims 83-99, wherein V or W of
said second polypeptide comprises a bone-targeting moiety.
101. The composition of any one of claims 83-100, wherein V or W of
said second polypeptide comprises a cathepsin cleavage
sequence.
102. The composition of any one of claims 83-101, wherein said
second polypeptide comprises the structure V-NP-W, NP is a
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), each of V and W is, independently, absent or is
an amino acid sequence of at least one amino acid, and said NP
comprises the amino acid sequence of any one of SEQ ID NOs: 17-29,
31-40, 42-94, 101-116, 119-122, 128-161, or 163-233, or V or W
comprises the amino acid sequence of any one of SEQ ID NOs:
304-313, 322-333, or 337-391.
103. The composition of any one of claims 83-102, wherein said
second polypeptide comprises the structure V-NP or NP-W, NP is said
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), and each of V and W comprises, independently,
the amino acid sequence of any one of SEQ ID NOs: 304-313, 322-333,
or 337-391.
104. The composition of any one of claims 83-103, wherein said
second polypeptide comprises the structure X-Fc-Y-NP-Z or the
structure X-NP-Y-Fc-Z, NP is said natriuretic peptide that is an
agonist of natriuretic peptide receptor B (NPR-B), and each of X,
Y, and Z is, independently, absent or is an amino acid sequence of
at least one amino acid.
105. The composition of claim 104, wherein Y comprises a
glycine-rich region.
106. The composition of claim 104 or 105, wherein X is absent, Z is
absent, or X and Z are both absent.
107. The composition of any one of claims 104-106, wherein X, Y, or
Z comprises a bone-targeting moiety.
108. The composition of any one of claims 104-107, wherein X, Y, or
Z comprises a cathepsin cleavage sequence.
109. The composition of any one of claims 83-108, wherein the amino
acid sequence of said second polypeptide comprises the amino acid
sequence of any one of SEQ ID NOs: 501-608.
110. The composition of claim 109, wherein the amino acid sequence
of said second polypeptide comprises the amino acid sequence of any
one of SEQ ID NOs: 502, 504, 506, 512, 514, 516, 530, 560, 562,
564, 572, 574, 576, 584, 586, 588, 596, 598, 600, or 608.
111. The composition of claim 110, wherein the amino acid sequence
of said second polypeptide comprises or consists of the amino acid
sequence of SEQ ID NO: 512.
112. The composition of any one of claims 83-111, wherein said
first polypeptide and/or said second polypeptide are in dimeric
form.
113. The composition of any one of claims 83-112, wherein said
first polypeptide and/or said second polypeptide are glycosylated
or pegylated.
114. The composition of any one of claims 83-113, wherein the
composition is a pharmaceutical composition comprising a
pharmaceutically acceptable excipient.
115. The composition of any one of claims 83-114, wherein said
composition is lyophilized.
116. The composition of any one of claims 83-115, wherein said
first polypeptide is present in a dosage between about 0.2 mg/kg to
about 20 mg/kg and said second polypeptide is present in a dosage
between about 0.5 mg/kg to about 500 mg/kg.
117. The composition of any one of claims 83-116, wherein the amino
acid sequence of said first polypeptide comprises the amino acid
sequence of SEQ ID NO: 1204 and wherein the amino acid sequence of
said second polypeptide comprises the amino acid sequence of SEQ ID
NOs: 504, 512, 530, or 572.
118. A method of treating a disease or a condition in a subject,
said method comprising administering to said subject a
therapeutically effective amount of a first polypeptide and a
second polypeptide, wherein a) said first polypeptide comprises the
structure A-sALP-B, wherein i) sALP is the extracellular domain of
an alkaline phosphatase, ii) A is absent or is an amino acid
sequence of at least one amino acid, and iii) B is absent or is an
amino acid sequence of at least one amino acid; and b) said second
polypeptide comprises the structure V-NP-W, wherein i) NP is a
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), ii) V is absent or is an amino acid sequence of
at least one amino acid, and iii) W is absent or is an amino acid
sequence of at least one amino acid; and said disease or condition
is selected from the group consisting of a neurocutaneous syndrome,
a disorder associated with overactivation of FGFR3, a bone or
cartilage disorder, a vascular smooth muscle disorder, and a
condition for elongation of bone, thereby treating said disease or
said condition in said subject.
119. The method of claim 118, wherein said first polypeptide and
said second polypeptide are administered within ten days, five
days, or twenty-four hours of each other.
120. The method of claim 118, wherein said first polypeptide and
said second polypeptide are administered simultaneously.
121. The method of any one of claims 118-120, wherein said first
polypeptide and said second polypeptide are formulated together in
a composition or each separately in a composition.
122. The method of claim 121, wherein said composition is a
pharmaceutical composition comprising a pharmaceutically acceptable
excipient.
123. The method of claim 122, wherein said composition is
lyophilized.
124. The method of any one of claims 121-123, wherein said
composition is the composition of any one of claims 83-117.
125. The method of any one of claims 118-124, wherein the amino
acid sequence of said first polypeptide comprises the amino acid
sequence of SEQ ID NO: 1204 and wherein the amino acid sequence of
said second polypeptide comprises the amino acid sequence of SEQ ID
NOs: 504, 512, 530, or 572.
126. The method of any one of claims 118-125, wherein said first
polypeptide is present in a dosage between about 0.2 mg/kg to about
20 mg/kg and said second polypeptide is present in a dosage between
about 0.5 mg/kg to about 500 mg/kg.
127. The method of any one of claims 118-125, wherein said first
polypeptide is present in a dosage between about 0.2 mg/kg to about
20 mg/kg and said second polypeptide is present in a dosage between
about 10 .mu.g/kg to about 1,000 .mu.g/kg.
128. The method of any one of claims 118-127, wherein said first
polypeptide and said second polypeptide are administered
subcutaneously.
129. The method of any one of claims 118-128, wherein said first
polypeptide and said second polypeptide are administered one time,
two times, or three times per week.
130. The method of any one of claims 118-129, wherein said subject
is human.
131. The method of any one of claims 118-130, wherein said disease
is said neurocutaneous syndrome.
132. The method of claim 131, wherein said neurocutaneous syndrome
is neurofibromatosis type I.
133. A kit comprising: a) a first polypeptide comprising the
structure A-sALP-B, wherein i) sALP is the extracellular domain of
an alkaline phosphatase, ii) A is absent or is an amino acid
sequence of at least one amino acid, and iii) B is absent or is an
amino acid sequence of at least one amino acid; and b) instructions
for administering said first polypeptide to a patient diagnosed
with or at risk of developing a neurocutaneous syndrome.
134. The kit of claim 133, further comprising: (c) a second
polypeptide comprising the structure V-NP-W, wherein i) NP is a
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), ii) V is absent or is an amino acid sequence of
at least one amino acid, and iii) W is absent or is an amino acid
sequence of at least one amino acid.
135. A kit comprising: a) a polypeptide comprising the structure
V-NP-W, wherein i) NP is a natriuretic peptide that is an agonist
of natriuretic peptide receptor B (NPR-B), ii) V is absent or is an
amino acid sequence of at least one amino acid, and iii) W is
absent or is an amino acid sequence of at least one amino acid; and
b) instructions for administering said polypeptide to a patient
diagnosed with or at risk of developing a neurocutaneous
syndrome.
136. A kit comprising: a) a first polypeptide comprising the
structure A-sALP-B, wherein i) sALP is the extracellular domain of
an alkaline phosphatase, ii) A is absent or is an amino acid
sequence of at least one amino acid, and iii) B is absent or is an
amino acid sequence of at least one amino acid; and b) a second
polypeptide comprising the structure V-NP-W, wherein i) NP is a
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), ii) V is absent or is an amino acid sequence of
at least one amino acid, and iii) W is absent or is an amino acid
sequence of at least one amino acid.
137. The kit of claim 136, wherein said first polypeptide and said
second polypeptide are formulated together.
138. The kit of claim 136, wherein said first polypeptide and said
second polypeptide are formulated separately and in individual
dosage amount.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application 61/649,717 filed on May 21, 2012, which is hereby
incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] In general, this invention relates to the treatment of
various diseases using alkaline phosphatase and/or natriuretic
peptide.
[0004] Numerous diseases and conditions involve abnormal skeletal
function, structure, or growth of bone or cartilage. For some
diseases, the etiology of these skeletal manifestations is known,
such as in hypophosphatasia (HPP) and achondroplasia (ACH), but
treatment options are limited. In other diseases, the etiology is
unknown. For example, neurofibromatosis type I (NF1 or Von
Recklinghausen disease) is an autosomal dominant genetic disorder
having an incidence of approximately 1 in 3,500 live births. NF1
encodes neurofibromin, a member of the GTPase Activating Protein
(GAP) family known to suppress the Ras kinase. Neurofibromin is a
specific suppressor of p21-RAS, and mutations in the NF1 gene cause
unsuppressed activation of RAS that lead to abnormal cell growth
and differentiation. Accordingly, the clinical features of NF1
include various oncogenic transformations, such as neurocutaneous
neurofibromas and optic pathway tumors, and other non-cancer
manifestations, such as cognitive defects and skeletal
abnormalities. Some of the NF1 skeletal manifestations have high
morbidity (e.g., dystrophic scoliosis, long bone bowing, and
pseudarthrosis) and unsatisfactory treatment options, which has
been complicated by the fact that the etiology of these
manifestations is unclear.
[0005] Many skeletal diseases arise from loss of function of one or
more proteins. For example, hypophosphatasia (HPP) is a rare,
heritable disease caused by one or more loss-of-function mutations
in the gene ALPL, which encodes tissue-nonspecific alkaline
phosphatase (TNALP; a.k.a. liver/bone/kidney type ALP). Alkaline
phosphatase deficiency in osteoblasts and chondrocytes impairs
skeletal mineralization, leading to symptoms of varying severity,
from rickets or osteomalacia to almost complete absence of bone
mineralization in utero. However, enzyme replacement therapy with
unmodified alkaline phosphatase (e.g., infusion of native alkaline
phosphatase) has been largely unsuccessful.
[0006] In another example, achondroplasia (ACH) is the most common
form of short limb dwarfism in human beings, affecting more than
250,000 individuals worldwide, and is caused by mutations in the
gene encoding fibroblast growth factor receptor 3 (FGFR3), which
cause gain of FGFR3 function. The severity of the clinical
phenotype is related to the capacity of the mutation to
overactivate FGFR3 signaling pathways in chondrocytes, such as the
MAP-kinase pathway. This pathway can be inhibited by activating the
natriuretic peptide receptor B (NPR-B), which produces the second
messenger cGMP, and cGMP, in turn, inhibits the MAP-kinase pathway
inside the cell. In the cellular environment, the immature and
mature forms of C-type natriuretic peptide (CNP), such as CNP53 and
CNP22, bind to NPR-B and induce cGMP production in a dose-dependent
and similar fashion. Thus, use of CNP or a CNP analog that could
activate the NPR-B signaling pathway for the treatment of skeletal
dysplasia has been considered. However, a major drawback of the
therapeutic use of CNP is its extremely short half-life.
[0007] There is thus a need in the art to develop therapeutic
molecules and methods for treating diseases having skeletal
manifestations, such as neurofibromatosis. In addition, more
therapeutic molecules are needed that have an appreciable half-life
and/or other favorable pharmacokinetic and therapeutic properties,
and these molecules can be used to treat a variety of disorders
that would benefit from their underlying mode of action, such as
neurofibromatosis, hypophosphatasia, and achondroplasia.
SUMMARY OF THE INVENTION
[0008] It has surprisingly been discovered that neurofibromatosis,
a neurocutaneous syndrome resulting in tumors in the nervous
system, results in bone manifestations that arise from accumulation
of inorganic pyrophosphate (PPi). As alkaline phosphatase-Fc fusion
proteins (either with or without a bone-targeting moiety) can
reduce PPi accumulation, the present invention provides a
polypeptide including a soluble alkaline phosphatase (sALP) domain
(i.e., an sALP polypeptide), as well as compositions and uses
thereof. In addition, bone manifestations in neurofibromatosis may
also arise from overactivation of the MAP-kinase pathway, and a
natriuretic peptide (NP) or NP analog could be used to inhibit this
pathway. Accordingly, the present invention provides a polypeptide
including an NP (e.g., a CNP) domain (i.e., an NP polypeptide) and
compositions and uses thereof.
[0009] Furthermore, the present invention includes a combination of
an sALP polypeptide and an NP polypeptide, where these polypeptides
can be administered separately or together. This combination would
be particularly useful in diseases that would benefit from
increased ALP levels (e.g., disorders associated with increased
levels of PPi, such as neurofibromatosis, or disorders associated
with ALP deficiency, such as hypophosphatasia) and/or diseases that
would benefit from inactivation of a signaling pathway involving
FGFR3 (e.g., disorders associated with overactivation of the
MAP-kinase pathway, such as neurofibromatosis or achondroplasia, or
disorders associated with overactivation of FGFR3, such as
achondroplasia or craniosynostosis or cancer) and/or diseases that
would benefit from increased NP levels (e.g., disorders associated
with CNP deficiency, such as skeletal dysplasia, or vascular smooth
muscle disorders). The polypeptides of the invention can also be
provided in kits, either separately or together.
[0010] In a first aspect, the invention features a method of
treating a neurocutaneous syndrome in a subject, the method
including administering to the subject a therapeutically effective
amount of a pharmaceutical composition including: (a) a polypeptide
including the structure A-sALP-B; and (b) a pharmaceutically
acceptable excipient, where sALP is the extracellular domain of an
alkaline phosphatase, A is absent or is an amino acid sequence of
at least one amino acid, and B is absent or is an amino acid
sequence of at least one amino acid. In some embodiments, the
syndrome in the subject is thereby treated.
[0011] In some embodiments, the amino acid sequence of the sALP
includes or consists of amino acid residues 23-508 of SEQ ID NO:
1215, amino acid residues 18-498 of SEQ ID NO: 1216, amino acid
residues 23-508 of SEQ ID NO: 1218, or amino acid residues 18-498
of SEQ ID NO: 1219, or the amino acid sequence of the sALP includes
or consists of amino acid residues 23-512 of SEQ ID NO: 1215, amino
acid residues 18-502 of SEQ ID NO: 1216, amino acid residues 23-512
of SEQ ID NO: 1218, or amino acid residues 18-502 of SEQ ID NO:
1219. In some embodiments, the amino acid sequence of the sALP
includes an amino acid sequence having at least 80%, 85%, 90%, 95%,
or 99% sequence identity to SEQ ID NO: 1205. In some embodiments,
the amino acid sequence of the sALP includes or consists of the
amino acid sequence of SEQ ID NO: 1205.
[0012] In some embodiments, the amino acid sequence of the sALP
includes or consists of amino acid residues 1-497, 1-498, 1-499,
1-500, 1-501, 1-502, 1-503, 1-504, 1-505, 1-506, 1-507, 1-508,
1-509, 1-510, 1-511, 1-512, 23-497, 23-498, 23-499, 23-500, 23-501,
23-502, 23-503, 23-504, 23-505, 23-506, 23-507, 23-508, 23-509,
23-510, 23-511, or 23-512 of SEQ ID NO: 1218, where X is any amino
acid but is not an amino acid corresponding to a pathogenic
mutation at that position of human TNALP, e.g., not an amino acid
corresponding to a pathogenic mutation provided in Table 1. In some
embodiments, the amino acid sequence of the sALP includes or
consists of amino acid residues 18-497, 18-498, 18-499, 18-500,
18-501, 18-502, 18-503, 18-504, 18-505, 18-506, 18-507, 18-508,
18-509, 18-510, 18-511, or 18-512 of SEQ ID NO: 1219, where X is
any amino acid but is not an amino acid corresponding to a
pathogenic mutation at that position of human TNALP, e.g., not an
amino acid corresponding to a pathogenic mutation provided in Table
1.
[0013] In some embodiments, the amino acid sequence of the sALP
includes an amino acid sequence having at least 80%, 85%, 90%, 95%,
or 99% sequence identity to SEQ ID NOs: 1218 or 1219, where X is
any amino acid but is not an amino acid corresponding to a
pathogenic mutation at that position of human TNALP, e.g., not an
amino acid corresponding to a pathogenic mutation provided in Table
1. In some embodiments, the amino acid sequence of the sALP
includes or consists of an amino acid sequence having at least 80%,
85%, 90%, 95%, or 99% sequence identity to amino acid residues
23-508 of SEQ ID NO: 1215, amino acid residues 18-498 of SEQ ID NO:
1216, amino acid residues 23-508 of SEQ ID NO: 1218, or amino acid
residues 18-498 of SEQ ID NO: 1219, or the amino acid sequence of
the sALP consists of an amino acid sequence having at least 80%,
85%, 90%, 95%, or 99% sequence identity to amino acid residues
23-512 of SEQ ID NO: 1215, amino acid residues 18-502 of SEQ ID NO:
1216, amino acid residues 23-512 of SEQ ID NO: 1218, or amino acid
residues 18-502 of SEQ ID NO: 1219, where X in SEQ ID NO: 1218 or
1219 is any amino acid but is not an amino acid corresponding to a
pathogenic mutation at that position of human TNALP, e.g., not an
amino acid corresponding to a pathogenic mutation provided in Table
1.
[0014] In some embodiments, A and/or B are absent.
[0015] In some embodiments, A and/or B includes a fragment
crystallizable region (Fc). In some embodiments, the Fc includes a
C.sub.H2 domain, a C.sub.H3 domain, and a hinge region, or the Fc
is a constant domain of an immunoglobulin selected from the group
consisting of IgG-1, IgG-2, IgG-3, and IgG-4, e.g., IgG-1. In some
embodiments, the amino acid sequence of the Fc includes an amino
acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence
identity to SEQ ID NO: 401. In some embodiments, the amino acid
sequence of the Fc includes or consists of the amino acid sequence
of SEQ ID NO: 401.
[0016] In some embodiments, A and/or B includes I.sub.n, where I
represents an aspartic acid or a glutamic acid and n=10 to 16,
e.g., n is 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the
I.sub.n is E.sub.10, E.sub.11, E.sub.12, E.sub.13, E.sub.14,
E.sub.15, E.sub.16, D.sub.10, D.sub.11, D.sub.12, D.sub.13,
D.sub.14, D.sub.15, or D.sub.16, e.g., E.sub.10 or D.sub.10.
[0017] In some embodiments, A and/or B includes a bone-targeting
moiety, e.g., including 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16
consecutive acidic residues, e.g., aspartic acid or glutamic acid.
In some embodiments, the bone-targeting moiety includes or consists
of E.sub.6, E.sub.7, E.sub.8, E.sub.9, E.sub.10, E.sub.11,
E.sub.12, E.sub.13, E.sub.14, E.sub.15, E.sub.16, D.sub.6, D.sub.7,
D.sub.8, D.sub.9, D.sub.10, D.sub.11, D.sub.12, D.sub.13, D.sub.14,
D.sub.15, or D.sub.16, e.g., E.sub.6, E.sub.10, D.sub.6, or
D.sub.10. In some embodiments, the polypeptide does not include a
polyaspartic acid or polyglutamic acid region longer than three
consecutive aspartic acid or glutamic acid residues, or the
polypeptide does not include a polyaspartic acid or polyglutamic
acid region longer than two consecutive aspartic acid or glutamic
acid residues.
[0018] In some embodiments, the polypeptide does not include a
bone-targeting moiety.
[0019] In some embodiments, the polypeptide includes the structure
C-sALP-D-Fc-E or the structure C-Fc-D-sALP-E, where C is absent or
is an amino acid sequence of at least one amino acid, D is absent
or is an amino acid sequence of at least one amino acid, and E is
absent or is an amino acid sequence of at least one amino acid.
[0020] In some embodiments, C and/or E are absent. In some
embodiments, D is two amino acid residues, e.g., leucine-lysine or
aspartic acid-isoleucine. In some embodiments, D is any linker
described herein, e.g., the amino acid sequence of any one of SEQ
ID NOs: 301-391.
[0021] In some embodiments, the polypeptide includes the structure
C-sALP-D-Fc-G-I.sub.n-H or the structure C-Fc-D-sALP-G-I.sub.n-H,
where C is absent or is an amino acid sequence of at least one
amino acid, D is absent or is an amino acid sequence of at least
one amino acid, G is absent or is an amino acid sequence of at
least one amino acid, H is absent or is an amino acid sequence of
at least one amino acid, I represents an aspartic acid or a
glutamic acid, and n=10 to 16, e.g., n is 10, 11, 12, 13, 14, 15,
or 16. In some embodiments, the I.sub.n is E.sub.10, E.sub.11,
E.sub.12, E.sub.13, E.sub.14, E.sub.15, E.sub.16, D.sub.10,
D.sub.11, D.sub.12, D.sub.13, D.sub.14, D.sub.15, or D.sub.16,
e.g., E.sub.10 or D.sub.10.
[0022] In some embodiments, C and/or H are absent. In some
embodiments, G is two amino acid residues, e.g., leucine-lysine or
aspartic acid-isoleucine, e.g., aspartic acid-isoleucine. In some
embodiments, I is an aspartic acid or a glutamic acid and n=10 to
16, e.g., n is 10, 11, 12, 13, 14, 15, or 16, e.g., I is aspartic
acid and n=10. In some embodiments, D is two amino acid residues,
e.g., leucine-lysine or aspartic acid-isoleucine, e.g.,
leucine-lysine. In some embodiments, D or G is any linker described
herein, e.g., the amino acid sequence of any one of SEQ ID NOs:
301-391.
[0023] In some embodiments, the amino acid sequence of the
polypeptide includes an amino acid sequence having at least 80%,
85%, 90%, 95%, or 99% sequence identity of any one of SEQ ID NOs:
1201, 1204, 1220, or 1221, e.g., SEQ ID NO: 1204. In some
embodiments, the amino acid sequence of the polypeptide includes or
consists of the amino acid sequence of any one of SEQ ID NOs: 1201,
1204, 1220, or 1221, e.g., SEQ ID NO: 1204. In some embodiments,
the amino acid sequence of the polypeptide consists of the amino
acid sequence of SEQ ID NO: 1204.
[0024] In a second aspect, the invention features a method of
treating a neurocutaneous syndrome in a subject, the method
including administering to the subject a therapeutically effective
amount of a pharmaceutical composition including: (a) a polypeptide
including the structure V-NP-W; and (b) a pharmaceutically
acceptable excipient, where NP is a natriuretic peptide that is an
agonist of natriuretic peptide receptor B (NPR-B), V is absent or
is an amino acid sequence of at least one amino acid, and W is
absent or is an amino acid sequence of at least one amino acid. In
some embodiments, the syndrome in the subject is thereby
treated.
[0025] In some embodiments, the NP includes the structure:
[N-terminal extension]-[short segment]-[ring domain]-[C-terminal
extension], where the ring domain includes the amino acid sequence
of SEQ ID NO: 6, amino acid residues 11-27 of SEQ ID NO: 30, or SEQ
ID NO: 95, and each of the N-terminal extension, short segment, and
C-terminal extension is, independently, absent or is an amino acid
sequence of at least one amino acid. In some embodiments, the ring
domain includes amino acid residues 6-22 of SEQ ID NO: 126. In some
embodiments, the amino acid at position 17 of SEQ ID NO: 126 is
Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro, Asp, Gly, Ala, Ser,
Val, Trp, Asn, Gln, His, or Lys, e.g., Phe, Leu, Ile, Thr, Val,
Ala, Ser, Glu, Arg, Tyr, Cys, Pro, or Asp, e.g., Phe, Leu, Ile,
Thr, Val, Ala, or Ser, e.g., Phe or Leu, e.g., Phe, e.g., Leu. In
some embodiments, the ring domain includes the amino acid sequence
of SEQ ID NO: 12. In some embodiments, the short segment and the
ring domain together include the amino acid sequence of any one of
SEQ ID NOs: 4, 13-30, 119-122, 126, or 156-161, e.g., SEQ ID NOs: 4
or 13-30. In some embodiments, the amino acid sequence of the short
segment includes or consists of amino acid residues 1-5, 2-5, 3-5,
4-5, or 5, e.g., consists of amino acid residues 1-5, of SEQ ID NO:
4; amino acid residues 1-10 of SEQ ID NO: 17; amino acid residues
1-5 of SEQ ID NO: 19; amino acid residues 1-3 of SEQ ID NO: 20;
amino acid residues 1-5 of SEQ ID NO: 21; or amino acid residues
1-6 of SEQ ID NO: 29. In some embodiments, the amino acid sequence
of the short segment and the ring domain together includes or
consists of the amino acid sequence of SEQ ID NO: 4. In some
embodiments, the amino acid sequence of the short segment and the
ring domain together includes or consists of the amino acid
sequence of any one of SEQ ID NOs: 119-122, 126, or 156-161 (e.g.,
where X in SEQ ID NO: 126 is Phe, Leu, Ile, Thr, Glu, Arg, Tyr,
Cys, Pro, Asp, Gly, Ala, Ser, Val, Trp, Asn, Gin, His, or Lys,
e.g., Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro, or Asp, e.g.,
Phe, Leu, Ile, Thr, Val, Ala, or Ser, e.g., Phe or Leu, e.g., Phe,
e.g., Leu).
[0026] In some embodiments, the amino acid sequence of the
N-terminal extension includes amino acid residues 1-31 or 17-31 of
SEQ ID NO: 11. In some embodiments, the amino acid sequence of the
N-terminal extension includes amino acid residues 17-31 of SEQ ID
NO: 11. In some embodiments, the amino acid sequence of the
N-terminal extension includes KGANKK (SEQ ID NO: 314) or KGANQK
(SEQ ID NO: 315). In some embodiments, the N-terminal extension,
short segment, and ring domain together include the amino acid
sequence of SEQ ID NO: 11. In some embodiments, the C-terminal
extension includes the amino acid sequence of SEQ ID NOs: 117 or
118 or includes amino acid residues 23-37 selected from any one of
SEQ ID NOs: 101-116. In some embodiments, the amino acid sequence
of the NP consists of SEQ ID NOs: 4 or 11, or the amino acid
sequence of any one of SEQ ID NOs: 31-94, or a fragment thereof
including at least a ring domain, or the amino acid sequence of any
one of SEQ ID NOs: 13-29, 100-116, 119-125, 127-233, or
1001-1155.
[0027] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Phe.
[0028] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Leu.
[0029] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Ile.
[0030] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Thr.
[0031] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Glu.
[0032] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Arg.
[0033] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Tyr.
[0034] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Cys.
[0035] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Pro.
[0036] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Asp.
[0037] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Gly.
[0038] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Ala.
[0039] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Ser.
[0040] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Val.
[0041] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Trp.
[0042] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Asn.
[0043] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Gln.
[0044] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be His.
[0045] In any of the aspects described herein, the amino acid
sequence of the NP may include amino acids 6-22 of SEQ ID NO: 126,
and the amino acid at position 17 of SEQ ID NO: 126 may be Lys.
[0046] In some embodiments, V and/or W are absent.
[0047] In some embodiments, V and/or W includes a fragment
crystallizable region (Fc). In some embodiments, the Fc includes a
C.sub.H2 domain, a C.sub.H3 domain, and a hinge region, or the Fc
is a constant domain of an immunoglobulin selected from the group
consisting of IgG-1, IgG-2, IgG-3, and IgG-4, e.g., IgG-1. In some
embodiments, the amino acid sequence of the Fc includes an amino
acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence
identity to SEQ ID NO: 401. In some embodiments, the amino acid
sequence of the Fc includes or consists of the amino acid sequence
of SEQ ID NO: 401.
[0048] In some embodiments, V and/or W includes a glycine-rich
region.
[0049] In some embodiments, the amino acid sequence of V or W
consists of one or more glycines and one or more serines. In some
embodiments, the amino acid sequence of V or W includes
[(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, where each of m, n, and p is,
independently, between 0 and 20. In some embodiments, m is between
1 and 6; n is between 1 and 10; and p is between 0 and 4, e.g., m
is 4 and n is 1-6. In some embodiments, combinations of m, n, and p
are selected from a single row of Table 2, or the amino acid
sequence of V or W includes the amino acid sequence of any one of
SEQ ID NOs: 301-391.
[0050] In some embodiments, V and/or W does not include a
bone-targeting moiety.
[0051] In some embodiments, V and/or W includes a bone-targeting
moiety, e.g., including 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16
consecutive acidic residues, e.g., aspartic acid or glutamic acid.
In some embodiments, the bone-targeting moiety includes or consists
of E.sub.6, E.sub.7, E.sub.8, E.sub.9, E.sub.10, E.sub.11,
E.sub.12, E.sub.13, E.sub.14, E.sub.15, E.sub.16, D.sub.6, D.sub.7,
D.sub.8, D.sub.9, D.sub.10, D.sub.11, D.sub.12, D.sub.13, D.sub.14,
D.sub.15, or D.sub.16, e.g., E.sub.6, E.sub.10, D.sub.6, or
D.sub.10.
[0052] In some embodiments, V and/or W includes a cathepsin (e.g.,
cathepsin K) cleavage sequence. In some embodiments, the cathepsin
cleavage sequence is HGPQG (SEQ ID NO: 374) or HKLRG (SEQ ID NO:
375).
[0053] In some embodiments, the polypeptide includes the structure
V-NP-W, where NP is a natriuretic peptide that is an agonist of
natriuretic peptide receptor B (NPR-B), each of V and W is,
independently, absent or is an amino acid sequence of at least one
amino acid, and the NP includes the amino acid sequence of any one
of SEQ ID NOs: 17-29, 31-40, 42-94, 101-116, 119-122, 128-161, or
163-233, or V or W includes the amino acid sequence of any one of
SEQ ID NOs: 304-313, 322-333, or 337-391.
[0054] In some embodiments, the polypeptide includes the structure
V-NP or NP-W, where NP is the natriuretic peptide that is an
agonist of natriuretic peptide receptor B (NPR-B), and each of V
and W includes, independently, the amino acid sequence of any one
of SEQ ID NOs: 304-313, 322-333, or 337-391.
[0055] In some embodiments, the polypeptide includes the structure
X-Fc-Y-NP-Z or the structure X-NP-Y-Fc-Z, where NP is the
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B) (e.g., any NP described herein), and each of X,
Y, and Z is, independently, absent or is an amino acid sequence of
at least one amino acid. In some embodiments, Y includes a
glycine-rich region. In some embodiments, the amino acid sequence
of Y consists of one or more glycines and one or more serines. In
some embodiments, the amino acid sequence of Y includes
[(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, where each of m, n, and p is,
independently, between 0 and 20. In some embodiments, m is between
1 and 6; n is between 1 and 10; and p is between 0 and 4, e.g., m
is 4 and n is 1-6. In some embodiments, combinations of m, n, and p
are selected from a single row of Table 2, or the amino acid
sequence of Y includes the amino acid sequence of any one of SEQ ID
NOs: 301-389. In some embodiments, the amino acid sequence of Y
consists of [(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, wherein combinations of m, n,
and p are selected from a single row of Table 2, or the amino acid
sequence of Y consists of the amino acid sequence of any one of SEQ
ID NOs: 301-389. In some embodiments, X is absent, Z is absent, or
X and Z are both absent. In some embodiments, X, Y, or Z includes a
bone-targeting moiety, e.g., including 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, or 16 consecutive acidic residues, e.g., aspartic acid or
glutamic acid. In some embodiments, the bone-targeting moiety
includes or consists of E.sub.6, E.sub.7, E.sub.8, E.sub.9,
E.sub.10, E.sub.11, E.sub.12, E.sub.13, E.sub.14, E.sub.15,
E.sub.16, D.sub.6, D.sub.7, D.sub.8, D.sub.9, D.sub.10, D.sub.11,
D.sub.12, D.sub.13, D.sub.14, D.sub.15, or D.sub.16, e.g., E.sub.6,
E.sub.10, D.sub.6, or D.sub.10.
[0056] In some embodiments, X, Y, or Z includes a cathepsin (e.g.,
cathepsin K) cleavage sequence. In some embodiments, the cathepsin
cleavage sequence includes or consists of HGPQG (SEQ ID NO: 374) or
HKLRG (SEQ ID NO: 375).
[0057] In some embodiments, the amino acid of the polypeptide
includes or consists of the amino acid sequence of any one of SEQ
ID NOs: 501-608, e.g., SEQ ID NOs: 502, 504, 506, 512, 514, 516,
530, 560, 562, 564, 572, 574, 576, 584, 586, 588, 596, 598, 600, or
608, e.g., SEQ ID NOs: 504, 512, 530, 554, 572, or 578, e.g., SEQ
ID NO: 512. In some embodiments, the amino acid of the polypeptide
includes a bone-targeting moiety, e.g., E.sub.6, E.sub.7, E.sub.8,
E.sub.9, E.sub.10, E.sub.11, E.sub.12, E.sub.13, E.sub.14,
E.sub.15, E.sub.16, D.sub.6, D.sub.7, D.sub.8, D.sub.9, D.sub.10,
D.sub.11, D.sub.12, D.sub.13, D.sub.14, D.sub.15, or D.sub.16,
e.g., E.sub.6, E.sub.10, D.sub.6, or D.sub.10.
[0058] In some embodiments, the amino acid sequence of the
polypeptide includes an amino acid sequence having at least 80%,
85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs:
504, 512, 530, 554, 572, or 578, e.g., SEQ ID NOs: 504, 512, 530,
or 572, e.g., SEQ ID NO: 512. In some embodiments, the amino acid
sequence of the polypeptide includes or consists of the amino acid
sequence of any one of SEQ ID NOs: 504, 512, 530, 554, 572, or 578,
e.g., SEQ ID NOs: 504, 512, 530, or 572, e.g., SEQ ID NO: 512. In
some embodiments, the amino acid sequence of the polypeptide
consists of the amino acid sequence of SEQ ID NOs: 504, 512, 530,
or 572, e.g., SEQ ID NO: 512.
[0059] In some embodiments, the polypeptide includes the structure
X-Fc-Y-NP-Z or the structure X-NP-Y-Fc-Z, wherein NP is a
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), and wherein either: (i) NP includes amino acids
6-22 of SEQ ID NO: 126, wherein the amino acid at position 17 is
not Met; and each of X, Y, and Z is, independently, absent or is an
amino acid sequence of at least one amino acid; or (ii) each of X
and Z is, independently, absent or is an amino acid sequence of at
least one amino acid; and the amino acid sequence of Y includes
[(Gly).sub.4(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.4].sub.n, wherein n is between 1 and 10
and p is between 0 and 4 or wherein combinations of m, n, and p are
selected from a single row of Table 2, or wherein the amino acid
sequence of Y includes the amino acid sequence of any one of SEQ ID
NOs: 304-313, 322-333, or 337-391.
[0060] In some embodiments, the polypeptide includes the structure
X-Fc-Y-NP-Z.
[0061] In some embodiments, (i) NP includes amino acids 6-22 of SEQ
ID NO: 126, wherein the amino acid at position 17 is not Met; and
each of X, Y, and Z is, independently, absent or is an amino acid
sequence of at least one amino acid. In some embodiments, the amino
acid at position 17 of SEQ ID NO: 126 is Phe, Leu, Ile, Thr, Val,
Ala, or Ser. In some embodiments, the amino acid at position 17 of
SEQ ID NO: 126 is Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro, Asp,
Gly, Ala, Ser, Val, Trp, Asn, Gln, His, or Lys, e.g., Phe, Leu,
Ile, Thr, Glu, Arg, Tyr, Cys, Pro, or Asp, e.g., Phe or Leu, e.g.,
Phe, e.g., Leu. In some embodiments, the NP includes the structure:
[N-terminal extension]-[short segment]-[ring domain]-[C-terminal
extension], wherein said ring domain comprises amino acids 6-22 of
SEQ ID NO: 126, wherein the amino acid at position 17 is not Met,
and each of said N-terminal extension, short segment, and
C-terminal extension is, independently, absent or is an amino acid
sequence of at least one amino acid. In some embodiments, the amino
acid sequence of said NP includes or consists of the amino acid
sequence of any one of SEQ ID NOs: 119-125 or 156-220, wherein
position 17 relative to SEQ ID NO: 126 is not Met, or the amino
acid sequence of any one of SEQ ID NOs: 221-233.
[0062] In some embodiments, (ii) each of X and Z is, independently,
absent or is an amino acid sequence of at least one amino acid; and
the amino acid sequence of Y comprises
[(Gly).sub.4(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.4].sub.n, wherein n is between 1 and 10
and p is between 0 and 4, or wherein the amino acid sequence of Y
comprises the amino acid sequence of any one of SEQ ID NOs:
304-313, 322-333, or 337-391. In some embodiments, the NP includes
the structure: [N-terminal extension]-[short segment]-[ring
domain]-[C-terminal extension], wherein the ring domain includes
the amino acid sequence of SEQ ID NO: 6, amino acids 11-27 of SEQ
ID NO: 30, or SEQ ID NO: 95, and each of the N-terminal extension,
short segment, and C-terminal extension is, independently, absent
or is an amino acid sequence of at least one amino acid. In some
embodiments, the ring domain includes amino acids 6-22 of SEQ ID
NO: 126. In some embodiments, the amino acid at position 17 of SEQ
ID NO: 126 is Phe, Leu, Ile, Thr, Val, Ala, or Ser. In some
embodiments, the amino acid at position 17 of SEQ ID NO: 126 is
Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro, Asp, Gly, Ala, Ser,
Val, Trp, Asn, Gln, His, or Lys, e.g., Phe, Leu, Ile, Thr, Glu,
Arg, Tyr, Cys, Pro, or Asp, e.g., Phe or Leu, e.g., Phe, e.g., Leu.
In some embodiments, the ring domain includes the amino acid
sequence of SEQ ID NO: 12. In some embodiments, the short segment
and the ring domain together include the amino acid sequence of any
one of SEQ ID NOs: 4 or 13-30. In some embodiments, the amino acid
sequence of the short segment and the ring domain together consists
of the amino acid sequence of SEQ ID NO: 4. In some embodiments,
the amino acid sequence of the short segment and the ring domain
together consists of the amino acid sequence of any one of SEQ ID
NOs: 119-122, 126, or 156-161 (e.g., where X in SEQ ID NO: 126 is
Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro, Asp, Gly, Ala, Ser,
Val, Trp, Asn, Gln, His, or Lys, e.g., Phe, Leu, Ile, Thr, Glu,
Arg, Tyr, Cys, Pro, or Asp, e.g., Phe or Leu, e.g., Phe, e.g.,
Leu). In some embodiments, the N-terminal extension, short segment,
and ring domain together include the amino acid sequence of SEQ ID
NO: 11. In some embodiments, the amino acid sequence of the NP
consists of SEQ ID NO: 4. In some embodiments, the amino acid
sequence of the NP consists of SEQ ID NO: 11. In some embodiments,
the amino acid sequence of the NP consists of the amino acid
sequence of any one of SEQ ID NOs: 31-94, or a fragment thereof
including at least a ring domain. In some embodiments, the amino
acid sequence of the NP includes or consists of the amino acid
sequence of any one of SEQ ID NOs: 13-29, 100-116, 119-125,
127-233, or 1001-1155.
[0063] In some embodiments, the amino acid sequence of the short
segment consists of amino acids 1-5 of SEQ ID NO: 4. In some
embodiments, the amino acid sequence of the short segment consists
of amino acids 1-5, 2-5, 3-5, 4-5, or 5 of SEQ ID NO: 4, amino
acids 1-10 of SEQ ID NO: 17, amino acids 1-5 of SEQ ID NO: 19,
amino acids 1-3 of SEQ ID NO: 20, amino acids 1-5 of SEQ ID NO: 21,
or amino acids 1-6 of SEQ ID NO: 29. In some embodiments, the amino
acid sequence of the N-terminal extension includes amino acids 1-31
of SEQ ID NO: 11. In some embodiments, the amino acid sequence of
the N-terminal extension includes amino acids 17-31 of SEQ ID NO:
11. In some embodiments, the amino acid sequence of the N-terminal
extension includes KGANKK (SEQ ID NO: 314) or KGANQK (SEQ ID NO:
315). In some embodiments, the C-terminal extension includes the
amino acid sequence of SEQ ID NO: 118, SEQ ID NO: 117, or amino
acids 23-37 selected from any one of SEQ ID NOs: 101-116.
[0064] In some embodiments, the NP is selective for NPR-B over
NPR-A, wherein the EC.sub.50(NPR-A)/EC.sub.50(NPR-B) ratio for the
NP, as determined in an in vivo pharmacokinetic assay, is at least
30.
[0065] In some embodiments, the Fc includes a C.sub.H2 domain, a
C.sub.H3 domain, and a hinge region. In some embodiments, the Fc is
a constant domain of an immunoglobulin selected from the group
consisting of IgG-1, IgG-2, IgG-3, IgG-3 and IgG-4. In some
embodiments, the Fc includes the amino acid sequence of SEQ ID NO:
401. In some embodiments, the immunoglobulin is IgG-1. In some
embodiments, the amino acid sequence of the Fc includes an amino
acid sequence having at least 80%, 85%, 90%, 95%, or 99% sequence
identity to SEQ ID NO: 401, or includes or consists of the amino
acid sequence of SEQ ID NO: 401.
[0066] In some embodiments, Y includes a glycine-rich region, or
the amino acid sequence of Y consists of one or more glycines and
one or more serines. For example, the amino acid sequence of Y may
include [(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, wherein each of m, n, and p
is, independently, between 0 and 20. In some embodiments, m is 0-20
(e.g., m is 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 3-14, 3-15, 3-16,
3-17, 3-18, 3-19, 3-20, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-14,
4-15, 4-16, 4-17, 4-18, 4-19, 4-20, 5-6, 5-7, 5-8, 5-9, 5-10, 5-11,
5-12, 5-14, 5-15, 5-16, 5-17, 5-18, 5-19, 5-20, 6-7, 6-8, 6-9,
6-10, 6-11, 6-12, 6-14, 6-15, 6-16, 6-17, 6-18, 6-19, 6-20, 7-8,
7-9, 7-10, 7-11, 7-12, 7-14, 7-15, 7-16, 7-17, 7-18, 7-19, 7-20,
8-9, 8-10, 8-11, 8-12, 8-14, 8-15, 8-16, 8-17, 8-18, 8-19, 8-20,
9-10, 9-11, 9-12, 9-14, 9-15, 9-16, 9-17, 9-18, 9-19, 9-20, 10-11,
10-12, 10-14, 10-15, 10-16, 10-17, 10-18, 10-19, or 10-20). In some
embodiments, m is 4 and n is 1-6. In some embodiments, combinations
of m, n, and p are selected from a single row of Table 2, or the
amino acid sequence of Y includes the amino acid sequence of any
one of SEQ ID NOs: 304-313, 322-333, or 337-391. In some
embodiments, the amino acid sequence of Y consists of
[(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, wherein combinations of m, n,
and p are selected from a single row of Table 2, or the amino acid
sequence of Y consists of the amino acid sequence of any one of SEQ
ID NOs: 304-313, 322-333, or 337-391.
[0067] In some embodiments, X is absent, Z is absent, or X and Z
are both absent.
[0068] In some embodiments, X, Y, or Z includes a bone-targeting
moiety, e.g., including 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16
consecutive acidic residues, e.g., aspartic acid or glutamic acid.
In some embodiments, the bone-targeting moiety includes or consists
of E.sub.6, E.sub.7, E.sub.8, E.sub.9, E.sub.10, E.sub.11,
E.sub.12, E.sub.13, E.sub.14, E.sub.15, E.sub.16, D.sub.6, D.sub.7,
D.sub.8, D.sub.9, D.sub.10, D.sub.11, D.sub.12, D.sub.13, D.sub.14,
D.sub.15, or D.sub.16, e.g., E.sub.6, E.sub.10, D.sub.6, or
D.sub.10.
[0069] In some embodiments, X, Y, or Z includes a cathepsin (e.g.,
cathepsin K) cleavage sequence. In some embodiments, the cathepsin
cleavage sequence includes or consists of HGPQG (SEQ ID NO: 374) or
HKLRG (SEQ ID NO: 375).
[0070] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of any one of SEQ ID NOs: 501-608, e.g.,
SEQ ID NOs: 502, 504, 506, 512, 514, 516, 530, 560, 562, 564, 572,
574, 576, 584, 586, 588, 596, 598, 600, or 608. In some
embodiments, the polypeptide includes a bone-targeting moiety,
e.g., E.sub.6, E.sub.7, E.sub.8, E.sub.9, E.sub.10, E.sub.11,
E.sub.12, E.sub.13, E.sub.14, E.sub.15, E.sub.16, D.sub.6, D.sub.7,
D.sub.8, D.sub.9, D.sub.10, D.sub.11, D.sub.12, D.sub.13, D.sub.14,
D.sub.15, or D.sub.16, e.g., E.sub.6, E.sub.10, D.sub.6, or
D.sub.10.
[0071] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of SEQ ID NO: 504.
[0072] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of SEQ ID NO: 512.
[0073] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of SEQ ID NO: 530.
[0074] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of SEQ ID NO: 554.
[0075] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of SEQ ID NO: 572.
[0076] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of SEQ ID NO: 578.
[0077] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of SEQ ID NO: 560.
[0078] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of SEQ ID NO: 566.
[0079] In some embodiments, the polypeptide includes or consists of
the amino acid sequence of SEQ ID NO: 538 (e.g., where X in SEQ ID
NO: 538 can be any amino acid, e.g., Phe, Leu, Ile, Thr, Glu, Arg,
Tyr, Cys, Pro, Asp, Gly, Ala, Ser, Val, Trp, Asn, Gln, His, or Lys,
e.g., Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro, or Asp, e.g.,
Phe or Leu, e.g., Phe, e.g., Leu).
[0080] In some embodiments, the polypeptide includes the structure
V-NP.
[0081] In some embodiments, any of the NPs or polypeptides
described herein may be used in conjunction with the method (e.g.,
NPs or polypeptides described in any of the aspects described
herein). In some embodiments, the amino acid sequence of V or W
includes [(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, wherein each of m, n, and p
is, independently, between 0 and 20. In some embodiments, m is 4
and n is 1-6. In some embodiments, V is absent, W is absent, or V
and W are both absent. In some embodiments, V and/or W includes a
bone-targeting moiety, e.g., E.sub.6, E.sub.7, E.sub.8, E.sub.9,
E.sub.10, E.sub.11, E.sub.12, E.sub.13, E.sub.14, E.sub.15,
E.sub.16, D.sub.6, D.sub.7, D.sub.8, D.sub.9, D.sub.10, D.sub.11,
D.sub.12, D.sub.13, D.sub.14, D.sub.15, or D.sub.16, e.g., E.sub.6,
E.sub.10, D.sub.6, or D.sub.10. In some embodiments, V and/or W
includes a cathepsin (e.g., cathepsin K) cleavage sequence, e.g.,
HGPQG (SEQ ID NO: 374) or HKLRG (SEQ ID NO: 375).
[0082] In some embodiments of any of the aspects described herein,
the polypeptide of the invention, e.g., the sALP polypeptide and/or
the NP polypeptide, is in dimeric form. In some embodiments, the
polypeptide is glycosylated or pegylated.
[0083] In some embodiments of any of the aspects described herein,
the pharmaceutical composition is administered in a dosage between
about 0.2 mg/kg to about 20 mg/kg of the polypeptide of the
invention, e.g., the sALP polypeptide and/or the NP polypeptide,
e.g., the sALP polypeptide. In some embodiments, the pharmaceutical
composition is administered to the subject in a dosage between
about 0.2 mg/kg to about 20 mg/kg once, twice, three times, or four
times daily. The dosage may be between, e.g., about 0.5 mg/kg to
about 10 mg/kg, e.g., about 1 mg/kg to about 5 mg/kg, once, twice,
three times, or four times daily. In some embodiments, the dosage
is about 1 mg/kg, about 2 mg/kg, or about 3 mg/kg once, twice, or
three times daily. In some embodiments, the pharmaceutical
composition is administered to the subject in a dosage between
about 0.2 mg/kg to about 20 mg/kg once, twice, three times, or four
times weekly. The dosage may be between, e.g., about 0.5 mg/kg to
about 10 mg/kg, e.g., about 1 mg/kg to about 5 mg/kg, once, twice,
three times, or four times weekly. In some embodiments, the dosage
is about 1 mg/kg, about 2 mg/kg, or about 3 mg/kg once, twice, or
three times weekly.
[0084] In some embodiments of any of the aspects described herein,
the pharmaceutical composition is administered in a dosage between
about 0.5 mg/kg to about 500 mg/kg of the polypeptide of the
invention, e.g., the sALP polypeptide and/or the NP polypeptide,
e.g., the NP polypeptide. In some embodiments, the pharmaceutical
composition is administered to the subject in a dosage between
about 0.5 mg/kg to about 500 mg/kg once, twice, three times, or
four times daily. The dosage may be between, e.g., about 5 mg/kg to
about 200 mg/kg, e.g., about 10 mg/kg to about 100 mg/kg, once,
twice, three times, or four times daily. In some embodiments, the
dosage is about 10 mg/kg or about 100 mg/kg twice daily. In some
embodiments, the pharmaceutical composition is administered to the
subject in a dosage between about 0.5 mg/kg to about 500 mg/kg
once, twice, three times, or four times weekly. The dosage may be
between, e.g., about 5 mg/kg to about 200 mg/kg, e.g., about 10
mg/kg to about 100 mg/kg, e.g., about 20 mg/kg to about 40 mg/kg,
once, twice, three times, or four times weekly. In some
embodiments, the dosage is about 10 mg/kg, about 30 mg/kg, or about
100 mg/kg, once, twice, or three times weekly.
[0085] In some embodiments of any of the aspects described herein,
the pharmaceutical composition is administered in a dosage between
about 10 .mu.g/kg to about 1,000 .mu.g/kg of the polypeptide of the
invention, e.g., the sALP polypeptide and/or the NP polypeptide,
e.g., the NP polypeptide. In some embodiments, the pharmaceutical
composition is administered to the subject in a dosage between
about 10 .mu.g/kg to about 1,000 .mu.g/kg once, twice, three times,
or four times weekly. The dosage may be between, e.g., about 20
.mu.g/kg to about 800 sg/kg, e.g., about 30 .mu.g/kg to about 600
.mu.g/kg, e.g., about 50 .mu.g/kg to about 500 sg/kg, e.g., about
100 .mu.g/kg to about 400 .mu.g/kg, e.g., about 200 .mu.g/kg to
about 300 r.mu.g/kg, once, twice, three times, or four times
weekly. In some embodiments, the dosage is about 30 .mu.g/kg, about
100 .mu.g/kg, about 300 .mu.g/kg, or about 500 r.mu.g/kg, once,
twice, or three times weekly.
[0086] In some embodiments of any of the aspects described herein,
the pharmaceutical composition is administered subcutaneously. In
some embodiments, the pharmaceutical composition is administered
one time, two times, or three times per week.
[0087] In a third aspect, the invention features a composition
including a first polypeptide and a second polypeptide, where a)
the first polypeptide includes the structure A-sALP-B, where i)
sALP is the extracellular domain of an alkaline phosphatase, ii) A
is absent or is an amino acid sequence of at least one amino acid,
and iii) B is absent or is an amino acid sequence of at least one
amino acid; and b) the second polypeptide includes the structure
V-NP-W, where i) NP is a natriuretic peptide that is an agonist of
natriuretic peptide receptor B (NPR-B), ii) V is absent or is an
amino acid sequence of at least one amino acid, and iii) W is
absent or is an amino acid sequence of at least one amino acid.
[0088] In some embodiments, the first polypeptide is any sALP
polypeptide described herein, e.g., as described for the first
aspect. In some embodiments, the amino acid sequence of the first
polypeptide includes an amino acid sequence having at least 80%,
85%, 90%, 95%, or 99% sequence identity of any one of SEQ ID NOs:
1201, 1204, 1220, or 1221, e.g., SEQ ID NO: 1204. In some
embodiments, the amino acid sequence of the first polypeptide
includes or consists of the amino acid sequence of SEQ ID NO:
1204.
[0089] In some embodiments, the second polypeptide is any NP
polypeptide described herein, e.g., as described for the second
aspect. In some embodiments, the amino acid sequence of the second
polypeptide includes an amino acid sequence having at least 80%,
85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs:
504, 512, 530, 554, 572, or 578, e.g., SEQ ID NO: 512. In some
embodiments, the amino acid sequence of the second polypeptide
includes or consists of the amino acid sequence of SEQ ID NO:
512.
[0090] In some embodiments, the amino acid sequence of the first
polypeptide includes or consists of the amino acid sequence of SEQ
ID NOs: 1204 or 1221, and the amino acid sequence of the second
polypeptide includes or consists of the amino acid sequence of SEQ
ID NOs: 504, 512, 530, or 572.
[0091] In some embodiments, the first polypeptide and/or the second
polypeptide are in dimeric form. In some embodiments, the first
polypeptide and/or the second polypeptide are glycosylated or
pegylated.
[0092] In some embodiments, the composition is a pharmaceutical
composition including a pharmaceutically acceptable excipient,
e.g., saline. In some embodiments, the composition is
lyophilized.
[0093] In some embodiments, the first polypeptide is present in a
dosage between about 0.2 mg/kg to about 20 mg/kg and the second
polypeptide is present in a dosage between about 0.5 mg/kg to about
500 mg/kg. In some embodiments, the first polypeptide is present in
a dosage between about 0.2 mg/kg to about 20 mg/kg, e.g., about 0.5
mg/kg to about 10 mg/kg, e.g., about 1 mg/kg to about 5 mg/kg, for
once daily, twice daily, three times daily, four times daily, once
weekly, twice weekly, three times weekly, or four times weekly
administration, e.g., about 1 mg/kg, about 2 mg/kg, or about 3
mg/kg for once daily, twice daily, four times daily, once weekly,
twice weekly, or three times weekly administration; and the second
polypeptide is present in a dosage between about 0.5 mg/kg to about
500 mg/kg, e.g., about 5 mg/kg to about 200 mg/kg, e.g., about 10
mg/kg to about 100 mg/kg, e.g., about 20 mg/kg to about 40 mg/kg,
for once daily, twice daily, three times daily, four times daily,
once weekly, twice weekly, three times weekly, or four times weekly
administration, e.g., about 10 mg/kg, about 30 mg/kg, or about 100
mg/kg, for once daily, twice daily, three times daily, once weekly,
twice weekly, or three times weekly administration.
[0094] In some embodiments, the amino acid sequence of the first
polypeptide includes the amino acid sequence of SEQ ID NO: 1204 and
the amino acid sequence of the second polypeptide includes the
amino acid sequence of SEQ ID NO: 512.
[0095] In a fourth aspect, the invention features a method of
treating a disease or a condition in a subject, the method
including administering to the subject a therapeutically effective
amount of a first polypeptide and a second polypeptide, where a)
the first polypeptide includes the structure A-sALP-B, where i)
sALP is the extracellular domain of an alkaline phosphatase, ii) A
is absent or is an amino acid sequence of at least one amino acid,
and iii) B is absent or is an amino acid sequence of at least one
amino acid; and b) the second polypeptide includes the structure
V-NP-W, where i) NP is a natriuretic peptide that is an agonist of
natriuretic peptide receptor B (NPR-B), ii) V is absent or is an
amino acid sequence of at least one amino acid, and iii) W is
absent or is an amino acid sequence of at least one amino acid; and
the disease or the condition is selected from the group consisting
of a neurocutaneous syndrome, a disorder associated with
overactivation of FGFR3, a bone or cartilage disorder, a vascular
smooth muscle disorder, and a condition for elongation of bone. In
some embodiments, the disease or the condition in the subject is
thereby treated.
[0096] In some embodiments, the first polypeptide is any
polypeptide described herein including sALP, e.g., any sALP
polypeptide described herein, e.g., as described for the first or
third aspect. In some embodiments, the amino acid sequence of the
first polypeptide includes an amino acid sequence having at least
80%, 85%, 90%, 95%, or 99% sequence identity of any one of SEQ ID
NOs: 1201, 1204, 1220, or 1221, e.g., SEQ ID NO: 1204. In some
embodiments, the amino acid sequence of the first polypeptide
includes or consists of the amino acid sequence of SEQ ID NO:
1204.
[0097] In some embodiments, the second polypeptide is any
polypeptide described herein including NP, e.g., any NP polypeptide
described herein, e.g., as described for the second or third
aspect. In some embodiments, the amino acid sequence of the second
polypeptide includes an amino acid sequence having at least 80%,
85%, 90%, 95%, or 99% sequence identity to any one of SEQ ID NOs:
504, 512, 530, 554, 572, or 578, e.g., SEQ ID NO: 512. In some
embodiments, the amino acid sequence of the second polypeptide
includes or consists of the amino acid sequence of SEQ ID NO:
512.
[0098] In some embodiments, the first polypeptide and the second
polypeptide are administered within ten days, five days, or
twenty-four hours of each other, e.g., within ten, nine, eight,
seven, six, five, four, three, or two days of each other or within
twenty-four, twelve, eleven, ten, nine, eight, seven, six, five,
four, three, two, or one hour(s) of each other.
[0099] In some embodiments, the first polypeptide and the second
polypeptide are administered simultaneously. In some embodiments,
the first polypeptide and the second polypeptide are formulated
together in a composition or each separately in a composition. In
some embodiments, the composition is a pharmaceutical composition
comprising a pharmaceutically acceptable excipient, e.g., saline.
In some embodiments, the composition is lyophilized. In some
embodiments, the composition is any composition, e.g., a
pharmaceutical composition, described herein, e.g., as described
for the first, second, or third aspect.
[0100] In a fifth aspect, the invention features a kit including:
a) a first polypeptide including the structure A-sALP-B, where i)
sALP is the extracellular domain of an alkaline phosphatase, ii) A
is absent or is an amino acid sequence of at least one amino acid,
and iii) B is absent or is an amino acid sequence of at least one
amino acid; and b) instructions for administering the first
polypeptide to a patient diagnosed with or at risk of developing a
neurocutaneous syndrome. In some embodiments, the first polypeptide
is any polypeptide described herein including sALP, e.g., any sALP
polypeptide described herein, e.g., as described in any of the
above aspects.
[0101] In some embodiments, the kit further includes (c) a second
polypeptide including the structure V-NP-W, where i) NP is a
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), ii) V is absent or is an amino acid sequence of
at least one amino acid, and iii) W is absent or is an amino acid
sequence of at least one amino acid. In some embodiments, the
second polypeptide is any polypeptide described herein including
NP, e.g., any NP polypeptide described herein, e.g., as described
in any of the above aspects.
[0102] In a sixth aspect, the invention features a kit including:
a) a polypeptide including the structure V-NP-W, where i) NP is a
natriuretic peptide that is an agonist of natriuretic peptide
receptor B (NPR-B), ii) V is absent or is an amino acid sequence of
at least one amino acid, and iii) W is absent or is an amino acid
sequence of at least one amino acid; and b) instructions for
administering the polypeptide to a patient diagnosed with or at
risk of developing a neurocutaneous syndrome. In some embodiments,
the polypeptide is any polypeptide described herein including NP,
e.g., any NP polypeptide described herein, e.g., as described in
any of the above aspects.
[0103] In a seventh aspect, the invention features a kit including:
a) a first polypeptide including the structure A-sALP-B, where i)
sALP is the extracellular domain of an alkaline phosphatase, ii) A
is absent or is an amino acid sequence of at least one amino acid,
and iii) B is absent or is an amino acid sequence of at least one
amino acid; and b) a second polypeptide including the structure
V-NP-W, where i) NP is a natriuretic peptide that is an agonist of
natriuretic peptide receptor B (NPR-B), ii) V is absent or is an
amino acid sequence of at least one amino acid, and iii) W is
absent or is an amino acid sequence of at least one amino acid. In
some embodiments, the first polypeptide is any polypeptide
described herein including sALP, e.g., any sALP polypeptide
described herein, e.g., as described in any of the above aspects;
and the second polypeptide is any polypeptide described herein
including NP, e.g., any NP polypeptide described herein, e.g., as
described in any of the above aspects. In some embodiments, the
first polypeptide and the second polypeptide are formulated
together. In some embodiments, the first polypeptide and the second
polypeptide are formulated separately and in individual dosage
amount.
[0104] In any of the aspects described herein, the amino acid
sequence of the sALP includes an amino acid sequence having at
least 80%, 85%, 90%, 95%, or 99% sequence identity to any one of
SEQ ID NOs: 1202, 1205, 1218, or 1219.
[0105] In any of the aspects described herein, the amino acid
sequence of the sALP includes or consists of an amino acid sequence
having at least 80%, 85%, 90%, 95%, or 99% sequence identity to
amino acid residues 23-508 of SEQ ID NO: 1215, amino acid residues
18-498 of SEQ ID NO: 1216, amino acid residues 23-508 of SEQ ID NO:
1218, amino acid residues 18-498 of SEQ ID NO: 1219, amino acid
residues 23-512 of SEQ ID NO: 1215, amino acid residues 18-502 of
SEQ ID NO: 1216, amino acid residues 23-512 of SEQ ID NO: 1218, or
amino acid residues 18-502 of SEQ ID NO: 1219, where X in SEQ ID
NO: 1218 or 1219 is any amino acid but is not an amino acid
corresponding to a pathogenic mutation at that position of human
TNALP, e.g., not an amino acid corresponding to a pathogenic
mutation provided in Table 1.
[0106] In any of the aspects described herein, the amino acid
sequence of the NP includes or consists of amino acid residues 6-22
of SEQ ID NO: 126, and the amino acid at position 17 of SEQ ID NO:
126 is Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro, Asp, Gly, Ala,
Ser, Val, Trp, Asn, Gln, His, or Lys, e.g., Phe, Leu, Ile, Thr,
Val, Ala, Ser, Glu, Arg, Tyr, Cys, Pro, or Asp, e.g., Phe, Leu,
Ile, Thr, Val, Ala, or Ser, e.g., e.g., Phe, Leu, Arg, Tyr, e.g.,
Phe or Leu, e.g., Phe, e.g., Leu.
[0107] In any of the aspects described herein, the amino acid
sequence of the NP comprises of consists of the amino acid sequence
of any one of SEQ ID NOs: 13-29, 100-116, 119-125, 127-233, or
1001-1155.
[0108] In any of the aspects described herein, the NP is selective
for NPR-B over NPR-A, where the EC.sub.50(NPR-A)/EC.sub.50(NPR-B)
ratio for the NP, as determined in an in vivo pharmacokinetic
assay, is at least 30, e.g., at least 35, 40, 35, 50, 55, or
60.
[0109] In any of the aspects described herein, the Fc includes a
C.sub.H2 domain, a C.sub.H3 domain, and a hinge region, or where
the Fc is a constant domain of an immunoglobulin selected from the
group consisting of IgG-1, IgG-2, IgG-3, and IgG-4, e.g., IgG-1. In
some embodiments, the amino acid sequence of the Fc includes an
amino acid sequence having at least 80%, 85%, 90%, 95%, or 99%
sequence identity to SEQ ID NO: 401. In some embodiments, the amino
acid sequence of the Fc includes or consists of SEQ ID NO: 401.
[0110] In any of the aspects described herein, the amino acids
sequence of D or Y includes a glycine-rich region, or the amino
acid sequence of D or Y consists of one or more glycines and one or
more serines. For example, the amino acid sequence of D or Y may
include or consist of [(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, where each of m, n, and p is,
independently, between 0 and 20. In some embodiments, m is between
1 and 6; n is between 1 and 10; and p is between 0 and 4, e.g., m
is 4 and n is 1-6. In some embodiments, combinations of m, n, and p
are selected from a single row of Table 2, or the amino acid
sequence of D or Y includes or consists of the amino acid sequence
of any one of SEQ ID NOs: 301-391.
[0111] In any of the aspects described herein, the amino acids
sequence of D, G, or Y is optionally two amino acid residues (e.g.,
leucine-lysine or aspartic acid-isoleucine). In some embodiments of
an sALP polypeptide, C and E may both be absent, and D may be
absent or may be an amino acid sequence of at least one amino acid.
For example, the polypeptide may consist of the structure sALP-D-Fc
or the structure Fc-D-sALP, where D may be any linker described
herein or may optionally consist of two amino acid residues, e.g.,
leucine-lysine. For example, the polypeptide may consist of the
structure sALP-D-Fc. Optionally, the amino acid sequence of sALP is
the amino acid sequence of SEQ ID NO: 1205, the amino acid sequence
of D is leucine-lysine, and/or the amino acid sequence of Fc is the
amino acid sequence of SEQ ID NO: 401. In other embodiments of an
sALP polypeptide, C may be absent, and D and E may both be absent
or may be an amino acid sequence of at least one amino acid. For
example, the polypeptide may include the structure sALP-D-Fc-E or
the structure Fc-D-sALP-E. In other embodiments of an sALP
polypeptide, C may be absent, and D, G, and H may all be absent or
may be an amino acid sequence of at least one amino acid. For
example, the polypeptide may include the structure
sALP-D-Fc-G-I.sub.n-H. In some embodiments of an NP polypeptide, X
and Z may both be absent, and Y may be absent or may be an amino
acid sequence of at least one amino acid. For example, the
polypeptide may consist of the structure NP-Y-Fc or the structure
Fc-Y-NP, where Y may be any linker described herein or may
optionally consist of two amino acid residues, e.g.,
leucine-lysine. For example, the polypeptide may consist of the
structure NP-Y-Fc.
[0112] In any of the aspects described herein, one or more of A, B,
C, D, E, G, H, V, W, X, Y, or Z are absent. In some embodiments, A
is absent, B is absent, or A and B are both absent. In some
embodiments, C is absent, E is absent, or C and E are both absent.
In some embodiments, C is absent, H is absent, or C and H are both
absent. In some embodiments, X is absent, Z is absent, or X and Z
are both absent.
[0113] In any of the aspects described herein, the polypeptide of
the invention, e.g., the sALP polypeptide and/or the NP
polypeptide, may include a bone-targeting moiety, e.g., including
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive acidic
residues, e.g., aspartic acid or glutamic acid, e.g., E.sub.6,
E.sub.7, E.sub.8, E.sub.9, E.sub.10, E.sub.11, E.sub.12, E.sub.13,
E.sub.14, E.sub.15, E.sub.16, D.sub.6, D.sub.7, D.sub.8, D.sub.9,
D.sub.10, D.sub.11, D.sub.12, D.sub.13, D.sub.14, D.sub.15, or
D.sub.16, e.g., E.sub.6, E.sub.10, D.sub.6, or D.sub.10. In some
embodiments of any of the above aspects, one or more of A, B, C, D,
E, V, W, X, Y, or Z includes a bone-targeting moiety, e.g.,
including 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive
acidic residues, e.g., aspartic acid or glutamic acid, e.g.,
E.sub.6, E.sub.7, E.sub.8, E.sub.9, E.sub.10, E.sub.11, E.sub.12,
E.sub.13, E.sub.14, E.sub.15, E.sub.16, D.sub.6, D.sub.7, D.sub.8,
D.sub.9, D.sub.10, D.sub.11, D.sub.12, D.sub.13, D.sub.14,
D.sub.15, or D.sub.16, e.g., E.sub.6, E.sub.10, D.sub.6, or
D.sub.10.
[0114] In any of the aspects described herein, the polypeptide of
the invention optionally does not include a bone-targeting moiety
(e.g., a polyaspartic acid or polyglutamic acid region longer than
two consecutive aspartic acid or glutamic acid residues).
[0115] In any of the aspects described herein, the polypeptide of
the invention may include a cathepsin (e.g., cathepsin K) cleavage
sequence, e.g., HGPQG (SEQ ID NO: 374) or HKLRG (SEQ ID NO: 375).
In some embodiments of any of the above aspects, A, B, C, D, E, G,
H, V, W, X, Y, or Z includes a cathepsin (e.g., cathepsin K)
cleavage sequence.
[0116] In some embodiments of any of the above aspects, the
polypeptide of the invention may include a polypeptide having
reduced (e.g., by about 20%, about 25%, about 30%, about 35%, about
40%, about 45%, about 50%, about 55%, about 60%, about 65%, about
70%, about 75%, about 80%, about 85%, about 90%, about 95%, about
96%, about 97%, about 98%, about 99%, or about 100%) degradation
(e.g., by neutral endopeptidase (NEP), insulin degrading enzyme
(IDE), or any other enzyme that cleaves a natriuretic peptide in
vivo), as compared to a control (e.g., CNP22, CNP53, or any
polypeptide described herein, such as a peptide described in
International Application Pub. No. WO2010/135541 or U.S.
Application Pub. No. 2010-0331256).
[0117] In some embodiments of any of the above aspects, the
polypeptide of the invention may have increased (e.g., by about
20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 55%, about 60%, about 65%, about 70%, about 75%, about
80%, about 85%, about 90%, about 95%, about 96%, about 97%, about
98%, about 99%, about 100%, or more) efficacy and/or reduced (e.g.,
by about 20%, about 25%, about 30%, about 35%, about 40%, about
45%, about 50%, about 55%, about 60%, about 65%, about 70%, about
75%, about 80%, about 85%, about 90%, about 95%, about 96%, about
97%, about 98%, about 99%, or about 100%) dose-dependent side
effects (e.g., decreased adverse hemodynamic effects, such as
decreased lowering of blood pressure), as compared to a control
(e.g., any polypeptide described herein, such as a peptide
described in International Application Pub. No. WO2010/135541 or
U.S. Application Pub. No. 2010-0331256).
[0118] In any of the aspects described herein, the polypeptide of
the invention (e.g., an sALP polypeptide or an NP polypeptide) is
glycosylated or pegylated. In some embodiments, the pharmaceutical
composition includes a dimer of one or more of the polypeptides of
the invention. In some embodiments, the pharmaceutically acceptable
excipient includes saline. In some embodiments, the pharmaceutical
composition is lyophilized. In some embodiments, the pharmaceutical
composition is administered subcutaneously, intravenously, orally,
nasally, intramuscularly, sublingually, intrathecally, or
intradermally, e.g., subcutaneously.
[0119] In some embodiments, the pharmaceutical composition is
administered to the subject in a dosage between about 0.5 mg/kg to
about 500 mg/kg once, twice, three times, or four times daily. The
dosage may be between, e.g., about 5 mg/kg to about 200 mg/kg,
e.g., about 10 mg/kg to about 100 mg/kg, once, twice, three times,
or four times daily. In some embodiments, the dosage is about 10
mg/kg or about 100 mg/kg twice daily.
[0120] In some embodiments, the pharmaceutical composition is
administered to the subject in a dosage between about 0.5 mg/kg to
about 500 mg/kg once or twice weekly. The dosage may be between,
e.g., about 5 mg/kg to about 200 mg/kg, e.g., about 10 mg/kg to
about 100 mg/kg, e.g., about 20 mg/kg to about 40 mg/kg, once or
twice weekly. In some embodiments, the dosage is about 10 mg/kg,
about 30 mg/kg, or about 100 mg/kg, once or twice weekly.
[0121] In some embodiments, the pharmaceutical composition is
administered to the subject in a dosage between about 10 .mu.g/kg
to about 1,000 .mu.g/kg once or twice weekly. The dosage may be
between, e.g., about 20 .mu.g/kg to about 800 .mu.g/kg, e.g., about
30 .mu.g/kg to about 600 .mu.g/kg, e.g., about 50 .mu.g/kg to about
500 .mu.g/kg, e.g., about 100 .mu.g/kg to about 400 .mu.g/kg, e.g.,
about 200 .mu.g/kg to about 300 .mu.g/kg, once or twice weekly. In
some embodiments, the dosage is about 30 .mu.g/kg, about 100
.mu.g/kg, about 300 .mu.g/kg, or about 500 .mu.g/kg, once or twice
weekly.
[0122] In any of the aspects described herein, the pharmaceutical
composition is administered to the subject in a dosage between
about 0.1 mg/kg to about 500 mg/kg of one or more of the
polypeptides of the invention in a dosage regimen of once, twice,
three times, or four times daily. The dosage may be between, e.g.,
about 1 mg/kg to about 200 mg/kg, e.g., about 2 mg/kg to about 100
mg/kg or about 10 mg/kg to about 100 mg/kg of one or more of the
polypeptides of the invention in a dosage regimen of once, twice,
three times, or four times daily. In some embodiments, the dosage
is about 10 mg/kg or about 100 mg/kg of one or more of the
polypeptides of the invention in a dosage regimen of twice daily.
For example, the methods of the invention may optionally include
administering a pharmaceutical composition including the
polypeptides of the invention (e.g., an sALP polypeptide and/or an
NP polypeptide) to the subject in a dosage of about 0.5 mg/kg/day
to about 10 mg/kg/day (e.g., about 2 mg/kg/day to about 3
mg/kg/day).
[0123] In any of the aspects described herein, the pharmaceutical
composition is administered to the subject between one and fourteen
times a week, or is administered at least once daily for at least
one month. In some embodiments, the pharmaceutical composition is
administered to the subject once weekly for at least one month. In
some embodiments, the pharmaceutical composition is administered
one time, two times, three times, or four times a week, e.g., three
times a week.
[0124] Any of the pharmaceutical compositions of the invention may
optionally be formulated for treating a disease or condition (e.g.,
any described herein) in a subject.
[0125] Any of the pharmaceutical composition of the invention
featuring an isolated nucleic acid may optionally include a
recombinant expression vector (e.g., a lentiviral vector) including
the isolated nucleic acid. In some embodiments, the pharmaceutical
composition includes about 0.1 mg to about 10 mg of the isolated
nucleic acid.
[0126] In any embodiment described herein, the polypeptide may or
may not be isolated.
[0127] In any embodiment described herein, the subject may be
human.
[0128] In any of the aspects described herein, the combination of
two or more polypeptides of the invention (e.g., any sALP
polypeptide and/or any NP polypeptide described herein) or two or
more compositions of the invention provides a synergistic effect.
In particular embodiments, the synergistic effect is a therapeutic
effect that is observed for the combination of two or more
polypeptides of the invention, wherein one or more of the
polypeptides of the invention is present at a dose that is normally
non-therapeutic; or a therapeutic effect that results in an
unexpected decrease in one or more adverse events (e.g.,
hemodynamic effects, such as a decrease in blood pressure, such as
systolic arterial blood pressure, diastolic arterial blood
pressure, or mean arterial blood pressure, that results in adverse
hypotensive effects); or a therapeutic effect that results in
reduced dose-dependent side effects, as compared to the level of
dose-dependent side effects observed for a single polypeptide of
the invention at a therapeutic dose.
[0129] In some embodiments of any of the methods described herein,
the disease is the neurocutaneous syndrome (e.g., neurofibromatosis
(e.g., classic von Recklinghausen type (type I), either with
gastrointestinal stromal tumors (i.e., as in intestinal
neurofibromatosis (type 3B)) or without such tumors; an acoustic
neuroma type (type II); a mixed type that combines the features of
types I and II with predominant features, such as bilateral
acoustic neuromas, posterior fossa and upper cervical meningiomas,
and spinal/paraspinal neurofibromas (type III, Riccardi type or
type 3A); an atypical type that is distinguished from by the lack
of iris Lisch nodules that are characteristic of type I (type VI);
segmental neurofibromatosis, which is a variant of type I having
lesions affecting a specific area of the body, such as a single
segment of the body or an area that crosses the midline (type V); a
type having only the symptoms of cafe au lait spots without other
manifestations of neurofibromatosis (type VI); familial spinal
neurofibromatosis, which is caused by mutation in the neurofibromin
gene NF1 and considered a distinguishable variant of type I; other
variants of type I, such as
neurofibromatosis-pheochromocytoma-duodenal carcinoid syndrome;
neurofibromatosis with manifestations of Noonan syndrome, such as
short stature, ptosis, midface hypoplasia, webbed neck, learning
disabilities, and muscle weakness; and schwannomatosis, where any
of these disorders can include or exclude one or more bone
manifestations); tuberous sclerosis; Sturge-Weber disease; ataxia
telangiectasia; von Hippel-Lindau disease; incontinentia pigmenti;
epidermal nevus syndromes, such as linear sebaceous nevus of
Jadassohn; nevoid basal cell carcinoma syndrome; hypomelanosis of
Ito; neurocutaneous melanosis; Klippel-Ternaunay syndrome; and
Waardenburg syndrome, including types I, II, III, and IV). In some
embodiments, the neurocutaneous syndrome has one or more bone
manifestations. In some embodiments, the neurocutaneous syndrome is
neurofibromatosis type I. In some embodiments of any of the methods
described herein, the disease is any syndrome (e.g., a
neurocutaneous syndrome) with overactivated RAS and/or ERK
signaling (e.g., Noonan syndrome, Costello syndrome, Noonan
syndrome with multiple lentigines/LEOPARD syndrome,
neurofibromatosis type 1, hereditary gingival fibromatosis type 1,
NF1-Noonan syndrome, capillary malformation-AV malformation
syndrome, Legius syndrome, Noonan syndrome-like disorder with loose
anagen hair, Noonan syndrome-like disorder with juvenile
myelomonocytic leukemia (JMML), cardio-facio-cutaneous syndrome, or
autoimmune lymphoproliferative syndrome, where any of these
disorders can include or exclude one or more bone manifestations).
In some embodiments of any of the methods described herein, the
disorder associated with overactivation of FGFR3 is a bone or
cartilage disorder, e.g., a skeletal dysplasia, such as any
described herein, e.g., achondroplasia or craniosynostosis. In some
embodiments of any of the methods described herein, the disorder is
a bone or cartilage disorder, e.g., a skeletal dysplasia, such as
any described herein. In some embodiments, the bone or cartilage
disorder is a skeletal dysplasia, e.g., achondroplasia, homozygous
achondroplasia, heterozygous achondroplasia, achondrogenesis,
acrodysostosis, acromesomelic dysplasia, atelosteogenesis,
camptomelic dysplasia, chondrodysplasia punctata, rhizomelic type
of chondrodysplasia punctata, cleidocranial dysostosis, congenital
short femur, craniosynostosis (e.g., Muenke syndrome, Crouzon
syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer
syndrome, or Crouzonodermoskeletal syndrome), dactyly,
brachydactyly, camptodactyly, polydactyly, syndactyly, diastrophic
dysplasia, dwarfism, dyssegmental dysplasia, enchondromatosis,
fibrochondrogenesis, fibrous dysplasia, hereditary multiple
exostoses, hypochondroplasia, hypophosphatasia, hypophosphatemic
rickets, Jaffe-Lichtenstein syndrome, Kniest dysplasia, Kniest
syndrome, Langer-type mesomelic dysplasia, Marfan syndrome,
McCune-Albright syndrome, micromelia, metaphyseal dysplasia,
Jansen-type metaphyseal dysplasia, metatrophic dysplasia, Morquio
syndrome, Nievergelt-type mesomelic dysplasia, neurofibromatosis
(e.g., type 1, e.g., with bone manifestations or without bone
manifestations; type 2; schwannomatosis; or any described herein),
osteoarthritis, osteochondrodysplasia, osteogenesis imperfecta,
perinatal lethal type of osteogenesis imperfecta, osteopetrosis,
osteopoikilosis, peripheral dysostosis, Reinhardt syndrome, Roberts
syndrome, Robinow syndrome, short-rib polydactyly syndromes, short
stature, spondyloepiphyseal dysplasia congenita,
spondyloepimetaphyseal dysplasia, or thanatophoric dysplasia. In
some embodiments, the bone or cartilage disorder is optionally
hypophosphatasia (e.g., infantile HPP, childhood HPP, perinatal
HPP, adult HPP, or odontohypophosphatasia). In some embodiments,
the pharmaceutical composition is administered in an amount that is
therapeutically effective to treat an achondroplasia phenotype
selected from the group consisting of growth retardation, skull
deformities, and orthodontic defects. In some embodiments, the
pharmaceutical composition is administered in an amount that is
therapeutically effective to treat an achondroplasia phenotype
selected from the group consisting of cervical cord compression,
spinal stenosis, hydrocephalus, hearing loss due to chronic otitis,
cardiovascular disease, neurological disease, and obesity. In some
embodiments of any of the methods described herein, the disorder
associated with overactivation of FGFR3 is cancer, e.g., multiple
myeloma, myeloproliferative syndrome, leukemia, plasma cell
leukemia, lymphoma, glioblastoma, prostate cancer, bladder cancer,
or mammary cancer. In some embodiments of any of the methods
described herein, the vascular smooth muscle disorder is
hypertension, restenosis, arteriosclerosis, acute decompensated
heart failure, congestive heart failure, cardiac edema, nephredema,
hepatic edema, acute renal insufficiency, or chronic renal
insufficiency. In some embodiments of any of the methods described
herein, the condition for elongation of bone is insufficient or
impaired bone growth arising from fractures, renal failure or
insufficiency, poor diet, vitamin deficiency, hormone deficiency,
or any skeletal dysplasia described herein.
[0130] In any method described herein, the neurocutaneous syndrome,
disorder associated with overactivation of FGFR3, bone or cartilage
disorder, vascular smooth muscle disorder, or condition for
elongation of bone in the subject is thereby treated.
[0131] In some embodiments of any of the methods described herein,
one or more polypeptides of the invention or one or more
compositions of the invention is optionally administered in an
amount that is therapeutically effective to treat a HPP phenotype
selected from the group consisting of HPP-related seizure,
premature loss of deciduous teeth, incomplete bone mineralization,
elevated blood and/or urine levels of inorganic pyrophosphate
(PPi), elevated blood and/or urine levels of phosphoethanolamine
(PEA), elevated blood and/or urine levels of pyridoxal 5'-phosphate
(PLP), inadequate weight gain, rickets, bone pain, calcium
pyrophosphate dihydrate crystal deposition, aplasia, hypoplasia,
and dysplasia of the dental cementum. In some embodiments, the
incomplete bone mineralization is incomplete femoral bone
mineralization, incomplete tibial bone mineralization, incomplete
metatarsal bone mineralization, or incomplete rib bone
mineralization.
Definitions
[0132] As used herein, the term "about" means.+-.10% of the recited
value.
[0133] By "area under the curve" or "AUC" in the context of an in
vivo pharmacokinetic assay is meant the area under the serum
concentration vs. time curve after administration in an animal.
[0134] By "bone or cartilage disorder" is meant any disorder,
disease, or other abnormality that affects the function, structure,
or growth of bone or cartilage.
[0135] By "bone-targeting moiety" is meant an amino acid sequence
of between 6 and 20 amino acid residues in length having a
sufficient affinity to the bone matrix such that the bone-targeting
moiety, taken alone, has an in vivo binding affinity to the bone
matrix that is at least 10.sup.-6 M or better (e.g., 10.sup.-7 M,
10.sup.-8 M, 10.sup.-9 M, or better).
[0136] By "cathepsin cleavage sequence" is meant an amino acid
sequence having a site that can be cleaved by cathepsin with a
k.sub.cat/K.sub.M rate constant of at least 10.sup.3
M.sup.-1s.sup.-1 (e.g., 10.sup.4 M.sup.-1s.sup.-1, 10.sup.5
M.sup.-1s.sup.-1, 106 M.sup.-1s.sup.-1, 10.sup.7 M.sup.-1, or
10.sup.8 M.sup.-1s.sup.-1) at 30.degree. C. or higher (e.g.,
37.degree. C.). In particular embodiments, the cathepsin cleavage
sequence is specific for cathepsin K. Exemplary cathepsin cleavage
sequences are P2-P1-P1', where cleavage by the enzyme would occur
at the P1-P1' peptide bond; P2 is preferentially composed of Pro,
Leu, Ile, but could also be Val, Norleucine, Met, or Ala; P1 is
preferentially Arg, Lys, Gln, but could also be Met, Norleucine,
Leu, Ile, or Thr; and P1' can be any amino acid but is
preferentially Gly. Additional cathepsin cleavage sequences are
provided in Choe et al., J. Biol. Chem. 281(18):12824-832, 2006,
which is incorporated herein by reference.
[0137] By "CNP22" is meant human CNP22 (SEQ ID NO: 4), unless a
different meaning is expressly indicated.
[0138] By "CNP53" is meant human CNP53 (SEQ ID NO: 11), unless a
different meaning is expressly indicated.
[0139] By "condition for elongation of bone" is meant any disorder,
disease, or other abnormality that would benefit from lengthening
of one or more segments of bone. After administration of any
polypeptide described herein, the lengthening of one or more
segments of bone can be increased by more than about 1%, about 2%,
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 55%, about 60%,
about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,
about 95%, about 96%, about 97%, about 98%, about 99%, or about
100%, or more.
[0140] By "disorder associated with overactivation of FGFR3" is
meant any disorder, disease, or other abnormality that is caused
by, or is associated with, overactivation of FGFR3, e.g., stemming
from a gain-of-function FGFR3 mutation.
[0141] By "efficacy" is meant the E.sub.max value of a compound in
a dose-response assay.
[0142] By "Fc" is meant a fragment crystallizable region of an
immunoglobulin, e.g., IgG-1, IgG-2, IgG-3, IgG-3 or IgG-4,
including the C.sub.H2 and C.sub.H3 domains of the immunoglobulin
heavy chain. Fc may also include any portion of the hinge region
joining the Fab and Fc regions. The Fc can be of any mammal,
including human, and may be post-translationally modified (e.g., by
glycosylation). In a non-limiting example, Fc can be the fragment
crystallizable region of human IgG-1 having the amino acid sequence
of SEQ ID NO: 401.
[0143] By "fragment" is meant a portion of a polypeptide or nucleic
acid molecule that contains, preferably, at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
more of the entire length of the reference nucleic acid molecule or
polypeptide. A fragment may contain, e.g., 10, 15, 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,
270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390,
400, 500, 600, 700, 800, 900, 1,000, 1100, 1200, 1300, 1400, 1500,
1600, 1700, 1800, 1900, 2000, 2100, or more nucleotides, up to the
entire length of the nucleic acid molecule, or 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,
130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,
260, 270, 280, 290, 300, 400, 500, 600, 700, or more amino acid
residues, up to the entire length of the polypeptide. Exemplary
sALP fragments have amino acid residues 18-498, 18-499, 18-500,
18-501, 18-502, 18-503, 18-504, 18-505, 18-506, 18-507, 18-508,
18-509, 18-510, 18-511, or 18-512 of an consensus sequence for ALP
(e.g., SEQ ID NOs: 1215, 1216, 1218, or 1219), and may include
additional N-terminal and/or C-terminal portions. Exemplary NP
fragments have at least a consensus ring domain, e.g., of SEQ ID
NOs: 6, 30, or 95, and may include additional N-terminal and/or
C-terminal portions.
[0144] By "homolog" is meant a polypeptide or nucleic acid molecule
exhibiting at least 50% identity to a reference amino acid sequence
or nucleic acid sequence. Such a sequence is generally at least,
e.g., 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identical at the amino acid level or nucleic acid to a reference
sequence. In general, for polypeptides, the length of comparison
sequences can be at least five amino acid residues, e.g., 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400,
500, 600, 700, or more amino acid residues, up to the entire length
of the polypeptide. For nucleic acids, the length of comparison
sequences can generally be at least 10, 20, 30, 40, 50, 60, 70, 80,
90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,
2000, 2100, or more nucleotides, up to the entire length of the
nucleic acid molecule. It is understood that for the purposes of
determining sequence identity when comparing a DNA sequence to an
RNA sequence, a thymine nucleotide is equivalent to a uracil
nucleotide.
[0145] As used herein, when a polypeptide or nucleic acid sequence
is referred to as having "at least X % sequence identity" to a
reference sequence, it is meant that at least X percent of the
amino acid residues or nucleotides in the polypeptide or nucleic
acid are identical to those of the reference sequence when the
sequences are optimally aligned. An optimal alignment of sequences
can be determined in various ways that are within the skill in the
art, for instance, the Smith Waterman alignment algorithm (Smith et
al., J. Mol. Biol. 147:195-7, 1981) and BLAST (Basic Local
Alignment Search Tool; Altschul et al., J. Mol. Biol. 215: 403-10,
1990). These and other alignment algorithms are accessible using
publicly available computer software such as "Best Fit" (Smith and
Waterman, Advances in Applied Mathematics, 482-489, 1981) as
incorporated into GeneMatcher Plus.TM. (Schwarz and Dayhof, Atlas
of Protein Sequence and Structure, Dayhoff, M. O., Ed pp 353-358,
1979), BLAST, BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2,
ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR). In addition, those
skilled in the art can determine appropriate parameters for
measuring alignment, including any algorithms needed to achieve
optimal alignment over the length of the sequences being
compared.
[0146] By "hybridize" is meant to pair to form a double-stranded
molecule between complementary polynucleotides, or portions
thereof, under various conditions of stringency. (See, e.g., Wahl,
G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.
R. (1987) Methods Enzymol. 152:507.) For example, high stringency
salt concentration will ordinarily be less than about 750 mM NaCl
and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM
trisodium citrate, or less than about 250 mM NaCl and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in
the absence of organic solvent, e.g., formamide, while high
stringency hybridization can be obtained in the presence of at
least about 35% formamide or at least about 50% formamide. High
stringency temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., 37.degree. C., or
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In
one embodiment, hybridization will occur at 30.degree. C. in 750 mM
NaCl, 75 mM trisodium citrate, and 1% SDS. In an alternative
embodiment, hybridization will occur at 37.degree. C. in 500 mM
NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100
.mu.g/ml denatured salmon sperm DNA (ssDNA). In a further
alternative embodiment, hybridization will occur at 42.degree. C.
in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and
200 .mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0147] For most applications, washing steps that follow
hybridization will also vary in stringency. Wash stringency
conditions can be defined by salt concentration and by temperature.
As above, wash stringency can be increased by decreasing salt
concentration or by increasing temperature. For example, high
stringency salt concentrations for the wash steps may be, e.g.,
less than about 30 mM NaCl and 3 mM trisodium citrate, or less than
about 15 mM NaCl and 1.5 mM trisodium citrate. High stringency
temperature conditions for the wash steps will ordinarily include a
temperature of, e.g., at least about 25.degree. C., 42.degree. C.,
or 68.degree. C. In one embodiment, wash steps will occur at
25.degree. C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS.
In an alternative embodiment, wash steps will occur at 42.degree.
C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a
further alternative embodiment, wash steps will occur at 68.degree.
C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.
Additional variations on these conditions will be readily apparent
to those skilled in the art. Hybridization techniques are well
known to those skilled in the art and are described, for example,
in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness
(Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al.
(Current Protocols in Molecular Biology, Wiley Interscience, New
York, 2001); Berger and Kimmel (Guide to Molecular Cloning
Techniques, 1987, Academic Press, New York); and Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, New York.
[0148] By "isolated" or "purified" is meant separated from other
naturally accompanying components. Typically, a compound (e.g.,
polypeptide, nucleic acid, or small molecule), factor, cell, or
other component is considered isolated when it is at least, e.g.,
50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or even 99%,
by weight, free from proteins, antibodies, naturally-occurring
organic molecules, and other components with which it is naturally
associated. In some instances, the component is at least 75%, 90%,
or even 99%, by weight, pure. An isolated component may be obtained
by chemical synthesis, separation of the factor from natural
sources, or production of the component in a recombinant host cell
that does not naturally produce the component. Proteins and small
molecules may be purified by one skilled in the art using standard
techniques such as those described by Ausubel et al. (Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
2000). The component is preferably at least, e.g., 2, 5, or 10
times as pure as the starting material, as measured using, e.g.,
polyacrylamide gel electrophoresis, column chromatography, optical
density, HPLC analysis, or Western analysis (Ausubel et al.,
supra). Exemplary methods of purification are column
chromatography, immunoprecipitation, and magnetic bead
immunoaffinity purification.
[0149] By "natriuretic peptide that is an agonist of natriuretic
peptide receptor B" (abbreviated "NP") is meant a natriuretic
peptide as described herein, e.g., human CNP22 (SEQ ID NO: 4), or
variant thereof, that is capable of agonizing NPR-B, e.g., human
NPR-B, with at least 0.000001, 0.000005, 0.00001, 0.00005, 0.0001,
0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 0.9, or 1 times the
potency, and at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 95%, or even 100% times the efficacy of CNP22
as measured in a standard NPR-B activation assay, e.g., a membrane
assay or whole cell assay, as described herein. Variant NPs may
include one or more substitutions, additions or deletions relative
to CNP22 and have the ability to agonize NPR-B. An NP as described
herein may include any other sequence or moiety, attached
covalently or non-covalently, provided that the NP has the ability
to agonize NPR-B.
[0150] By "neurocutaneous syndrome" is meant a neurological
disorder with one or more cutaneous manifestations, such as lesions
on the skin and/or the eye. Such syndromes can optionally be
accompanied by benign or malignant tumors in multiple sites of the
body.
[0151] By "NP polypeptide" is meant any sequence including an NP
sequence, as defined herein. Exemplary NP polypeptides include
those having the structure V--NP-W, wherein each of V and W is
absent or is an amino acid sequence of at least one amino acid
(e.g., any NP fusion polypeptide described herein).
[0152] By "nucleic acid molecule" is meant a molecule, e.g., RNA or
DNA, having a sequence of two or more covalently bonded, naturally
occurring or modified nucleotides. The nucleic acid molecule may
be, e.g., single or double stranded, and may include modified or
unmodified nucleotides, or mixtures or combinations thereof.
Various salts, mixed salts, and free acid forms are also
included.
[0153] The terms "peptide," "polypeptide," and "protein" are used
interchangeably and refer to any chain of two or more natural or
unnatural amino acid residues, regardless of post-translational
modification (e.g., glycosylation or phosphorylation), constituting
all or part of a naturally-occurring or non-naturally occurring
polypeptide or peptide, as is described herein.
[0154] As used herein, a natural amino acid is a natural
.alpha.-amino acid having the L-configuration, such as those
normally occurring in natural polypeptides. Unnatural amino acid
refers to an amino acid that normally does not occur in
polypeptides, e.g., an epimer of a natural .alpha.-amino acid
having the L configuration, that is to say an amino acid having the
unnatural D-configuration; or a (D,L)-isomeric mixture thereof; or
a homolog of such an amino acid, for example, a .beta.-amino acid,
an .alpha.,.alpha.-disubstituted amino acid, or an .alpha.-amino
acid wherein the amino acid side chain has been shortened by one or
two methylene groups or lengthened to up to 10 carbon atoms, such
as an .alpha.-amino alkanoic acid with 5 up to and including 10
carbon atoms in a linear chain, an unsubstituted or substituted
aromatic (.alpha.-aryl or .alpha.-aryl lower alkyl), for example, a
substituted phenylalanine or phenylglycine.
[0155] By "pharmaceutically acceptable carrier" or
"pharmaceutically acceptable excipient" is meant a carrier or
excipient that is physiologically acceptable to the treated patient
while retaining the therapeutic properties of the compound with
which it is administered. One exemplary pharmaceutically acceptable
carrier substance is physiological saline. Other physiologically
acceptable carriers and their formulations are known to those
skilled in the art and described, for example, in Remington's
Pharmaceutical Sciences, (20th edition), ed. A. Gennaro, 2000,
Lippincott, Williams & Wilkins, Philadelphia, Pa.
[0156] By "pharmaceutical composition" is meant a composition
containing a polypeptide or nucleic acid molecule as described
herein formulated with a pharmaceutically acceptable excipient, and
manufactured or sold with the approval of a governmental regulatory
agency as part of a therapeutic regimen for the treatment or
prevention of a disease or event in a subject. Pharmaceutical
compositions can be formulated, for example, for subcutaneous
administration, intravenous administration (e.g., as a sterile
solution free of particulate emboli and in a solvent system
suitable for intravenous use), for oral administration (e.g., a
tablet, capsule, caplet, gelcap, or syrup), or any other
formulation described herein, e.g., in unit dosage form.
[0157] By "potency" is meant the reciprocal of the EC.sub.50 value
of a compound in a dose-response assay. When comparing potency
between a compound and a control or between an assay and a control
assay, decreased potency indicates an increased EC.sub.50 value,
and increased potency indicates a decreased EC.sub.50 value, as
compared to the EC.sub.50 value for the control or the control
assay.
[0158] By "reduced degradation" is meant having a lower percentage
of degraded peptide after exposure to an enzyme for at least 5, 10,
15, 20, 25, 30, 60, 120, 180, or 240 minutes, or higher, or any
range between any two of these values, as compared to a percentage
of degraded control, such as CNP22, CNP53, or any polypeptide
described herein, such as a peptide described in International
Application Pub. No. WO2010/135541 or U.S. Application Pub. No.
2010-0331256. The percentage of degraded peptide can be lower by
about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,
about 50%, about 55%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,
about 98%, about 99%, or about 100%, where the percentage of
degraded peptide can be determined by measuring the percentage of
degraded peptide directly or indirectly by measuring the percentage
of remaining peptide after exposure to an enzyme (e.g., neutral
endopeptidase, insulin degrading enzyme, and any other enzyme that
cleaves a natriuretic peptide in vivo) and subtracting this
percentage of remaining peptide from 100%. Percentage of degraded
peptide or remaining peptide can be measured by any useful method,
such as liquid chromatography (e.g., high performance liquid
chromatography (HPLC)), mass spectrometry (MS), or combined
analytic techniques (e.g., LC-MS).
[0159] By "reduced dose-dependent side effect" is meant a decrease
in one or more adverse effects as a function of a dosage of a
compound, as compared to a control (e.g., any polypeptide described
herein, such as a peptide described in International Application
Pub. No. WO2010/135541 or U.S. Application Pub. No. 2010-0331256).
The decrease in one or more adverse effects can be by about 20%,
about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,
about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,
about 85%, about 90%, about 95%, about 96%, about 97%, about 98%,
about 99%, or about 100%, as determined by any useful assay for
detecting the adverse effect. Exemplary adverse effects include
hemodynamic effects, such as a decrease in blood pressure, such as
systolic arterial blood pressure, diastolic arterial blood
pressure, or mean arterial blood pressure, that results in adverse
hypotensive effects, and assays to detect such hemodynamic effects
include a sphygmomanometer or an implanted pressure transducer.
[0160] The terms "sALP," "soluble alkaline phosphatase," and
"extracellular domain of an alkaline phosphatase" are used
interchangeably and mean a soluble, non-membrane-bound alkaline
phosphatase or a domain, biologically active fragment, or
biologically active variant thereof. sALPs include, for example, an
alkaline phosphatase lacking a C-terminal GPI signal sequence,
e.g., a polypeptide including or consisting of the amino acid
residues 18-502 of human TNALP (SEQ ID NO: 1208). sALPs further
include, for example, soluble, non-membrane-bound forms of
mammalian orthologs of human TNALP (e.g., polypeptides including or
consisting of amino acid residues 16-502 or 18-502 of SEQ ID NO:
1206, amino acid residues 18-502 of SEQ ID NO: 1207, amino acid
residues 18-502 of SEQ ID NO: 1209, amino acid residues 18-502 of
SEQ ID NO: 1210, or amino acid residues 1-480 of SEQ ID NO: 1211),
soluble, non-membrane-bound forms of human IALP, GALP, and PLALP
(e.g., polypeptides including or consisting of amino acid residues
20-503 of SEQ ID NO: 1212, amino acid residues 20-503 of SEQ ID NO:
1213, or amino acid residues 23-506 of SEQ ID NO: 1214), and
additional variants and analogs thereof which retain alkaline
phosphatase activity, e.g., the ability to hydrolyze PPi.
[0161] By "sALP polypeptide" is meant any sequence including an
sALP sequence, as defined herein. Exemplary sALP polypeptides
include those having the structure A-sALP-B, wherein each of A and
B is absent or is an amino acid sequence of at least one amino acid
(e.g., any sALP fusion polypeptide described herein).
[0162] By "selective for NPR-B over NPR-A" is meant having an
EC.sub.50(NPR-A)/EC.sub.50(NPR-B) ratio that is at least 1.25, 1.5,
2, 2.5, 3, 3.5, 4, 4.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125,
130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350,
400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000,
1,100, 1,200, 1,250, 1,300, 1,400, 1,500, 1,750, 2,000, 2,500,
3,000, 4,000, 5,000, 10,000, or higher, or any range between any
two of these values, in an in vivo or in vitro dose-response assay,
e.g., measuring cGMP production, as described herein.
Alternatively, or in addition, the term "selective for NPR-B over
NPR-A" means having an AUC.sub.(NPR-B)/AUC.sub.(NPR-A) ratio that
is at least 1.1, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.75, 1.8,
1.9, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 4.5, 5, 7.5, 10, 12.5, 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
110, 120, 125, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250,
275, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,
900, 950, 1,000, 1,100, 1,200, 1,250, 1,300, 1,400, 1,500, 1,750,
2,000, 2,500, 3,000, 4,000, 5,000, 10,000, or higher, or any range
between any two of these values, as described herein.
[0163] By "signal peptide" or "signal sequence" is meant an amino
acid sequence that directs a polypeptide to the cellular membrane
such that the polypeptide is secreted. Alternatively, the signal
sequence may direct the polypeptide to an intracellular compartment
or organelle, such as the Golgi apparatus. A signal sequence may be
identified by homology, or biological activity, to a peptide
sequence with the known function of targeting a polypeptide to a
particular region of the cell. One of ordinary skill in the art can
identify a signal sequence by using readily available software
(e.g., Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705, BLAST, or PILEUP/PRETTYBOX
programs). A signal sequence can be one that is, for example,
substantially identical to amino acid residues 1-25 of SEQ ID NO:
501 or to amino acid residues 1-17 of SEQ ID NO: 1201.
[0164] By "skeletal dysplasia" is meant a bone or cartilage
disorder characterized by short stature or dwarfism.
[0165] By "subject" is meant a mammal, including, but not limited
to, a human or non-human mammal, such as a bovine, equine, canine,
ovine, or feline.
[0166] By a "synergistic" effect is meant a therapeutic effect
observed following administration of two or more agents that is
greater than the sum of the therapeutic effects observed following
the administration of each single agent. In one example of synergy,
a therapeutic effect is observed for the combination of two or more
agents, wherein one or more of the agents is present at a dose that
is normally non-therapeutic. In another example of synergy, the
combination of two or more agents results in an unexpected decrease
in one or more adverse events (i.e., a level or number of adverse
events that is less than the sum of adverse events observed
following administration of the single agents). In another example,
the combination of two or more agents at a therapeutic dose results
in reduced dose-dependent side effects, as compared to the level of
dose-dependent side effects observed for a single agent at a
therapeutic dose.
[0167] By "therapeutically effective amount" is meant an amount of
a polypeptide or nucleic acid molecule described herein that is
sufficient to substantially treat, prevent, delay, suppress, or
arrest any symptom of a neurocutaneous syndrome, a disorder
associated with overactivation of FGFR3, a bone or cartilage
disorder (e.g., achondroplasia), or a vascular smooth muscle
disorder, or that is sufficient to substantially elongate bone. A
therapeutically effective amount of a composition described herein
may depend on the severity of the disorder being treated and the
condition, weight, and general state of the subject and can be
determined by an ordinarily-skilled artisan with consideration of
such factors. A therapeutically effective amount of a composition
described herein can be administered to a subject in a single dose
or in multiple doses administered over a period of time.
[0168] By "treating," "treat," or "treatment" is meant the medical
management of a patient with the intent to cure, ameliorate,
stabilize, reduce the likelihood of, or prevent a neurocutaneous
syndrome, a disorder associated with overactivation of FGFR3, a
bone or cartilage disorder (e.g., achondroplasia), or a vascular
smooth muscle disorder, or management of a healthy subject with the
intent to elongate bone, e.g., by administering a pharmaceutical
composition. This term includes active treatment, that is,
treatment directed specifically toward the improvement or
associated with the cure of a disease, pathological condition,
disorder, or event, and also includes causal treatment, that is,
treatment directed toward removal of the cause of the associated
disease, pathological condition, disorder, or event. In addition,
this term includes palliative treatment, that is, treatment
designed for the relief of symptoms rather than the curing of the
disease, pathological condition, disorder, or event; symptomatic
treatment, that is, treatment directed toward constitutional
symptoms of the associated disease, pathological condition,
disorder, or event; preventative treatment, that is, treatment
directed to minimizing or partially or completely inhibiting the
development of the associated disease, pathological condition,
disorder, or event, e.g., in a patient who is not yet ill, but who
is susceptible to, or otherwise at risk of, a particular disease,
pathological condition, disorder, or event; and supportive
treatment, that is, treatment employed to supplement another
specific therapy directed toward the improvement of the associated
disease, pathological condition, disorder, or event.
[0169] By "vascular smooth muscle disorder" is meant any disorder,
disease, or other abnormality that affects the function, structure,
or growth of vascular smooth muscle.
[0170] By "vector" is meant a DNA molecule, usually derived from a
plasmid or bacteriophage, into which fragments of DNA may be
inserted or cloned. A recombinant vector will contain one or more
unique restriction sites, and may be capable of autonomous
replication in a defined host or vehicle organism such that the
cloned sequence is reproducible. A vector contains a promoter
operably linked to a gene or coding region such that, upon
transfection into a recipient cell, an RNA is expressed.
[0171] Other features and advantages of the invention will be
apparent from the detailed description and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0172] In figures showing a multiple sequence alignment, "*"
represents identity; ":" represents a conserved substitution; and
"." represents a semi-conserved substitution.
[0173] FIG. 1A is a graph showing levels of pyrophosphate
(PP.sub.i) in osteoblasts from wild-type mice (left) and
NF1.sub.co2.sup.-/- mice (right).
[0174] FIG. 1B is a graph showing levels of progressive ankylosis
gene (Ank) mRNA expression in osteoblasts from wild-type mice
(labeled "WT") and NF1.sub.col2.sup.-/- mice (labeled "KO").
Osteoblasts were treated with vehicle, a MEK1/MEK2 kinase inhibitor
U0126 (1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)
butadiene), or bone morphogenetic protein 2 (BMP2).
[0175] FIG. 2 is a schematic showing a non-limiting, hypothetical
working model for defective bone matrix mineralization in
NF1.sub.col2.sup.-/- mice.
[0176] FIG. 3A shows the effect of recombinant human bone
morphogenetic protein 2 (rhBMP2) in osteoblasts from wild-type mice
(labeled "WT") and NF1.sub.col2.sup.-/- mice. Cell plates are shown
with histological staining for alkaline phosphatase (ALP) alone or
in combination with alizarin red S (ALP/Alizarin) and for cell
number with crystal violet.
[0177] FIG. 3B shows the effect of an sALP fusion polypeptide
(sTNALP-FcD.sub.10, SEQ ID NO: 1204) in bone marrow stromal cells
from wild-type mice (labeled "WT") and NF1.sub.col2.sup.-/- mice.
Cells were then treated for 8 days with increasing doses of
sTNALP-FcD.sub.10. Cell plates are shown with histological staining
for alizarin red S to determine the presence of calcific deposition
(top), and the relative intensity of staining with alizarin red S
was quantified (bottom).
[0178] FIG. 3C shows the effect of an sALP fusion polypeptide
(sTNALP-FcD.sub.10, SEQ ID NO: 1204) in bone marrow stromal cells
(BMSCs) from wild-type mice (labeled "WT") and NF1.sub.col2.sup.-/-
mice. BMSCs were plated in culture and differentiation was induced
for 14 days using vitamin C and beta-glycerophosphate. Cells were
then treated with 0.5 .mu.g/mL of sTNALP-FcD.sub.10 and stained
with alizarin red to assess level of mineralization, with
picrosirius red to demonstrate presence of extracellular matrix, or
with crystal violet to stain cells.
[0179] FIG. 3D shows the effect of an sALP fusion polypeptide
(sTNALP-FcD.sub.10, SEQ ID NO: 1204) in bone marrow stromal cells
(BMSCs) from floxed NF1 gene mice. BMSCs were plated in culture,
and differentiation was induced for 14 days using vitamin C and
beta-glycerophosphate. Cells were treated for 8 days with an
adenovirus coding for GFP ("Ad-GFP," as control) or an adenovirus
coding for CRE recombinase ("Ad-Cre") either with vehicle or with
0.5 .mu.g/mL of sTNALP-FcD.sub.10. Cells were then stained with
alizarin red to assess level of mineralization (first and second
columns) or with picrosirius red to demonstrate presence of
extracellular matrix (third and fourth columns).
[0180] FIG. 3E shows the in vivo effect of an sALP fusion
polypeptide (sTNALP-FcD.sub.10, SEQ ID NO: 1204) in wild-type mice
(labeled "WT") and NF1.sub.col2.sup.-/- mice. NF1.sub.col2.sup.-/-
mice were treated from day 1 to day 18 with 8.2 mg/kg of
sTNALP-FcD.sub.10 and compared to untreated or WT mice. Mice were
sacrificed at day 19, and vertebrae were analysed with micro-CT
imaging to measure bone volume over total volume (BV/TV). Treatment
of mice with sTNALP-FcD.sub.10 increased bone mineral density
deficit in NF1.sub.col2.sup.-/- mice compared to vehicle (*
p<0.5).
[0181] FIG. 4A shows the expression of the NPR-B gene in
NF1.sub.col2.sup.-/- mice. Bone marrow stromal cells were cultured
for 3 weeks under osteogenic conditions (with ascorbate acetate)
and lysed. RT-PCR was performed using NPR-B and housekeeping gene
GAPDH primers.
[0182] FIG. 4B shows the effect of an NP fusion polypeptide
(NC2-KGANKK, SEQ ID NO: 512) in chondrocytes from wild-type mice
(labeled "WT") and NF1.sub.col2.sup.-/- mice. Western blot analysis
provides levels of ERK and phosphorylated ERK (p-ERK) for
chondrocytes from wild-type mice or from NF1.sub.col2.sup.-/- mice.
Primary chondrocytes extracted from ribs of newborn
NF1.sub.col2.sup.-/- mice or WT mice were cultured and then treated
for 30 minutes with increasing concentrations of NC2-KGANKK. Cells
were then lysed, and western blotting was performed on lysates
using anti-ERK or anti-phospho-ERK specific antibodies.
[0183] FIG. 4C shows the in vivo effect of an NP fusion polypeptide
(NC2B, SEQ ID NO: 504) on naso-anal length in wild-type mice
(labeled "WT") and NF1.sub.col2.sup.-/- mice. NF1.sub.col2.sup.-/-
mice were treated from day 1 to day 18 with 100 or 300 mg/kg of
NC2B and compared to vehicle-treated or WT mice. Mice were measured
for naso-anal length at day 19.
[0184] FIG. 4D shows the in vivo effect of an NP fusion polypeptide
(NC2B, SEQ ID NO: 504) on tibia length in wild-type mice (labeled
"WT") and NF1.sub.col2.sup.-/- mice. NF1.sub.col2.sup.-/- mice were
treated from day 1 to day 18 with 100 or 300 mg/kg of NC2B and
compared to vehicle-treated or WT mice. Mice were measured for
tibia length at day 19.
[0185] FIG. 4E shows the in vivo effect of an NP fusion polypeptide
(NC2B, SEQ ID NO: 504) on growth plates in wild-type mice (labeled
"WT") and NF1.sub.col2.sup.-/- mice. NF1.sub.col2.sup.-/- mice were
treated from day 1 to day 18 with 300 mg/kg of NC2B and compared to
vehicle-treated or WT mice. At day 19, tibias were used to analyze
proximal growth plates in histology and to measure the size of
chondrocyte zones.
[0186] FIG. 5A is a schematic diagram of the structure of human
tissue nonspecific alkaline phosphatase (hsTNALP) as described
herein, which includes a polypeptide having a TNALP ectodomain, an
N-terminal signaling sequence, and a GPI signal sequence (top); a
hsTNALP-FcD.sub.10 having a TNALP ectodomain, an N-terminal
signaling sequence, an IgG.sub.1-Fc sequence, and a bone-targeting
D.sub.10 moiety (middle); and a hsTNALP-FcD.sub.10 without a signal
sequence having a TNALP ectodomain, an IgG.sub.1-Fc sequence, and a
bone-targeting D.sub.10 moiety (bottom).
[0187] FIG. 5B shows the amino acid sequence of hsTNALP-FcD.sub.10
(SEQ ID NO: 1201), including the N-terminal signal sequence (the
first 17 amino acid residues, underlined and italicized; position
2, a valine, differs from the wild-type residue in that position,
isoleucine); and the amino acid sequence of secreted
hsTNALP-FcD.sub.10 (SEQ ID NO: 1204), which lacks the N-terminal
signal sequence. Amino acid residues of the hsTNALP portion of the
polypeptides, which correspond to amino acid residues 18-502 of
hsTNALP (SEQ ID NO: 1205), are italicized. The signal sequence in
SEQ ID NO: 1201 is italicized and underlined. The Fc portions of
the polypeptides (SEQ ID NO: 401) are underlined. A dipeptide
leucine-lysine (LK) linker between the hsTNALP and Fc portions and
a dipeptide aspartic acid-isoleucine (DI) linker between the
hsTNALP and bone-targeting moiety are shown in bold.
[0188] FIG. 5C shows the amino acid sequence of sALP fusion
proteins lacking a bone-targeting moiety: hsTNALP-Fc (SEQ ID NO:
1220) and secreted hsTNALP-FcD.sub.10 (SEQ ID NO: 1221), which
lacks the N-terminal signal sequence. Amino acid residues of the
hsTNALP portion of the polypeptides, which correspond to amino acid
residues 18-502 of hsTNALP (SEQ ID NO: 1205), are italicized. The
signal sequence in SEQ ID NO: 1201 is italicized and underlined.
The Fc portions of the polypeptides (SEQ ID NO: 401) are
underlined. A dipeptide leucine-lysine (LK) linker between the
hsTNALP and Fc portions are shown in bold.
[0189] FIG. 5D shows a nucleic acid sequence (SEQ ID NO: 1217)
encoding the hsTNALP-Fc polypeptide depicted in FIG. 5B.
[0190] FIG. 6 shows amino acid sequences for human soluble tissue
nonspecific alkaline phosphatase (hsTNALP), including the sequence
of hsTNALP having the N-terminal signal sequence (amino acid
residues 1-502) (SEQ ID NO: 1202) and the sequence of secreted
hsTNALP lacking the signal sequence (amino acid residues 18-502)
(SEQ ID NO: 1205). The signal sequence is underlined.
[0191] FIG. 7 is a listing of the amino acid sequence of an
exemplary Fc from human IgG-1 (SEQ ID NO: 401).
[0192] FIG. 8 shows a CLUSTAL.TM. W (1.82) multiple sequence
alignment of mammalian tissue nonspecific alkaline phosphatase
(TNALP) orthologs. Mammalian TNALP orthologs include cow TNALP
("P09487|PPBT_BOVIN"; Accession No. P09487; SEQ ID NO: 1206); cat
TNALP ("Q29486|PPBT_FELCA"; Accession No. Q29486; SEQ ID NO: 1207),
human TNALP ("P05186|PPBT_HUMAN"; Accession No. P05186; SEQ ID NO:
1208), mouse TNALP ("P09242|PPBT_MOUSE"; Accession No. P09242; SEQ
ID NO: 1209), rat TNALP ("P08289|PPBT_RAT"; Accession No. P08289;
SEQ ID NO: 1210), and a partial sequence of dog TNALP
("Q9N0V0|Q9N0V0_CANFA"; Accession No. Q9N0V0; SEQ ID NO: 1211). A
consensus sequence is derived from this alignment ("Consensus"; SEQ
ID NO: 1216), where X denotes degenerate positions and can be any
amino acid.
[0193] FIG. 9 shows a CLUSTAL.TM. (2.0.5) multiple sequence
alignment of mammalian tissue nonspecific alkaline phosphatase
(TNALP) orthologs and human alkaline phosphatase (ALP) isozymes.
Mammalian TNALP orthologs include those shown in FIG. 8, including
rat TNALP ("TNALPrn," SEQ ID NO: 1210), mouse TNALP ("TNALPmm," SEQ
ID NO: 1209), human TNALP ("TNALPhs," SEQ ID NO: 1208), a partial
sequence of dog TNALP ("TNALPcf," SEQ ID NO: 1211), cat TNALP
("TNALPfc," SEQ ID NO: 1207), and cow TNALP ("TNALPbt," SEQ ID NO:
1206). Human ALP isozymes include a human gastrointestinal ALP
("GALPhs"; Accession No. P10696; SEQ ID NO: 1213), a human
placental ALP ("PLALPhs"; Accession No. 05187; SEQ ID NO: 1214),
and a human intestinal ALP ("IALPhs"; Accession No. P09923; SEQ ID
NO: 1212). A consensus sequence is derived from this alignment
("Consensus"; SEQ ID NO: 1215), where X denotes degenerate
positions and can be any amino acid.
[0194] FIG. 10 is a consensus sequence (SEQ ID NO: 1218) for
mammalian tissue nonspecific alkaline phosphatase (TNALP) orthologs
and human alkaline phosphatase (ALP) isozymes excluding pathogenic
mutations, where X can be any amino acid but not an amino acid
corresponding to one or more pathogenic mutations provided in Table
1.
[0195] FIG. 11 is a consensus sequence (SEQ ID NO: 1219) for
mammalian tissue nonspecific alkaline phosphatase (TNALP) orthologs
excluding pathogenic mutations, where X can be any amino acid but
not an amino acid corresponding to one or more pathogenic mutations
provided in Table 1.
[0196] FIG. 12 is a multiple sequence alignment of human ANP (SEQ
ID NO: 1), human urodilatin (SEQ ID NO: 2), human BNP (SEQ ID NO:
3), human CNP22 (SEQ ID NO: 4), and DNP (SEQ ID NO: 5). The
17-amino acid ring domain for each natriuretic peptide is shown in
bold and enclosed in a box. A consensus sequence (SEQ ID NO: 6) is
shown below, wherein each X represents any amino acid, or
optionally represents any amino acid at the corresponding position
in one of SEQ ID NOs: 1-5.
[0197] FIG. 13 is an alignment of human CNP53 (SEQ ID NO: 11),
human CNP22, and human CNP (ring domain only) (SEQ ID NO: 12).
[0198] FIG. 14 is a multiple sequence alignment of various CNP22
homologs. The 17-amino acid ring domain for each NP is shown in
bold and enclosed in a box. A consensus sequence (SEQ ID NO: 30) is
shown below, wherein each X within the ring domain represents any
amino acid, or optionally represents any amino acid at the
corresponding position in one of SEQ ID NOs: 4 and 13-29. Each X
outside the ring domain represents any amino acid or may be absent,
or optionally represents any amino acid at the corresponding
position in one of SEQ ID NOs: 4 and 13-29.
[0199] FIGS. 15A-15G are a multiple sequence alignment of various
CNP homologs, in some cases including the N-terminal pre- and
pro-sequences. The 17-amino acid ring domain for each NP is shown
in bold and enclosed in a box. A consensus sequence (SEQ ID NO: 95)
is shown below, wherein each X represents any amino acid, or
optionally represents any amino acid at the corresponding position
in one of SEQ ID NOs: 31-94.
[0200] FIG. 16 is a schematic diagram of the structure of a
natriuretic peptide as described herein, which includes an optional
N-terminal extension, an optional short segment, a required ring
domain, and an optional C-terminal extension.
[0201] FIGS. 17A-17E are schematic diagrams of exemplary Fc-NP or
NP-Fc constructs. FIG. 17A depicts an Fc-NP dimer. FIG. 17B depicts
an NP-Fc dimer. FIG. 17C depicts an Fc:Fc-NP monomer-dimer hybrid.
FIG. 17D depicts an NP-Fc:Fc monomer-dimer hybrid. FIG. 17E depicts
an NP-Fc:Fc-NP hybrid dimer.
[0202] FIG. 18A is a listing of the amino acid sequence of the
immature NC2 Streptag ("NC2st") fusion protein (SEQ ID NO: 501),
together with a table providing a summary of protein regions. The
N-terminal signal sequence, which is cleaved during translation, is
underlined. Various linker sequences are shown in italics. The Fc
domain is shown in bold. The CNP domain is shown in gray
highlighting. FIG. 18B is a listing of the amino acid sequence of
the NC2st fusion protein (SEQ ID NO: 502) without the signal
sequence. FIG. 18C is a listing of the nucleic acid sequence (SEQ
ID NO: 801) encoding the NC2st fusion protein.
[0203] FIG. 19A is a listing of the NC2B amino acid sequence, both
with the signal sequence (SEQ ID NO: 503) and without the signal
sequence (SEQ ID NO: 504), and the D10-NC2 amino acid sequence
having a D.sub.10 tag, both with the signal sequence (SEQ ID NO:
607) and without the signal sequence (SEQ ID NO: 608).
[0204] FIG. 19B is a listing of a nucleic acid sequence (SEQ ID NO:
802) encoding NC2B.
[0205] FIG. 20A is a listing of amino acid sequences for NC2B-22,
NC2B-28, and NC2B-34, both with the signal sequence (SEQ ID NOs:
505, 507, and 509, respectively) and without the signal sequence
(SEQ ID NOs: 506, 508, and 510, respectively). Signal sequences are
underlined. The Fc domain is shown in bold. Linker sequences are
shown in italics. The CNP domain is shown in gray highlighting.
FIG. 20B is a listing of a nucleic acid sequence (SEQ ID NO: 803)
encoding NC2B-22. FIG. 20C is a listing of a nucleic acid sequence
(SEQ ID NO: 804) encoding NC2B-28. FIG. 20D is a listing of a
nucleic acid sequence (SEQ ID NO: 805) encoding NC2B-34.
[0206] FIG. 21 is a listing of amino acid sequences for NC2-KGANKK
and NC2-KGANQK, both with the signal sequence (SEQ ID NOs: 511 and
513, respectively) and without the signal sequence (SEQ ID NOs: 512
and 514, respectively). Signal sequences are underlined. The Fc
domain is shown in bold. Linker sequences are shown in italics. The
CNP domain is shown in gray highlighting.
[0207] FIG. 22 is a listing of amino acid sequences for
NC2-CNP53mut2, both with the signal sequence (SEQ ID NO: 515) and
without the signal sequence (SEQ ID NOs: 516). Signal sequence is
underlined. The Fc domain is shown in bold. Linker sequences are
shown in italics. The CNP domain is shown in gray highlighting.
[0208] FIG. 23 is a listing of amino acid sequences for Fc-CNP53-A
(also referred to as Fc-CNP53 wt) and Fc-CNP53-AAA (also referred
to as Fc-CNP53mut), both with the signal sequence (SEQ ID NOs: 517
and 519, respectively) and without the signal sequence (SEQ ID NOs:
518 and 520, respectively). Signal sequences are underlined. The Fc
domain is shown in bold. Linker sequences are shown in italics. The
CNP domain is shown in gray highlighting.
[0209] FIG. 24 is a multiple sequence alignment of various NPs and
homologs, including CDNP. The boxed region is the most conserved
region of the DNP tail among NPRA-binding peptides. The sequences
of numerous CDNP variants are shown in the bottom half of the
figure, and a consensus sequence (SEQ ID NO: 118) for the DNP
C-terminal tail is also shown. Each X in the consensus sequence
represents any amino acid, or optionally represents any amino acid
at the corresponding position in one of SEQ ID NOs: 100-116.
[0210] FIG. 25A is a listing of amino acid sequences for
CNP-16AAlinker-Fc-His10 (NC1) (SEQ ID NO: 521),
CNP-6AAlinker-Fc-His10 (NC3) (SEQ ID NO: 522), CNP-6AAlinker-Fc
(SEQ ID NO: 523), CDNP-Fc (SEQ ID NO: 524), CDNP-A17saa-Fc (SEQ ID
NO: 525), and CDNP-A17sra-Fc (SEQ ID NO: 526). The CNP domain is
shown in gray highlighting. Linker sequences are shown in italics.
The Fc domain is shown in bold. FIG. 25B is a listing of the
nucleic acid sequence (SEQ ID NO: 806) of NC1.
[0211] FIG. 26 is a listing of various point mutants (SEQ ID NOs:
119-125) each having a mutation at position 17 of CNP22, together
with a consensus sequence (SEQ ID NO: 126). X represents any amino
acid, or optionally represents any amino acid at the corresponding
position in one of SEQ ID NOs: 119-125.
[0212] FIG. 27 is a listing of amino acid sequences for several CNP
variants (SEQ ID NOs: 4 and 127-150). The 17-amino acid ring domain
for each variant is shown in bold. The linker region is shown in
italics.
[0213] FIGS. 28A-28E are a listing of amino acid sequences for
additional CNP variants (SEQ ID NOs: 1001-1155).
[0214] FIG. 29 is a listing of amino acid sequences for CNP22 (SEQ
ID NO: 4), CNP-L17 (SEQ ID NO: 120), CNP-F17 (SEQ ID NO: 119),
CNP-T17 (SEQ ID NO: 122), D6-14AAlinker-CNP [C3] (SEQ ID NO: 147),
CNP-14AAlinker-D6 [C4] (SEQ ID NO: 148), CNP-Nterm2 [C5] (SEQ ID
NO: 150), CDNP-S3A4A5R6 [C13] (SEQ ID NO: 115), CDNP29-S3A4A5R6
[C14] (SEQ ID NO: 151), C1(E6) [BC1] (SEQ ID NO: 129), C2(E6) [BC2]
(SEQ ID NO: 130), C3 (E6) [BC3] (SEQ ID NO: 131), C4(E6) [BC4] (SEQ
ID NO: 132), C5(E6) [BC5](SEQ ID NO: 133), C6(E6) [BC6] (SEQ ID NO:
134), C7(E6) [BC7] (SEQ ID NO: 135), C8(E6) [BC8] (SEQ ID NO: 136),
C9(E6) [BC9] (SEQ ID NO: 137), C10(E6) [BC10] (SEQ ID NO: 138),
C11(E6) [BC11] (SEQ ID NO: 139), PGCNP37(E6) (SEQ ID NO: 128), KA1
(SEQ ID NO:152), KA1(E6) (SEQ ID NO:153), KB1 (SEQ ID NO:154), and
KB1(E6) (SEQ ID NO: 155). The 17-amino acid ring domain for each
variant is shown in bold. The linker sequences are shown in
italics. The cathepsin cleavage sequences are shown in
underline.
[0215] FIG. 30 is a listing of amino acid sequences for CNP
variants having a point mutation at position 17 relative to CNP22
(SEQ ID NOs: 126, 119-122, and 156-172). For SEQ ID NOs: 126 and
162, X can be any amino acid, including but not limited to F, L, I,
T, E, R, Y, C, P, or D. The 17-amino acid ring domain for each
variant is shown in bold. The linker sequences are shown in
italics.
[0216] FIGS. 31A-31B are listings of amino acid sequences for
additional CNP variants having a point mutation at position 17
relative to CNP22 (SEQ ID NOs: 173-220). X can be any amino acid,
including but not limited to F, L, I, T, E, R, Y, C, P, or D. The
17-amino acid ring domain for each variant is shown in bold. The
linker sequences are shown in italics. The cathepsin cleavage
sequences are shown in underline.
[0217] FIG. 32 is a listing of amino acid sequences for CNP
variants having a point mutation at position 17 relative to CNP22,
where the methionine at position 17 has been substituted with a
leucine (SEQ ID NOs: 221-233). The 17-amino acid ring domain for
each variant is shown in bold. The linker sequences are shown in
italics. The cathepsin cleavage sequences are shown in
underline.
[0218] FIGS. 33A-33E are listings of amino acid sequences for
constructs having a point mutation at position 17 relative to CNP22
(SEQ ID NOs: 527-552). X can be any amino acid, including but not
limited to F, L, I, T, E, R, Y, C, P, or D. Signal sequences are
underlined. The Fc domain is shown in bold. Linker sequences are
shown in italics. The CNP domain is shown in gray highlighting.
[0219] FIGS. 34A-34J are listings of amino acid sequences for NC2
variants (SEQ ID NOs: 511-516 and 553-606) with or without the
signal sequence and either with or without a D.sub.10
bone-targeting moiety at the N-terminal. Signal sequences are
underlined. The Fc domain is shown in bold. Linker sequences are
shown in italics. The CNP domain is shown in gray highlighting.
DETAILED DESCRIPTION OF THE INVENTION
[0220] The present invention features soluble alkaline phosphatase
(sALP) polypeptides, e.g., fused to an Fc domain of an
immunoglobulin, nucleic acid encoding such, and their uses to treat
any disease or condition described herein (e.g., neurocutaneous
syndromes (e.g., neurofibromatosis, e.g., type 1), disorders
associated with overactivation of FGFR3, bone or cartilage
disorders (e.g., hypophosphatasia or achondroplasia), vascular
smooth muscle disorders, as well as to elongate bone). The present
invention also features natriuretic (NP) polypeptides, e.g., fused
to an Fc domain of an immunoglobulin, nucleic acid encoding such,
and their uses to treat any disease or condition described herein
(e.g., neurocutaneous syndromes (e.g., neurofibromatosis, e.g.,
type 1), disorders associated with overactivation of FGFR3, bone or
cartilage disorders (e.g., hypophosphatasia or achondroplasia),
vascular smooth muscle disorders, as well as to elongate bone). The
present invention also features a combination of such sALP
polypeptides with such NP polypeptides, as described herein, and
uses of this combination to treat any disease or condition
described herein (e.g., neurocutaneous syndromes, disorders
associated with overactivation of FGFR3, bone or cartilage
disorders (e.g., hypophosphatasia or achondroplasia), vascular
smooth muscle disorders, as well as to elongate bone). Additional
details of the invention are provided below.
Alkaline Phosphatase
[0221] Alkaline phosphatases encompass a group of enzymes that
share the property of being able to cleave phosphate in a variety
of contexts (e.g., hydrolysis of pyrophosphate, PPi). There are
four known mammalian alkaline phosphatase (ALP) isozymes: tissue
nonspecific alkaline phosphatase (TNALP; described further below),
placental alkaline phosphatase (PLALP) (e.g., Accession Nos.
P05187, NP_112603, and NP_001623), germ cell alkaline phosphatase
(GALP) (e.g., Accession No. P10696), and intestinal alkaline
phosphatase (IALP) (e.g., Accession Nos. P09923 and NP_001622).
These isozymes possess very similar three dimensional structures.
Each of their catalytic sites contains four metal binding domains
that bind to metal ions necessary for enzymatic activity, including
two zinc ions and one magnesium ion. These enzymes catalyze the
hydrolysis of monoesters of phosphoric acid and also catalyze a
transphosphorylation reaction in the presence of high
concentrations of phosphate acceptors. It has been shown that PLALP
is physiologically active toward phosphoethanolamine (PEA),
inorganic pyrophosphate (PPi), and pyridoxal 5'-phosphate (PLP),
all three being known natural substrate for TNALP (Whyte, 1995). An
alignment between these isozymes is shown in FIG. 9. Additional
alkaline phosphatases are described, e.g., in WO 2008/138131 and in
U.S. Publication No. 2006/0014687, which are hereby incorporated by
reference.
[0222] Tissue nonspecific phosphatases are a family of proteins,
encoded by a single gene, that differ from each other by
post-translational modification. TNALPs are present predominantly
in the liver, kidneys, and bone, but can occur throughout the body.
Known TNALPs in mammals include, e.g., human TNALP (Accession Nos.
NP_000469, AAI10910, AAH90861, AAH66116, AAH21289, and AAI26166);
rhesus TNALP (Accession No. XP_01109717); rat TNALP (Accession No.
NP_037191); dog TNALP (Accession No. AAF64516); pig TNALP
(Accession No. AAN64273), mouse (Accession No. NP_031457), cow
TNALP (Accession Nos. NP_789828, NP_776412, AAM 8209, and
AAC33858), and cat TNALP (Accession No. NP_001036028), in addition
to other examples provided herein.
Soluble Alkaline Phosphatase
[0223] The soluble alkaline phosphatases (sALP) of the invention
include, for example, soluble (e.g., extracellular or non
membrane-bound) forms of any of the alkaline phosphatases described
herein. The soluble alkaline phosphatase of the invention can be,
for example, a soluble form of human TNALP. A schematic
representation of the domains of human TNALP (hTNALP) is shown in
FIG. 5A (top). TNALP is a membrane-bound protein anchored through a
glycolipid bound to its C-terminal (Swiss-Prot, P05186). This
glycolipid anchor (GPI) is added post translationally after the
removal of a hydrophobic C-terminal end, which serves both as a
temporary membrane anchor and as a signal for the addition of the
GPI. This GPI anchor is buried in the cell membrane, and the
remaining portions of the protein are extracellular. TNALP,
including hTNALP, can be engineered to replace the first amino acid
of the hydrophobic C-terminal sequence (an alanine) with a stop
codon. The engineered hTNALP so formed contains all amino acid
residues of the native anchored form of TNALP but lacks the GPI
membrane anchor. An hTNALP which is soluble is herein referred to
as "hsTNALP." One skilled in the art will appreciate that the
position of the GPI membrane anchor will vary in different alkaline
phosphatases and may include, for example, the last 10, 12, 14, 16,
18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 36, 38, 40,
45, 50, or more amino acid residues on the C-terminus of the
polypeptide. For example, the GPI membrane anchor of the hTNALP
(SEQ ID NO: 1208) is amino acid residues 503-524. The amino acid
sequence of this hsTNALP (with one variation at position 2 of the
signal sequence), as fused to Fc, is shown in FIG. 5B. The sequence
of a nucleic acid encoding this hsTNALP-Fc fusion polypeptide is
shown in FIG. 5D (SEQ ID NO: 1217).
[0224] In addition to the C-terminal GPI anchor, TNALP also has an
N-terminal signal peptide sequence. The N-terminal signal peptide
is initially present on the protein when it is synthesized, but is
cleaved after translocation into the ER. Thus, the N-terminal
signal peptide is absent from the secreted form of TNALP. The sALPs
of the invention include both secreted (i.e., lacking the
N-terminal signal) and non-secreted (i.e., having the N-terminal
signal) forms thereof. One skilled in the art will appreciate that
the position of the N-terminal signal peptide will vary in
different alkaline phosphatases and may include, for example, the
first 5, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 27, 30, or more amino acid residues on the N-terminus of
the polypeptide. For example, the N-terminal signal peptide of the
hTNALP of SEQ ID NO: 1208 is its first 17 amino acid residues, as
shown in FIG. 6. Thus, a secreted, soluble form of this hTNALP is
amino acid residues 18-502 of SEQ ID NO: 1208 (SEQ ID NO: 1205), as
shown in FIG. 6. The amino acid sequence of this secreted hsTNALP,
as fused to Fc, is shown in FIG. 5B. The sALPs of the invention
include both secreted and non-secreted forms thereof. One of skill
in the art can predict the position of a signal sequence cleavage
site, e.g., by an appropriate computer algorithm such as that
described in Bendtsen et al. (J. Mol. Biol. 340(4):783-795, 2004)
and available on the Web at
http://www.cbs.dtu.dk/services/SignalP/.
[0225] The sALPs of the invention also include, for example,
polypeptide sequences satisfying a consensus sequence derived from
the ALP extracellular domain of human ALP isozymes and of mammalian
TNALP orthologs (human, mouse, rat, cow, cat, and dog) (SEQ ID NO:
1215, as shown in FIG. 9), or a consensus derived from the ALP
extracellular domain of just mammalian TNALP orthologs (human,
mouse, rat, cow, cat, and dog) (SEQ ID NO: 1216, as shown in FIG.
8). In some embodiments, the sALP includes amino acid residues
18-498, 18-499, 18-500, 18-501, 18-502, 18-503, 18-504, 18-505,
18-506, 18-507, 18-508, 18-509, 18-510, 18-511, or 18-512 of SEQ ID
NO: 1216. In some embodiments, the sALP includes amino acid
residues 23-498, 23-499, 23-500, 23-501, 23-502, 23-503, 23-504,
23-505, 23-506, 23-507, 23-508, 23-509, 23-510, 23-511, or 23-512
of SEQ ID NO: 1215.
[0226] The sALPs of the invention also include those which satisfy
similar consensus sequences derived from various combinations of
these TNALP orthologs or human ALP isozymes. Such consensus
sequences are given, for example, in WO 2008/138131, herein
incorporated by reference.
[0227] Furthermore, it has been shown that recombinant hsTNALP
retaining original amino acid residues 1 to 501 (18 to 501 when
secreted) (Oda et al., J. Biochem. 126: 694-699, 1999), amino acid
residues 1 to 502 (18 to 502 when secreted) (WO 2008/138131), amino
acid residues 1 to 504 (18 to 504 when secreted) (U.S. Pat. No.
6,905,689, which is herein incorporated by reference), and amino
acid residues 1 to 505 (18-505 when secreted) (U.S. Pat. Pub. No.
2007/0081984, which is herein incorporated by reference), are
enzymatically active. This indicates that certain amino acid
residues can be truncated from the C-terminal end of the soluble
hsTNALP polypeptide without affecting its enzymatic activity. This
also indicates that certain amino acid residues of the GPI membrane
anchor, when present, do not significantly affect the solubility of
the polypeptide. Hence, the sALPs of the invention also include
those where, e.g., up to five (e.g., one, two, three, four, or
five) amino acid residues are truncated on its C-terminal end, and
those where, e.g., up to five (e.g., one, two, three, four, or
five) amino acid residues of the GPI membrane anchor are present.
For example, non-secreted sALPs of the invention include those
containing amino acid residues 1-497, 1-498, 1-499, 1-500, 1-501,
1-502, 1-503, 1-504, 1-505, 1-506, or 1-507 of SEQ ID NO: 1208, as
well as variants thereof where the amino acid at position 2 is a
valine, and secreted sALPs of the invention include those
containing amino acid residues 18-497, 18-498, 18-499, 18-500,
18-501, 18-502, 18-503, 18-504, 18-505, 18-506, or 18-507 of SEQ ID
NO: 1208.
[0228] One skilled in the art will appreciate that many mutations
in the amino acid sequence of an enzyme will not significantly
disrupt the catalytic function of the enzyme. In some cases,
certain mutation may even benefit the catalytic function of the
enzyme in the context of therapy for any disorder or condition
described herein (e.g., a neurocutaneous syndrome or a bone or
cartilage disorder). Therefore, the sALPs of the invention include
not only the wild-type sequence of the alkaline phosphatases
described above, but also include any polypeptide having at least
50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to these alkaline phosphatases. It is known, however, that
specific mutations in TNALP are known to cause HPP. Such pathogenic
mutations are preferably absent in the sALPs of the invention.
Thus, the sALPs of the invention include those having an amino acid
sequence including a consensus sequence for multiple mammalian
TNALP orthologs and human ALP isozymes that lack pathogenic
mutations (SEQ ID NO: 1218, as shown in FIG. 10) or including a
consensus sequence for multiple mammalian TNALP orthologs that lack
pathogenic mutations (SEQ ID NO: 1219, as shown in FIG. 11).
Exemplary sALPs of the invention include those containing amino
acid residues 1-497, 1-498, 1-499, 1-500, 1-501, 1-502, 1-503,
1-504, 1-505, 1-506, 1-507, 1-508, 1-509, 1-510, 1-511, 1-512,
23-497, 23-498, 23-499, 23-500, 23-501, 23-502, 23-503, 23-504,
23-505, 23-506, 23-507, 23-508, 23-509, 23-510, 23-511, or 23-512
of SEQ ID NO: 1218, and secreted sALPs of the invention include
those containing amino acid residues 18-497, 18-498, 18-499,
18-500, 18-501, 18-502, 18-503, 18-504, 18-505, 18-506, 18-507,
18-508, 18-509, 18-510, 18-511, or 18-512 of SEQ ID NO: 1219. In
these consensus sequences (SEQ ID NOs: 1218 and 1219), X is any
amino acid but is not an amino acid corresponding to a pathogenic
mutation at that position of human TNALP. Examples of such
pathogenic mutations are listed below and provided in Table 1.
TABLE-US-00001 TABLE 1 Pathogenic mutations for human TNALP* Amino
acid change Non- standardized Standardized Clinical form in
Genotype of Exon Base change nomenclature nomenclature Reference
patient patient % WT ref. E. coli Total number of mutations 188 1
c.-195C > T Taillandier et al. 2000 perinatal c.- na Affects
transcription 195C > T/C184Y start site 2 c.17T > A L-12X
p.L6X Taillandier et al. 2000 childhood L-12X/? na Nonsense
mutation 2 c.50C > T S-1F p.S17F Mornet et al. 1998 infantile
S-1F/G58S 19.0 1 na 3 c.83A > G Y11C p.Y28C Taillandier et al.
2001 infantile Y11C/R119H 7.2 2 - 3 c.98C > T A16V p.A33V
Henthorn et al. 1992 childhood A16V/Y419H - 3 c.110T > C L20P
p.L37P Versailles lab October 2003 perinatal L20P/L20P + 3 c.119C
> T A23V p.A40V Mornet et al. 1998 perinatal A23V/G456S 2.3 1 +
3 c.132C > T Q27X p.Q44X Mornet E, unpublished perinatal
Q27X/c.662insG na Nonsense mutation 3 c.151G > T A34S p.A51S
Mumm et al. 2002 infantile A34S/T117H + 3 c.152G > T A34V p.A51V
Taillandier et al. 2001 infantile A34V/V442M + 4 c.184A > T M45L
p.M62L Taillandier et al. 1999 infantile M45L/c.1172delC 27.4 1 + 4
c.184A > G M45V p.M62V Spentchian et al. 2003 infantile
M45V/M45V + 4 c.186G > C M45I p.M62I Taillandier et al. 2005
childhood M45I/E174K 0 16 + 4 c.187G > C G46R p.G63R Spentchian
et al. 2003 infantile G46R/G46R + 4 c.188G > T G46V p.G63V
Lia-Baldini et al. 2001 infantile G46V/N 0.8 3 + 4 c.203C > T
T51M p.T68M Orimo et al. 2002 childhood T51M/A160T 5.2 4 + 4 c.211C
> T R54C p.R71C Henthorn et al. 1992 infantile R54C/D277A 0 17 +
4 c.211C > A R54S p.R71S Orimo et al. 2002 childhood R54S/? 2.9
4 + 4 c.212G > C R54P p.R71P Henthorn et al. 1992 perinatal
R54P/Q190P + 4 c.212G > A R54H p.R71H Taillandier et al. 2001
perinatal A23V/R54H + 4 c.219T > C I55T p.I72T Versailles lab
October 2004 odonto I55T/N - 4 c.223G > A G58S p.G75S Mornet et
al. 1998 infantile S-1F/G58S 3.5 1 + 4 c.227A > G Q59R p.Q76R
Mornet et al. 2001 infantile Q59R/T117N - IVS4 c.298-2A > G
Taillandier et al. 2000 perinatal c.298- na This mutation affects
2A > G/c.997 + 3A > C splicing and not coding sequence 5
c.299C > T T83M p.T100M Mornet et al. 2001 infantile T83M/E174K
+ 5 c.303_311del N85_N87del p.N102_N104del Versailles lab July 2007
perinatal c.303_311del/ na Deletion G474R 5 c.323C > T P91L
p.P108L Herasse et al. 2003 odonto P91L/N 0.4 unp. - 5 c.331G >
A A94T p.A111T Goseki-Sone et al. odonto A94T/? + 1998 5 c.334G
> A G95S p.G112S Witters et al. 2004 infantile G95S/R374C - 5
c.340G > A A97T p.A114T Mumm et al. 2001 infantile A97T/D277A +
5 c.341C > G A97G p.A114G Draguet et al. 2004 perinatal A97G +
c.348_349insACCGTC/ + G309R 5 c.348_349insACCGTC Draguet et al.
2004 perinatal A97G + c.348_349insACCGTC/ na Two missense G309R
mutations and insertion 5 c.346G > T A99S p.A116S Versailles lab
July 2007 adult A99S/N400S + 5 c.346G > A A99T p.A116T Hu et al.
2000 adult A99T/N 0.8 3 + 5 c.358G > A G103R p.G120R Mornet et
al. 1998 perinatal G103R/648 + 1G > A + 5 c.368C > A A106D
p.A123D Spentchian et al. 2006 perinatal A106D/S249_H250del - 5
c.382G > A V111M p.V128M Mumm et al. 2002 perinatal V111M/R206W
- 5 c.385G > A G112R p.G129R Mornet et al. 1998 perinatal
G112R/G474R + 5 c.388_391delGTAA Spentchian et al. 2003 perinatal
E294K/388_391delGTAA na Frameshift mutation 5 c.389delT Spentchian
et al. 2003 perinatal c.389delT/c.389delT na Frameshift mutation 5
c.392delG Mumm et al. 2002 perinat/infant c.392delG/A331T na
Frameshift mutation 5 c.394G > A A115T p.A132T Versailles lab
July 2006 adult A115T/E174K 5 c.395C > T A115V p.A132V Watanabe
et al. 2001 adult A115V/? 16.9 14 - 5 c.400_401AC > T117H
p.T134H Mumm et al. 2002 perinatal T117H/F310del - CA 5 c.401C >
A T117N p.T134N Taillandier et al. 2000 perinatal T117N/T117N 20.5
5 - 5 c.406C > T R119C p.R136C Versailles lab October 2003
odonto R119C/R119H - 5 c.407G > A R119H p.R136H Taillandier et
al. 1999 infantile R119H/G145V 33.4 1 - 5 c.442A > G T131A
p.T148A Michigami et al. 2005 perinatal T131A/? - 5 c.443C > T
T131I p.T148I Spentchian et al. 2003 infantile T131I/G145S - 6
c.480delT Versailles lab. January perinatal c.480delT/R206W na
deletion 2008 6 c.484G > A G145S p.G162S Spentchian et al. 2003
infantile T131I/G145S + 6 c.485G > T G145V p.G162V Taillandier
et al. 1999 infantile R119H/G145V 1.3 1 + 6 c.500C > T T150M
p.T167M Versailles lab October 2003 infantile T150M/E174K 0 + 6
c.508A > G N153D p.N170D Mornet et al. 1998 perinatal
N153D/N153D 0 13 - 6 c.511C > T H154Y p.H171Y Taillandier et al.
1999 infantile H154Y/E174K 2.1 1 - 6 c.512A > G H154R p.H171R
Mornet E, unPublished adult H154R/E174K - 6 c.526G > A A159T
p.A176T Taillandier et al. 2000 childhood A159T/R229S 45.4 5 + 6
c.529G > A A160T p.A177T Goseki-Sone et al. adult A160T/F310L
83.8 4 - 1998 6 c.535G > A A162T p.A179T Weiss et al. 1988
perinatal A162T/A162T 18 6 + 6 c.542C > T S164L p.S181L
Lia-Baldini et al. 2001 infantile S164L/del(ex12) 1.3 3 - 6
c.544delG Taillandier et al. 1999 perinatal G232V/544delG na
Frameshift mutation 6 c.550C > T R167W p.R184W Mornet et al.
1998 perinatal R167W/W253X 0.6 3 + 6 c.567C > A D172E p.D189E
Spentchian et al. 2003 perinatal D172E/D172E - 6 c.568_570delAAC
N173del p.N190del Michigami et al. 2005 perinatal
c.1559delT/N173del - Deletion of 1 a.a. 6 c.571G > A E174K
p.E191K Henthorn et al. 1992 infantile E174K/D361V 88.0 1 - 6
c.572A > G E174G p.E191G Goseki-Sone et al. odonto
E174G/c.1559delT - 1998 6 c.575T > C M175T p.M192T Versailles
lab July 2007 infantile M175T/E294K - 6 c.577C > G P176A p.P193A
Mumm et al. 2002 adult A97T/P176A + 6 c.602G > A C184Y p.C201Y
Taillandier et al. 1999 perinatal c.- - 195C > T/C184Y 6 c.609C
> G D186E p.D203E Versailles lab October 2004 perinatal
D186E/D186E - 6 c.620A > C Q190P p.Q207P Henthorn et al. 1992
perinatal R54P/Q190P + 6 c.631A > G N194D p.N211D Taillandier et
al. 2001 infantile A99T/N194D + 6 c.634A > T I195F p.I212F Souka
et al. 2002 perinatal I195F/E337D - IVS6 c.648 + 1G > T
Brun-Heath et al. 2005 perinatal c.648 + 1G > T/ Affects
splicing D277A IVS6 c.648 + 1G > A Mornet et al. 1998 perinatal
G103R/c.648 + na Affects splicing 1G > A IVS6 c.649- Versailles
lab July 2006 perinatal c.649- Frameshift mutation 1_3delinsAA
1_3delinsAA/c.649- 1_3delinsAA 7 c.653T > C I201T p.I218T Utsch
et al., 2005, perinatal I201T/R374C 3.7 unp. - contact 7 659G >
T G203V p.G220V Taillandier et al. 2001 odonto E174K/G203V + 7 659G
> C G203A p.G220A Spentchian et al. 2003 perinatal G203A/G203A +
7 662insG Mornet E, unpublished perinatal Q27X/662insG na
Frameshift mutation 7 c.662delG Spentchian et al. 2003 perinatal
R255L/c.662delG na Frameshift mutation 7 c.662G > T G204V
p.G221V Versailles lab October 2004 perinatal G204V/M338T + 7
c.667C > T R206W p.R223W Mornet et al. 1998 perinatal R206W/?
2.8 3 - 7 c.668G > A R206Q p.R223Q Mumm et al. 2002 perinatal
R206Q/deletion - 7 c.670A > G K207E p.K224E Mochizuki et al.
2000 infantile K207E/G409C 43 15 + 7 c.677T > C M209T p.M226T
Baumgartner-Sigl et al. infantile M209T/T354I - 2007 7 c.704A >
G E218G p.E235G Taillandier et al. 2001 adult E218G/A382S 3.6 7 + 7
c.738G > T R229S p.R246S Taillandier et al. 2000 childhood
A159T/R229S 4.4 5 - 7 c.746G > T G232V p.G249V Fedde et al. 1996
perinatal G232V/N 34.5 3 + 7 c.971A > G K247R p.K264R Versailles
lab January 2007 perinatal K247R/D361V - 8 c.797_802del
S249_H250del p.S266_H267del Spentchian et al. 2006 perinatal
A106D/S249_H250del Deletion of 2 a.a. 8 c.809G > A W253X p.W270X
Mornet et al. 1998 perinatal R167W/W253X na Nonsense mutation 8
c.814C > T R255C p.R272C Spentchian et al. 2006 perinatal
R255C/T117H - 8 c.815G > T R255L p.R272L Spentchian et al. 2003
perinatal R255L/c.662delG - 8 c.815G > A R255H p.R272H
Brun-Heath et al. 2005 infantile R255H/R255H 6.8 16 - 8 c.824T >
C L258P p.L275P Orimo et al. 2002 childhood L258P/A160T 3.3 4 - 8
c.853_854insGATC Y268X p.Y285X Michigami et al. 2005 perinatal
c1559delT/Y268X na Nonsense mutation IVS8 c.862 + 5G > A
Taillandier et al. 1999 infantile c.862 + 5G > A/c.862 + na
Affects splicing 5G > A 9 c.865C > T L272F p.L289F Sugimoto
et al. 1998 infantile L272F/? 50 8 - 9 c.871G > A E274K p.E291K
Mornet et al. 1998 infantile E174K/E274K 8.3 1 - 9 c.871G > T
E274X p.E291X Taillandier et al. 2000 perinatal A94T/E274X -
Nonsense mutation 9 c.874C > A P275T p.P292T Brun-Heath et al.
2005 infantile P275T/A16V 4.0 16 + 9 c.876_881delAGGGGA
G276_D277del Spentchian et al. 2003 perinatal G276_D277del/ na
c.962delG 9 c.880G > T D277Y p.D294Y Taillandier et al. 2001
infantile A159T/D277Y - 9 c.881A > C D277A p.D294A Henthorn et
al. 1992 infantile R54C/D277A 0 17 - 9 c.883A > G M278V p.M295V
Mornet et al. 2001 childhood E174K/M278V - 9 c.884T > C M278T
p.M295T Brun-Heath et al. 2005 perinatal M278T/R206W 8.5 16 - 9
c.885G > A M278I p.M295I Michigami et al. 2005 perinatal
M278I/c.1559delT - 9 c.889T > G Y280D p.Y297D Brun-Heath et al.
2005 childhood R119H/Y280D 1.3 16 - 9 c.892G > A E281K p.E298K
Orimo et al. 1994 infantile E281K/1559delT - 9 c.896T > C L282P
p.L299P Versailles lab October 2003 infantile L282P/L282P 9.7 15 -
9 c.917A > T D289V p.D306V Taillandier et al. 1999 infantile
D289V/D289V 0 12 - 9 c.919C > T P290S p.P307S Versailles lab
October 2004 infantile P290S/M450T + 9 c.920C > T P290L p.P307L
Versailles lab July 2006 childhood P290L/S164L 10 c.1062G > C
E337D p.E354D Souka et al. 2002 perinatal I195F/E337D + 10 c.1064A
> C M338T p.M355T Versailles lab October 2004 perinatal
G204V/M338T - 10 c.1065G > A M338I p.M355I Versailles lab.
January infantile M338I/R374C - 2008 10 c.1101_1103delCTC S351del
p.S368del Versailles lab October 2004 perinatal c.1101_1103delCTC/
Deletion of 1 a.a. T372I 10 c.1112C > T T354I p.T371I
Baumgartner-Sigl et al. infantile
M209T/T354I - 2007 10 c.1120G > A V357M p.V374M Versailles lab
October 2004 adult V357M/E281K + 10 c.1130C > T A360V p.A377V
Mornet et al. 2001 perinatal A360V/A360V + 10 c.1133A > T D361V
p.D378V Henthorn et al. 1992 infantile E174K/D361V 1.2 3 + 10
c.1142A > G H364R p.H381R Taillandier et al. 2000 infantile
A23V/H364R + 10 c.1144G > A V365I p.V382I Goseki-Sone et al.
childhood F310L/V365I 0 11 + 1998 10 c.1166C > T T372I p.T389I
Versailles lab October 2004 perinatal T372I/S351del - 10 c.1171C
> T R374C p.R391C Zurutuza et al. 1999 childhood E174K/R374C
10.3 1 - 10 c.1172G > A R374H p.R391H Orimo et al. 2002
childhood R374H/? 3.7 14 - 10 c.1172delC Taillandier et al. 1999
infantile M45L/c.1172delC na Frameshift mutation 10 c.1175G > C
G375A p.G392A Versailles lab. January perinatal G375A/R119C - 2008
10 c.1182T > C I378T p.I395T Versailles lab July 2006 perinatal
I378T/E174K 11 c.1195G > T A382S p.A399S Taillandier et al. 2001
adult E218G/A382S - 11 c.1196C > T A382V p.A399V Spentchian et
al. 2006 adult A382V/A16V - 11 c.1199C > T P383L p.P400L
Spentchian et al. 2006 infantile P383L/P383L + 11 c.1214_1215delCA
Versailles lab July 2006 adult c.1214_1215delCA/ Frameshift
mutation E174K 11 c.1216_1219delGACA Brun-Heath et al. 2005
perinatal c.1216_1219delGACA/? 11 c.1217A > G D389G p.D406G
Taillandier et al. 2000 odonto. D389G/R433H 14.9 5 + 11 c.1228T
> C F393L p.F410L Versailles lab October 2004 infantile
F393L/E174K - 11 c.1231A > G T394A p.T411A Brun-Heath et al.
2005 perinatal T394A/c.926_927delTC 0.3 16 - 11 c.1240C > A
L397M p.L414M Mumm et al. 2002 perinatal L397M/D277A - 11 c.1250A
> G N400S p.N417S Sergi et al. 2001 perinatal N400S/c.648 + 3
unp. + 1G > A 11 c.1256delC Taillandier et al. 2000 perinatal
c.1256delC/? na Frameshift mutation 11 c.1258G > A G403S p.G420S
Glaser et al. 2004 perinatal G403S/G403S 0.4 unp. - 11 c.1268T >
C V406A p.V423A Taillandier et al. 2001 perinatal A99T/V406A 15.7 2
- 11 c.1270G > A V407M p.V424M Versailles lab January 2007 adult
V407M/V407M - 11 c.1276G > T G409C p.G426C Mochizuki et al. 2000
infantile K207A/G409C 18.5 15 - 11 c.1277G > A G409D p.G426D
Mumm et al. 2002 childhood G409D/E174K - 11 c.1282C > T R411X
p.R428X Taillandier et al. 1999 perinatal R411X/R411X na Nonsense
mutation 11 c.1283G > C R411P p.R428P Spentchian et al. 2006
perinatal R411P/c.997 + - 2T > A 11 c.1285G > A E412K p.E429K
Versailles lab July 2006 odonto. E412K/? 11 c.1306T > C Y419H
p.Y436H Henthorn et al. 1992 childhood A16V/Y419H na 12 c.1333T
> C S428P p.S445P Mornet et al. 1998 infantile S428P/? 2.1 1 -
12 c.1349G > A R433H p.R450H Taillandier et al. 2000 odonto.
D389G/R433H - 12 c.1348C > T R433C p.R450C Mornet et al. 1998
infantile R433C/R433C 4.0 1 - 12 c.1354G > A E435K p.E452K
Spentchian et al. 2003 perinatal A94T/E435K + 12 c.1361A > G
H437R p.H454R Versailles lab October 2003 childhood E174K/H437R +
12 c.1363G > A G438S p.G455S Draguet et al. 2004 adult
G438S/G474R - 12 c.1364G > A G438D p.G455D Versailles lab
January 2007 perinatal G438D/G438D - 12 c.1366G > T G439W
p.G456W Versailles lab October 2003 childhood G439W/? + 12 c.1366G
> A G439R p.G456R Ozono et al. 1996 infantile G439R/? 1.5 unp. +
12 c.1375G > A V442M p.V459M Taillandier et al. 2000 infantile
A34V/V442M + 12 c.1375G > T V442L p.V459L Versailles lab October
2004 perinatal V442L/E435K - 12 c.1396C > T P449L p.P466L
Versailles lab October 2003 perinatal P449L/? + 12 c.1400T > C
M450T p.M467T Versailles lab October 2004 infantile M450T/P290S -
12 c.1402G > A A451T p.A468T Spentchian et al. 2003 perinatal
A451T/A451T + 12 c.1417G > A G456S p.G473S Mornet et al. 1998
perinatal A23V/G456S + 12 c.1426G > A E459K p.E476K Taillandier
et al. 1999 perinatal A94T/E459K + 12 c.1427A > G E459G p.E476G
Mornet et al. 2001 perinatal E459G/E459G + 12 c.1433A > T N461I
p.N478I Taillandier et al. 2000 childhood N461I/N 1.1 3 - 12
c.1444_1445insC Brun-Heath et al. 2005 perinatal c.1444_1445insC/
Frameshift mutation G317D 12 c.1456G > C C472S p.C489S
Taillandier et al. 2000 perinatal C472S/c.997 + 9.4 5 - 2T > A
12 c.1468A > T I473F p.I490F Lia-Baldini et al. 2001 adult
I473F/? 37.1 3 - 12 c.1471G > A G474R p.G491R Mornet et al. 1998
perinatal G112R/G474R - 12 c.1471delG Brun-Heath et al. 2005 odonto
c.1471delG/R119H Frameshift mutation 12 c.1559delT Orimo et al.
1994 infantile E281K/c.1559delT 28 18 na Frameshift mutation Large
deletions deletion of Spentchian et al. 2006 perinatal homozygote
exons 3-5 deletion of Spentchian et al. 2006 infantile compound
exon 12 heterozygote (3' part) with S164L *In the column labeled
"Non-standardized nomenclature" under "Amino acid change," the
position of the mutation is provided with respect to mature human
TNALP lacking the N-terminal signal sequence. In the column labeled
"Standardized nomenclature" under "Amino acid change," the position
is provided with respect to full length human TNALP having the
17-amino acid N-terminal signal sequence (SEQ ID NO: 1208).
[0229] In some embodiments, the sALP polypeptides of the invention
do not include any of the mutations provided in Table 1. In
particular, the sALP polypeptides of the invention, using the
numbering of the consensus sequence of SEQ ID NO: 1218 (FIG. 10),
the amino acid at position 22 is not a phenylalanine residue; the
amino acid at position 33 (position 11 in the sequence without
signal peptide) is not a cysteine residue; the amino acid at
position 38 (position 16 in the sequence without signal peptide) is
not a valine residue; the amino acid at position 42 (position 20 in
the sequence without signal peptide) is not a proline residue; the
amino acid at position 45 (position 23 in the sequence without
signal peptide) is not a valine residue; the amino acid residue at
position 56 (position 34 in the sequence without signal peptide) is
not a serine or a valine residue; the amino acid residue at
position 67 (position 45 in the sequence without signal peptide) is
not a leucine, an isoleucine or a valine residue; the amino acid
residue at position 68 (position 46 in the sequence without signal
peptide) is not a valine residue; the amino acid residue at
position 73 (position 51 in the sequence without signal peptide) is
not a methionine residue; the amino acid residue at position 76
(position 54 in the sequence without signal peptide) is not a
cysteine, a serine, a proline or a histidine residue; the amino
acid residue at position 77 (position 55 in the sequence without
signal peptide) is not a threonine residue; the amino acid residue
at position 80 (position 58 in the sequence without signal peptide)
is not a serine residue; the amino acid residue at position 81
(position 59 in the sequence without signal peptide) is not an
asparagine residue; the amino acid residue at position 105
(position 83 in the sequence without signal peptide) is not a
methionine residue; the amino acid residue at position 113
(position 89 in the sequence without signal peptide) is not a
leucine residue; the amino acid residue at position 116 (position
94 in the sequence without signal peptide) is not a threonine
residue; the amino acid residue at position 117 (position 95 in the
sequence without signal peptide) is not a serine residue; the amino
acid residue at position 119 (position 97 in the sequence without
signal peptide) is not a glycine residue; the amino acid residue at
position 121 (position 99 in the sequence without signal peptide)
is not a serine or a threonine residue; the amino acid residue at
position 125 (position 103 in the sequence without signal peptide)
is not an arginine residue; the amino acid residue at position 128
(position 106 in the sequence without signal peptide) is not a
aspartic acid residue; the amino acid residue at position 133
(position 111 in the sequence without signal peptide) is not a
methionine residue; the amino acid residue at position 134
(position 112 in the sequence without signal peptide) is not an
arginine residue; the amino acid residue at position 137 (position
115 in the sequence without signal peptide) is not a threonine or a
valine residue; the amino acid residue at position 139 (position
117 in the sequence without signal peptide) is not a histidine or
an asparagine residue; the amino acid residue at position 141
(position 119 in the sequence without signal peptide) is not a
histidine residue; the amino acid residue at position 153 (position
131 in the sequence without signal peptide) is not an alanine or an
isoleucine residue; the amino acid residue at position 167
(position 145 in the sequence without signal peptide) is not a
serine or a valine residue; the amino acid residue at position 172
(position 150 in the sequence without signal peptide) is not a
methionine residue; the amino acid residue at position 175
(position 153 in the sequence without signal peptide) is not an
aspartic acid residue; the amino acid residue at position 176
(position 154 in the sequence without signal peptide) is not a
tyrosine or an arginine residue; the amino acid residue at position
181 (position 159 in the sequence without signal peptide) is not a
threonine residue; the amino acid residue at position 182 (position
160 in the sequence without signal peptide) is not a threonine
residue; the amino acid residue at position 184 (position 162 in
the sequence without signal peptide) is not a threonine residue;
the amino acid residue at position 186 (position 164 in the
sequence without signal peptide) is not a leucine residue; the
amino acid residue at position 189 (position 167 in the sequence
without signal peptide) is not a tryptophan residue; the amino acid
residue at position 194 (position 172 in the sequence without
signal peptide) is not a glutamate residue; the amino acid residue
at position 196 (position 174 in the sequence without signal
peptide) is not a lysine or a glycine residue; the amino acid
residue at position 197 (position 175 in the sequence without
signal peptide) is not a threonine residue; the amino acid residue
at position 198 (position 176 in the sequence without signal
peptide) is not an alanine residue; the amino acid residue at
position 206 (position 184 in the sequence without signal peptide)
is not a tyrosine residue; the amino acid residue at position 208
(position 186 in the sequence without signal peptide) is not a
glutamate residue; the amino acid residue at position 207 (position
190 in the sequence without signal peptide) is not a proline
residue; the amino acid residue at position 216 (position 194 in
the sequence without signal peptide) is not a aspartic acid
residue; the amino acid residue at position 217 (position 195 in
the sequence without signal peptide) is not a phenylalanine
residue; the amino acid residue at position 223 (position 201 in
the sequence without signal peptide) is not a threonine residue;
the amino acid residue at position 225 (position 203 in the
sequence without signal peptide) is not a valine or an alanine
residue; the amino acid residue at position 226 (position 204 in
the sequence without signal peptide) is not a valine residue; the
amino acid residue at position 228 (position 206 in the sequence
without signal peptide) is not a tryptophan or a glutamine residue;
the amino acid residue at position 229 (position 207 in the
sequence without signal peptide) is not a glutamate residue; the
amino acid residue at position 231 (position 209 in the sequence
without signal peptide) is not a threonine residue; the amino acid
residue at position 240 (position 218 in the sequence without
signal peptide) is not a glycine residue; the amino acid residue at
position 251 (position 229 in the sequence without signal peptide)
is not a serine residue; the amino acid residue at position 254
(position 232 in the sequence without signal peptide) is not a
valine residue; the amino acid residue at position 269 (position
247 in the sequence without signal peptide) is not an arginine
residue; the amino acid residue at position 277 (position 255 in
the sequence without signal peptide) is not a cysteine, a leucine
or a histidine residue; the amino acid residue at position 280
(position 258 in the sequence without signal peptide) is not a
proline residue; the amino acid residue at position 295 (position
273 in the sequence without signal peptide) is not a phenylalanine
residue; the amino acid residue at position 297 (position 275 in
the sequence without signal peptide) is not a lysine residue; the
amino acid residue at position 298 (position 276 in the sequence
without signal peptide) is not a threonine residue; the amino acid
residue at position 300 (position 278 in the sequence without
signal peptide) is not a tyrosine or an alanine residue; the amino
acid residue at position 301 (position 279 in the sequence without
signal peptide) is not a valine, a threonine or an isoleucine
residue; the amino acid residue at position 303 (position 281 in
the sequence without signal peptide) is not an aspirate residue;
the amino acid residue at position 304 (position 282 in the
sequence without signal peptide) is not a lysine residue; the amino
acid residue at position 305 (position 283 in the sequence without
signal peptide) is not a proline residue; the amino acid residue at
position 312 (position 290 in the sequence without signal peptide)
is not a valine residue; the amino acid residue at position 313
(position 291 in the sequence without signal peptide) is not a
serine or a leucine residue; the amino acid residue at position 317
(position 295 in the sequence without signal peptide) is not a
lysine residue; the amino acid residue at position 332 (position
310 in the sequence without signal peptide) is not an arginine
residue; the amino acid residue at position 333 (position 311 in
the sequence without signal peptide) is not a cysteine, a glycine
or a leucine residue; the amino acid residue at position 334
(position 312 in the sequence without signal peptide) is not a
leucine residue; the amino acid residue at position 340 (position
318 in the sequence without signal peptide) is not an aspartic acid
residue; the amino acid residue at position 345 (position 323 in
the sequence without signal peptide) is not an arginine or a
glutamate residue; the amino acid residue at position 354 (position
332 in the sequence without signal peptide) is not a threonine
residue; the amino acid residue at position 360 (position 338 in
the sequence without signal peptide) is not an aspartic acid
residue; the amino acid residue at position 361 (position 339 in
the sequence without signal peptide) is not a threonine or an
isoleucine residue; the amino acid residue at position 377
(position 355 in the sequence without signal peptide) is not a
leucine residue; the amino acid residue at position 380 (position
358 in the sequence without signal peptide) is not a methionine
residue; the amino acid residue at position 383 (position 361 in
the sequence without signal peptide) is not a valine residue; the
amino acid residue at position 384 (position 362 in the sequence
without signal peptide) is not a valine residue; the amino acid
residue at position 387 (position 365 in the sequence without
signal peptide) is not an arginine residue; the amino acid residue
at position 388 (position 366 in the sequence without signal
peptide) is not a leucine residue; the amino acid residue at
position 395 (position 373 in the sequence without signal peptide)
is not a leucine residue; the amino acid residue at position 397
(position 375 in the sequence without signal peptide) is not a
cysteine or a histidine residue; the amino acid residue at position
398 (position 376 in the sequence without signal peptide) is not an
alanine residue; the amino acid residue at position 401 (position
379 in the sequence without signal peptide) is not a threonine
residue; the amino acid residue at position 405 (position 383 in
the sequence without signal peptide) is not a serine or a valine
residue; the amino acid residue at position 406 (position 384 in
the sequence without signal peptide) is not a leucine residue; the
amino acid residue at position 412 (position 390 in the sequence
without signal peptide) is not a glycine residue; the amino acid
residue at position 416 (position 394 in the sequence without
signal peptide) is not a leucine residue; the amino acid residue at
position 417 (position 395 in the sequence without signal peptide)
is not an alanine residue; the amino acid residue at position 420
(position 398 in the sequence without signal peptide) is not a
methionine residue; the amino acid residue at position 423
(position 401 in the sequence without signal peptide) is not a
serine residue; the amino acid residue at position 426 (position
404 in the sequence without signal peptide) is not a serine
residue; the amino acid residue at position 429 (position 407 in
the sequence without signal peptide) is not an alanine residue; the
amino acid residue at position 430 (position 408 in the sequence
without signal peptide) is not a methionine residue; the amino acid
residue at position 432 (position 410 in the sequence without
signal peptide) is not a cysteine or an aspartic acid residue;
amino acid residue at position 434 (position 412 in the sequence
without signal peptide) is not a proline residue; amino acid
residue at position 435 (position 413 in the sequence without
signal peptide) is not a lysine residue; amino acid residue at
position 442 (position 420 in the sequence without signal peptide)
is not a histidine residue; amino acid residue at position 451
(position 429 in the sequence without signal peptide) is not a
proline residue; amino acid residue at position 456 (position 434
in the sequence without signal peptide) is not a histidine or a
cysteine residue; amino acid residue at position 458 (position 436
in the sequence without signal peptide) is not a lysine residue;
amino acid residue at position 460 (position 438 in the sequence
without signal peptide) is not an arginine residue; amino acid
residue at position 461 (position 439 in the sequence without
signal peptide) is not a serine or an aspartic acid residue; amino
acid residue at position 462 (position 440 in the sequence without
signal peptide) is not a tryptophan or an arginine residue; amino
acid residue at position 465 (position 443 in the sequence without
signal peptide) is not a methionine or a leucine residue; amino
acid residue at position 472 (position 450 in the sequence without
signal peptide) is not a leucine residue; amino acid residue at
position 473 (position 451 in the sequence without signal peptide)
is not a threonine residue; amino acid residue at position 474
(position 452 in the sequence without signal peptide) is not a
threonine residue; amino acid residue at position 479 (position 457
in the sequence without signal peptide) is not a serine residue;
amino acid residue at position 482 (position 460 in the sequence
without signal peptide) is not a lysine or a glycine residue; amino
acid residue at position 484 (position 462 in the sequence without
signal peptide) is not a leucine residue; amino acid residue at
position 495 (position 473 in the sequence without signal peptide)
is not a serine residue; amino acid residue at position 496
(position 474 in the sequence without signal peptide) is not a
phenylalanine residue; and amino acid residue at position 497
(position 475 in the sequence without signal peptide) is not an
arginine residue.
Also more specifically, when a sTNALP is used in the bone targeted
sALPs of the present invention, using the numbering of the human
TNALP sequence, the amino acid at position 17 is not a
phenylalanine residue; the amino acid at position 28 (position 11
in the sequence without signal peptide) is not a cysteine residue;
the amino acid at position 33 (position 16 in the sequence without
signal peptide) is not a valine residue; the amino acid at position
37 (position 20 in the sequence without signal peptide) is not a
proline residue; the amino acid at position 40 (position 23 in the
sequence without signal peptide) is not a valine residue; the amino
acid residue at position 51 (position 34 in the sequence without
signal peptide) is not a serine or a valine residue; the amino acid
residue at position 62 (position 45 in the sequence without signal
peptide) is not a leucine, an isoleucine or a valine residue; the
amino acid residue at position 63 (position 46 in the sequence
without signal peptide) is not a valine residue; the amino acid
residue at position 68 (position 51 in the sequence without signal
peptide) is not a methionine residue; the amino acid residue at
position 71 (position 54 in the sequence without signal peptide) is
not a cysteine, a serine, a proline or a histidine residue; the
amino acid residue at position 72 (position 55 in the sequence
without signal peptide) is not a threonine residue; the amino acid
residue at position 75 (position 58 in the sequence without signal
peptide) is not a serine residue; the amino acid residue at
position 76 (position 59 in the sequence without signal peptide) is
not an asparagine residue; the amino acid residue at position 100
(position 83 in the sequence without signal peptide) is not a
methionine residue; the amino acid residue at position 108
(position 89 in the sequence without signal peptide) is not a
leucine residue; the amino acid residue at position 111 (position
94 in the sequence without signal peptide) is not a threonine
residue; the amino acid residue at position 112 (position 95 in the
sequence without signal peptide) is not a serine residue; the amino
acid residue at position 114 (position 97 in the sequence without
signal peptide) is not a glycine residue; the amino acid residue at
position 116 (position 99 in the sequence without signal peptide)
is not a serine or a threonine residue; the amino acid residue at
position 120 (position 103 in the sequence without signal peptide)
is not an arginine residue; the amino acid residue at position 123
(position 106 in the sequence without signal peptide) is not a
aspartic acid residue; the amino acid residue at position 128
(position 111 in the sequence without signal peptide) is not a
methionine residue; the amino acid residue at position 129
(position 112 in the sequence without signal peptide) is not an
arginine residue; the amino acid residue at position 132 (position
115 in the sequence without signal peptide) is not a threonine or a
valine residue; the amino acid residue at position 134 (position
117 in the sequence without signal peptide) is not a histidine or
an asparagine residue; the amino acid residue at position 136
(position 119 in the sequence without signal peptide) is not a
histidine residue; the amino acid residue at position 148 (position
131 in the sequence without signal peptide) is not an alanine or an
isoleucine residue; the amino acid residue at position 162
(position 145 in the sequence without signal peptide) is not a
serine or a valine residue; the amino acid residue at position 167
(position 150 in the sequence without signal peptide) is not a
methionine residue; the amino acid residue at position 170
(position 153 in the sequence without signal peptide) is not an
aspartic acid residue; the amino acid residue at position 171
(position 154 in the sequence without signal peptide) is not a
tyrosine or an arginine residue; the amino acid residue at position
176 (position 159 in the sequence without signal peptide) is not a
threonine residue; the amino acid residue at position 177 (position
160 in the sequence without signal peptide) is not a threonine
residue; the amino acid residue at position 179 (position 162 in
the sequence without signal peptide) is not a threonine residue;
the amino acid residue at position 181 (position 164 in the
sequence without signal peptide) is not a leucine residue; the
amino acid residue at position 184 (position 167 in the sequence
without signal peptide) is not a tryptophan residue; the amino acid
residue at position 189 (position 172 in the sequence without
signal peptide) is not a glutamate residue; the amino acid residue
at position 191 (position 174 in the sequence without signal
peptide) is not a lysine or a glycine residue; the amino acid
residue at position 192 (position 175 in the sequence without
signal peptide) is not a threonine residue; the amino acid residue
at position 193 (position 176 in the sequence without signal
peptide) is not an alanine residue; the amino acid residue at
position 201 (position 184 in the sequence without signal peptide)
is not a tyrosine residue; the amino acid residue at position 203
(position 186 in the sequence without signal peptide) is not a
glutamate residue; the amino acid residue at position 207 (position
190 in the sequence without signal peptide) is not a proline
residue; the amino acid residue at position 211 (position 194 in
the sequence without signal peptide) is not a aspartic acid
residue; the amino acid residue at position 212 (position 195 in
the sequence without signal peptide) is not a phenylalanine
residue; the amino acid residue at position 218 (position 201 in
the sequence without signal peptide) is not a threonine residue;
the amino acid residue at position 220 (position 203 in the
sequence without signal peptide) is not a valine or an alanine
residue; the amino acid residue at position 221 (position 204 in
the sequence without signal peptide) is not a valine residue; the
amino acid residue at position 223 (position 206 in the sequence
without signal peptide) is not a tryptophan or a glutamine residue;
the amino acid residue at position 224 (position 207 in the
sequence without signal peptide) is not a glutamate residue; the
amino acid residue at position 226 (position 209 in the sequence
without signal peptide) is not a threonine residue; the amino acid
residue at position 235 (position 218 in the sequence without
signal peptide) is not a glycine residue; the amino acid residue at
position 246 (position 229 in the sequence without signal peptide)
is not a serine residue; the amino acid residue at position 249
(position 232 in the sequence without signal peptide) is not a
valine residue; the amino acid residue at position 264 (position
247 in the sequence without signal peptide) is not an arginine
residue; the amino acid residue at position 272 (position 255 in
the sequence without signal peptide) is not a cysteine, a leucine
or a histidine residue; the amino acid residue at position 275
(position 258 in the sequence without signal peptide) is not a
proline residue; the amino acid residue at position 289 (position
272 in the sequence without signal peptide) is not a phenylalanine
residue; the amino acid residue at position 291 (position 274 in
the sequence without signal peptide) is not a lysine residue; the
amino acid residue at position 292 (position 275 in the sequence
without signal peptide) is not a threonine residue; the amino acid
residue at position 294 (position 277 in the sequence without
signal peptide) is not a tyrosine or an alanine residue; the amino
acid residue at position 295 (position 278 in the sequence without
signal peptide) is not a valine, a threonine or an isoleucine
residue; the amino acid residue at position 297 (position 280 in
the sequence without signal peptide) is not an aspirate residue;
the amino acid residue at position 298 (position 281 in the
sequence without signal peptide) is not a lysine residue; the amino
acid residue at position 299 (position 282 in the sequence without
signal peptide) is not a proline residue; the amino acid residue at
position 306 (position 289 in the sequence without signal peptide)
is not a valine residue; the amino acid residue at position 307
(position 290 in the sequence without signal peptide) is not a
serine or a leucine residue; the amino acid residue at position 311
(position 294 in the sequence without signal peptide) is not a
lysine residue; the amino acid residue at position 326 (position
309 in the sequence without signal peptide) is not an arginine
residue; the amino acid residue at position 327 (position 310 in
the sequence without signal peptide) is not a cysteine, a glycine
or a leucine residue; the amino acid residue at position 328
(position 311 in the sequence without signal peptide) is not a
leucine residue; the amino acid residue at position 334 (position
317 in the sequence without signal peptide) is not an aspartic acid
residue; the amino acid residue at position 339 (position 322 in
the sequence without signal peptide) is not an arginine or a
glutamate residue; the amino acid residue at position 348 (position
331 in the sequence without signal peptide) is not a threonine
residue; the amino acid residue at position 354 (position 337 in
the sequence without signal peptide) is not an aspartic acid
residue; the amino acid residue at position 355 (position 338 in
the sequence without signal peptide) is not a threonine or an
isoleucine residue; the amino acid residue at position 371
(position 354 in the sequence without signal peptide) is not a
leucine residue; the amino acid residue at position 374 (position
357 in the sequence without signal peptide) is not a methionine
residue; the amino acid residue at position 377 (position 360 in
the sequence without signal peptide) is not a valine residue; the
amino acid residue at position 378 (position 361 in the sequence
without signal peptide) is not a valine residue; the amino acid
residue at position 381 (position 364 in the sequence without
signal peptide) is not an arginine residue; the amino acid residue
at position 382 (position 365 in the sequence without signal
peptide) is not a leucine residue; the amino acid residue at
position 389 (position 372 in the sequence without signal peptide)
is not a leucine residue; the amino acid residue at position 391
(position 374 in the sequence without signal peptide) is not a
cysteine or a histidine residue; the amino acid residue at position
392 (position 375 in the sequence without signal peptide) is not an
alanine residue; the amino acid residue at position 395 (position
378 in the sequence without signal peptide) is not a threonine
residue; the amino acid residue at position 399 (position 382 in
the sequence without signal peptide) is not a serine or a valine
residue; the amino acid residue at position 400 (position 383 in
the sequence without signal peptide) is not a leucine residue; the
amino acid residue at position 406 (position 389 in the sequence
without signal peptide) is not a glycine residue; the amino acid
residue at position 410 (position 393 in the sequence without
signal peptide) is not a leucine residue; the amino acid residue at
position 411 (position 394 in the sequence without signal peptide)
is not an alanine residue; the amino acid residue at position 414
(position 397 in the sequence without signal peptide) is not a
methionine residue; the amino acid residue at position 417
(position 400 in the sequence without signal peptide) is not a
serine residue; the amino acid residue at position 420 (position
403 in the sequence without signal peptide) is not a serine
residue; the amino acid residue at position 423 (position 406 in
the sequence without signal peptide) is not an alanine residue; the
amino acid residue at position 424 (position 407 in the sequence
without signal peptide) is not a methionine residue; the amino acid
residue at position 426 (position 409 in the sequence without
signal peptide) is not a cysteine or an aspartic acid residue;
amino acid residue at position 428 (position 411 in the sequence
without signal peptide) is not a proline residue; amino acid
residue at position 429 (position 412 in the sequence without
signal peptide) is not a lysine residue; amino acid residue at
position 436 (position 419 in the sequence without signal peptide)
is not a histidine residue; amino acid residue at position 445
(position 428 in the sequence without signal peptide) is not a
proline residue; amino acid residue at position 450 (position 433
in the sequence without signal peptide) is not a histidine or a
cysteine residue; amino acid residue at position 452 (position 435
in the sequence without signal peptide) is not a lysine residue;
amino acid residue at position 454 (position 437 in the sequence
without signal peptide) is not an arginine residue; amino acid
residue at position 455 (position 438 in the sequence without
signal peptide) is not a serine or an aspartic acid residue; amino
acid residue at position 456 (position 439 in the sequence without
signal peptide) is not a tryptophan or an arginine residue; amino
acid residue at position 459 (position 442 in the sequence without
signal peptide) is not a methionine or a leucine residue; amino
acid residue at position 466 (position 449 in the sequence without
signal peptide) is not a leucine residue; amino acid residue at
position 467 (position 450 in the sequence without signal peptide)
is not a threonine residue; amino acid residue at position 468
(position 451 in the sequence without signal peptide) is not a
threonine residue; amino acid residue at position 473 (position 456
in the sequence without signal peptide) is not a serine residue;
amino acid residue at position 476 (position 459 in the sequence
without signal peptide) is not a lysine or a glycine residue; amino
acid residue at position 478 (position 461 in the sequence without
signal peptide) is not a leucine residue; amino acid residue at
position 489 (position 472 in the sequence without signal peptide)
is not a serine residue; amino acid residue at position 490
(position 473 in the sequence without signal peptide) is not a
phenylalanine residue; and amino acid residue at position 491
(position 474 in the sequence without signal peptide) is not an
arginine residue. In other specific embodiments, one or more Xs are
defined as being any of the amino acid residues found at that
position in the sequences of the alignment or a residue that
constitutes a conserved or semi-conserved substitution of any of
these amino acid residues. In other specific embodiments, X's are
defined as being any of the amino acid residues found at that
position in the sequences of the alignment. For instance, the amino
acid residue at position 51 (position 34 in the sequence without
signal peptide) is an alanine or a valine residue; the amino acid
residue at position 177 (position 160 in the sequence without
signal peptide) is an alanine or a serine residue; the amino acid
residue at position 212 (position 195 in the sequence without
signal peptide) is an isoleucine or a valine residue; the amino
acid residue at position 291 (position 274 in the sequence without
signal peptide) is a glutamic acid or an aspartic acid residue; and
the amino acid residue at position 374 (position 357 in the
sequence without signal peptide) is a valine or an isoleucine
residue.
[0231] An sALP may optionally be glycosylated at any appropriate
one or more amino acid residues.
[0232] In addition, an sALP may have at least 50% (e.g., 55%, 60%,
65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to any of the sALPs described herein.
[0233] An sALP may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
additions, deletions, or substitutions relative to any of the sALPs
described herein.
NPs
[0234] Any natriuretic peptide or variant thereof that is an
agonist of natriuretic peptide receptor B ("NPR-B"), e.g., human
NPR-B, may be used in any of the methods and compositions described
herein.
[0235] Natriuretic peptides as described herein are peptides that
are capable of agonizing NPR-B. Natriuretic peptides include atrial
natriuretic peptide (ANP), brain natriuretic peptide (BNP), and
C-type natriuretic peptide (CNP). These peptides bind to three
types of receptors that signal intracellularly to modulate
physiological functions. ANP and BNP bind preferentially to
natriuretic peptide receptor A (NPR-A) (also known as guanylyl
cyclase A (GC-A)), and CNP binds preferentially to natriuretic
peptide receptor B (NPR-B) (also known as guanylyl cyclase B
(GC-B)). All three peptides have similar affinity for natriuretic
peptide receptor C (NPR-C), which has both signaling and peptide
clearance functions. Clearance of natriuretic peptides also occurs
through the action of membrane-bound neutral endopeptidase (NEP).
Peptide binding to NPR-A or NPR-B activates the intracellular
guanylyl cyclase domain of these receptors, which produces the
second messenger cGMP. cGMP activates or inhibits multiple
signaling pathways inside the cell.
[0236] Natriuretic peptides, including CNP, which primarily
agonizes NPR-B, and ANP and BNP, which primarily agonize NPR-A,
have important roles in multiple biological processes. Multiple
sequence alignments of various NP family members and consensus
sequences are shown in FIGS. 12-14 and 15A-15G.
[0237] A key downstream effect of CNP22 and CNP53, and variants
thereof as described herein, in agonizing NPR-B is endochondral
ossification. Thus, the NPs described herein are useful, e.g., for
treating a wide array of disorders associated with overactivation
of FGFR3 and vascular smooth muscle disorders.
[0238] NPs include the schematic structure shown in FIG. 16,
wherein the ring domain is required and each of the N-terminal
extension, short segment, and C-terminal extension is optional. The
ring domain is 17 amino acid residues long, with cysteine residues
at each terminus of the ring domain (positions 1 and 17) that form
a disulfide bond. In some embodiments, the ring domain has an amino
acid sequence that falls within one of the consensus sequences
shown in FIG. 12, 14, or 15A-15G (SEQ ID NO: 6, amino acid residues
11-27 of SEQ ID NO: 30, or SEQ ID NO: 95, respectively). Any of the
ring domains shown in FIGS. 12-14 and 15A-15G may be used in an NP
as described herein.
[0239] The short segment is a segment immediately N-terminal to the
ring domain that is between 0 and 10 amino acid residues (e.g., 0,
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues) in length.
Exemplary short segments are shown immediately N-terminal to the
boxed region in FIG. 12 or FIG. 14, e.g., residues 1-5 of SEQ ID
NO: 4, or the 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues
immediately N-terminal to the conserved ring domain in any of the
species shown in FIGS. 14 and 15A-15G. In some embodiments, the
short segment consists of the 5-amino acid portion immediately
N-terminal to the conserved ring domain in any of the species shown
in FIG. 12, 14, or 15A-15G. In some embodiments, the short segment
confers increased selectivity for NPR-B relative to NPR-A.
[0240] The N-terminal extension is a region immediately N-terminal
to the short segment (if the short segment is present) or the ring
domain (if the short segment is not present) and may be of any
length, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200,
250, 300, 350, 400, 450, 500, or even more amino acid residues.
This region is absent in CNP22 but is present in CNP53 (residues
1-31 of SEQ ID NO: 11). Exemplary N-terminal extensions are the 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 225, 250, or more residues
immediately N-terminal to the short segment, e.g., of 5 amino acid
residues (if short segment is present), or immediately N-terminal
to the ring domain (if short segment is not present), of any of the
species shown in FIGS. 15A-15G. In some embodiments, the N-terminal
extension provides increased selectivity for NPR-B relative to
NPR-A.
[0241] The C-terminal extension is a region immediately C-terminal
to the ring domain and may be of any length, e.g., 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500,
or even more amino acid residues. This region is absent in CNP22
and CNP53 but is present in the hybrid peptide CDNP (SEQ ID NO:
100, FIG. 24). Additional variants of CDNP include those that have
one or more mutations that provide reduced NEP degradation, such as
those provided in FIG. 24: CDNP-N1 (SEQ ID NO: 101), CDNP-G1 (SEQ
ID NO: 102), CDNP-H1 (SEQ ID NO: 103), and CDNP-K1 (SEQ ID NO:
104).
[0242] Exemplary C-terminal extensions are shown immediately
C-terminal to the boxed region in FIG. 12, e.g., amino acid
residues 24-28 of SEQ ID NO: 1, amino acid residues 28-32 of SEQ ID
NO: 2, amino acid residues 27-32 of SEQ ID NO: 3, or amino acid
residues 24-38 of SEQ ID NO: 5. In some embodiments, the C-terminal
tail includes, or consists of, the DNP C-terminal tail (SEQ ID NO:
117), or a variant thereof having one or more addition, deletion,
or substitution mutations (e.g., SEQ ID NO: 118). For example, a
C-terminal tail of an NP may include any of the DNP C-terminal tail
mutations shown in FIG. 24. In particular, residues 1, 3, 4, 5, 6,
and/or 7 of the DNP C-terminal tail (SEQ ID NO: 117) may be
mutated, e.g., as in any of the mutations shown in FIG. 24. In some
embodiments, the C-terminal extension confers increased selectivity
for NPR-B relative to NPR-A.
[0243] An NP may optionally be glycosylated at any appropriate one
or more amino acid residues.
[0244] In addition, an NP may have at least 50% (e.g., 55%, 60%,
65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to any of the NPs described herein, or to one or more of
the ring domain, the short segment, the C-terminal extension, or
the N-terminal extension.
[0245] An NP may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
additions, deletions, or substitutions relative to any of the NPs
described herein, or to one or more of the ring domain, the short
segment, the C-terminal extension, or the N-terminal extension.
[0246] In one example, the NP can have one or more mutations that
are less sensitive to oxidation without substantially reducing
potency or efficacy. For example, residue 17 of CNP22 is one of the
less well-conserved positions in CNP22, with naturally-occurring
homologs having (without limitation) Phe, Leu, Ile, Thr, Val, or
Ser at this position (see, e.g., FIG. 14). Exemplary CNP22 variants
include CNP-F17 (SEQ ID NO: 119); CNP-L17 (SEQ ID NO: 120); CNP-I17
(SEQ ID NO: 121); CNP-T17 (SEQ ID NO: 122); CNP-V17 (SEQ ID NO:
123); CNP-A17 (SEQ ID NO: 124); CNP-S17 (SEQ ID NO: 125);
CNP-E.sub.17 (SEQ ID NO: 156); CNP-R17 (SEQ ID NO: 157); and
CNP-Y17 (SEQ ID NO: 158), where the consensus sequence is shown in
SEQ ID NO: 126 (where X can be any amino acid, including, without
limitation, Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro, Asp, Val,
Ala, or Ser) (FIG. 26). Additional exemplary CNP22 variants include
those having a point mutation at position 17, as shown in FIG. 30
(SEQ ID NOs: 126, 119-122, and 156-172), where X in SEQ ID NOs: 126
or 162 can be any amino acid, e.g., F, L, I, T, E, R, Y, C, P, or
D.
[0247] In another example, the NP can have one or more mutations
that provide increased resistance to one or more enzymes that
cleave CNP in vivo (e.g., neutral endopeptidase (NEP) and/or
insulin degrading enzyme (IDE)). Exemplary molecules are shown in
FIG. 27 (SEQ ID NOs: 127-150), FIGS. 28A-28E (SEQ ID NOs:
1001-1155), and FIG. 29 (SEQ ID NOs: 4, 115, 119, 120, 122,
128-139, 147, 148, and 150-155).
[0248] An NP as described herein may include any other sequence or
moiety, attached covalently or non-covalently, provided that the NP
has the ability to agonize NPR-B.
[0249] In some embodiments, an NP as described herein may be no
more than 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,
31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90,
100, 110, or 120 amino acid residues in length. Furthermore, in
some embodiments, an NP as described herein may be no more than
1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2,
4.4, 4.6, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8,
7.0, 7.2, 7.4, 7.6, 7.8, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.2, 9.4,
9.6, 9.8, or 10.0 kilodaltons (kDa) in molecular weight.
[0250] NPs that are suitable for use in the compositions and
methods described herein include those described, e.g., in U.S.
Pat. Nos. 5,352,770; 5,434,133; 6,020,168; 6,034,231; 6,407,211;
6,743,425; 6,818,619; 7,276,481; 7,384,917; and 7,754,852; U.S.
Application Pub. Nos. 2007-0197434; 2008-0181903; 2008-0312142;
2009-0170756; 2010-0055150; and 2010-0297021; International
Application Pub. Nos. WO 94/20534; WO 02/047871; WO 2004/047871; WO
2005/098490; WO 2008/154226; and WO 2009/067639; European
Application Pub. Nos. EP 0497368 and EP 0466174; Furuya et al.,
Biochem. Biophys. Res. Comm. 183: 964-969 (1992); Takano et al.,
Zool. Sci., 11: 451-454 (1994); Plater et al., Toxicon., 36(6):
847-857 (1998); and Inoue et al., Proc. Nat. Acad. Sci., 100(17):
10079-10084 (2003), each of which is hereby incorporated by
reference in its entirety, including all formulas, structures, and
sequences for natriuretic peptides and variants thereof. In
alternative embodiments, the NPs referenced in the present
paragraph are excluded from the compositions and methods described
herein.
[0251] In some embodiments, any of the NPs described or
incorporated by reference herein may be used in the compositions
and methods described herein without fusion to an Fc domain or to a
linker, or alternatively may be fused to any of the linkers
described herein but not to an Fc domain. Such NPs may be used to
treat a neurocutaneous syndrome, a disorder associated with
overactivation of FGFR3, e.g., achondroplasia, a bone or cartilage
disorder, a vascular smooth muscle disorder, or a condition for
elongation of bone, as described herein.
[0252] In other embodiments, any of the NPs described or
incorporated by reference herein may include a point mutation at
position 17 relative to CNP22. Wild-type CNP22 has a methionine at
position 17 relative to CNP22, which can be oxidized in vivo and/or
which can provide a peptide that is degradable by a protease. As
described herein, point mutations at position 17 relative to CNP22
could provide polypeptides having decreased degradation, while
maintaining potency. Exemplary amino acid residues at position 17
relative to CNP22 are Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro,
Asp, Gly, Ala, Ser, Val, Trp, Asn, Gln, His, or Lys, e.g., Phe,
Leu, Ile, Thr, Glu, Arg, Tyr, Cys, Pro, or Asp, e.g., Phe or Leu,
e.g., Phe, e.g., Leu. For example, the amino acid at position 17
relative to CNP22 could be Phe, Leu, Ile, Thr, Glu, Arg, Tyr, Cys,
Pro, and Asp, e.g., Phe or Leu, e.g., Phe, e.g., Leu. In another
example, the amino acid at position 17 relative to CNP22 could be
Phe, Leu, Ile, Thr, Val, Ala, or Ser. Alternatively, exemplary
amino acid residues at position 17 relative to CNP22 are Gly, Ala,
Ser, Val, Trp, Asn, Gln, His, or Lys.
[0253] Furthermore, included in the compositions and methods
described herein are nucleic acid molecules encoding any of the NPs
and fusion polypeptides described herein, as well as nucleic acid
molecules that hybridize under high stringency conditions to at
least a portion, e.g., to 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99%, or even 100%, of a nucleic acid
molecule that encodes any of the NPs or fusion polypeptides
described herein.
Fragment Crystallizable Region (Fc) Fragments
[0254] The fusion polypeptides of the invention may include an
N-terminal or C-terminal domain such as Fc, a fragment
crystallizable region of an immunoglobulin. For example, an sALP
polypeptide and/or an NP polypeptide of the invention can be a
fusion polypeptide including an Fc. An immunoglobulin molecule has
a structure that is well known in the art. It includes two light
chains (.about.23 kD each) and two heavy chains (.about.50-70 kD
each) joined by inter-chain disulfide bonds. Immunoglobulins are
readily cleaved proteolytically (e.g., by papain cleavage) into Fab
(containing the light chain and the VH and CH1 domains of the heavy
chain) and Fc (containing the C.sub.H2 and C.sub.H3 domains of the
heavy chain, along with adjoining sequences). Cleavage typically
occurs in a flexible hinge region joining the Fab and Fc regions.
For example, papain cleaves the hinge region immediately before the
disulfide bonds joining the two heavy chains.
[0255] Useful Fc fragments as described herein include the Fc
fragment of any immunoglobulin molecule, including IgG, IgM, IgA,
IgD, or IgE, and their various subclasses (e.g., IgG-1, IgG-2,
IgG-3, IgG-4, IgA-1, IgA-2), taken from any mammal (e.g., human).
The Fc fragments of the invention may include, for example, the
C.sub.H2 and C.sub.H3 domains of the heavy chain, as well as any
portion of the hinge region. Furthermore, the Fc region may
optionally be glycosylated at any appropriate one or more amino
acid residues, e.g., various amino acid residues known to those
skilled in the art. In some embodiments, the Fc fragment is of
human IgG-1. In particular embodiments, the Fc fragment of the
fusion polypeptide has the amino acid sequence of SEQ ID NO: 401,
or has at least 50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more) sequence identity to SEQ ID NO: 401
(FIG. 7).
[0256] In some embodiments, engineered, e.g., non-naturally
occurring, Fc regions may be utilized in the compositions and
methods of the invention, e.g., as described in International
Application Pub. No. WO2005/007809, which is hereby incorporated by
reference.
[0257] An Fc fragment as described herein may have 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,
40, 50, or more additions, deletions, or substitutions relative to
any of the Fc fragments described herein.
Linkers
[0258] The fusion proteins described herein may include a peptide
linker region between the Fc fragment and the sALP or between the
Fc fragment and the NP. In addition, a peptide linker region may be
included between the Fc fragment and the optional bone-targeting
moiety. The linker region may be of any sequence and length that
allows the sALP or the NP to remain biologically active, e.g., not
sterically hindered. Exemplary linker lengths are between 1 and 200
amino acid residues, e.g., 1-5, 6-10, 11-15, 16-20, 21-25, 26-30,
31-35, 36-40, 41-45, 46-50, 51-55, 56-60, 61-65, 66-70, 71-75,
76-80, 81-85, 86-90, 91-95, 96-100, 101-110, 111-120, 121-130,
131-140, 141-150, 151-160, 161-170, 171-180, 181-190, or 191-200
amino acid residues. Additional exemplary linker lengths are 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, or 200 amino acid residues.
Additional exemplary linker lengths are 14-18, 20-24, 26-30, 32-36,
38-42, and 44-48 amino acid residues.
[0259] In some embodiments, linkers include or consist of flexible
portions, e.g., regions without significant fixed secondary or
tertiary structure. Exemplary flexible linkers are glycine-rich
linkers, e.g., containing at least 50%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95%, or even 100% glycine residues. Linkers may also
contain, e.g., serine residues. In some cases, the amino acid
sequence of linkers consists only of glycine and serine
residues.
[0260] In some cases, the amino acid sequence of the linker
sequence includes or consists of a sequence according to the
formula [(Gly).sub.m(Ser)].sub.n(Gly).sub.p, where each of m, n,
and p is, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, m=1, 2, 3,
4, 5, or 6; n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p=0, 1, 2, 3,
or 4. Alternatively, the linker sequence includes or consists of a
sequence according to the formula
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n, where each of m, n, and p is,
independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, or 20. In some embodiments, m=1, 2, 3, 4, 5, or
6; n=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and p=0, 1, 2, 3, or 4.
[0261] Exemplary combinations of m, n, and p values for either of
the preceding two formulae are listed in Table 2.
TABLE-US-00002 TABLE 2 m n p N/A 0 1 N/A 0 2 N/A 0 3 N/A 0 4 N/A 0
5 N/A 0 6 N/A 0 7 N/A 0 8 N/A 0 9 N/A 0 10 1 1 0 1 2 0 1 3 0 1 4 0
1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 1 10 0 2 1 0 2 2 0 2 3 0 2 4 0 2 5 0
2 6 0 2 7 0 2 8 0 2 9 0 2 10 0 3 1 0 3 2 0 3 3 0 3 4 0 3 5 0 3 6 0
3 7 0 3 8 0 3 9 0 3 10 0 4 1 0 4 2 0 4 3 0 4 4 0 4 5 0 4 6 0 4 7 0
4 8 0 4 9 0 4 10 0 5 1 0 5 2 0 5 3 0 5 4 0 5 5 0 5 6 0 5 7 0 5 8 0
5 9 0 5 10 0 6 1 0 6 2 0 6 3 0 6 4 0 6 5 0 6 6 0 6 7 0 6 8 0 6 9 0
6 10 0 1 1 1 1 2 1 1 3 1 1 4 1 1 5 1 1 6 1 1 7 1 1 8 1 1 9 1 1 10 1
2 1 1 2 2 1 2 3 1 2 4 1 2 5 1 2 6 1 2 7 1 2 8 1 2 9 1 2 10 1 3 1 1
3 2 1 3 3 1 3 4 1 3 5 1 3 6 1 3 7 1 3 8 1 3 9 1 3 10 1 4 1 1 4 2 1
4 3 1 4 4 1 4 5 1 4 6 1 4 7 1 4 8 1 4 9 1 4 10 1 5 1 1 5 2 1 5 3 1
5 4 1 5 5 1 5 6 1 5 7 1 5 8 1 5 9 1 5 10 1 6 1 1 6 2 1 6 3 1 6 4 1
6 5 1 6 6 1 6 7 1 6 8 1 6 9 1 6 10 1 1 1 2 1 2 2 1 3 2 1 4 2 1 5 2
1 6 2 1 7 2 1 8 2 1 9 2 1 10 2 2 1 2 2 2 2 2 3 2 2 4 2 2 5 2 2 6 2
2 7 2 2 8 2 2 9 2 2 10 2 3 1 2 3 2 2 3 3 2 3 4 2 3 5 2 3 6 2 3 7 2
3 8 2 3 9 2 3 10 2 4 1 2 4 2 2 4 3 2 4 4 2 4 5 2 4 6 2 4 7 2 4 8 2
4 9 2 4 10 2 5 1 2 5 2 2 5 3 2 5 4 2 5 5 2 5 6 2 5 7 2 5 8 2 5 9 2
5 10 2 6 1 2 6 2 2 6 3 2 6 4 2 6 5 2 6 6 2 6 7 2 6 8 2 6 9 2 6 10 2
1 1 3 1 2 3 1 3 3 1 4 3 1 5 3 1 6 3 1 7 3 1 8 3 1 9 3 1 10 3 2 1 3
2 2 3 2 3 3 2 4 3 2 5 3 2 6 3 2 7 3 2 8 3 2 9 3 2 10 3 3 1 3 3 2 3
3 3 3 3 4 3 3 5 3 3 6 3 3 7 3 3 8 3 3 9 3 3 10 3 4 1 3 4 2 3 4 3 3
4 4 3 4 5 3 4 6 3 4 7 3 4 8 3 4 9 3 4 10 3 5 1 3 5 2 3 5 3 3 5 4 3
5 5 3 5 6 3 5 7 3 5 8 3 5 9 3 5 10 3 6 1 3 6 2 3 6 3 3 6 4 3 6 5
3
6 6 3 6 7 3 6 8 3 6 9 3 6 10 3 1 1 4 1 2 4 1 3 4 1 4 4 1 5 4 1 6 4
1 7 4 1 8 4 1 9 4 1 10 4 2 1 4 2 2 4 2 3 4 2 4 4 2 5 4 2 6 4 2 7 4
2 8 4 2 9 4 2 10 4 3 1 4 3 2 4 3 3 4 3 4 4 3 5 4 3 6 4 3 7 4 3 8 4
3 9 4 3 10 4 4 1 4 4 2 4 4 3 4 4 4 4 4 5 4 4 6 4 4 7 4 4 8 4 4 9 4
4 10 4 5 1 4 5 2 4 5 3 4 5 4 4 5 5 4 5 6 4 5 7 4 5 8 4 5 9 4 5 10 4
6 1 4 6 2 4 6 3 4 6 4 4 6 5 4 6 6 4 6 7 4 6 8 4 6 9 4 6 10 4
[0262] In some embodiments, the amino acid sequence of the linker
(e.g., between the Fc and the sALP, or between the Fc and the NP,
or between the Fc and the optional bone-targeting moiety) includes
or consists of a sequence in Table 3.
TABLE-US-00003 TABLE 3 SEQ ID Linker sequence NO. G 301 GG 302 GGG
303 GGGG 304 GGGGS 305 GGGGSG 306 GGGGSGGGGSGGGG 307
GGGGSGGGGSGGGGSG 308 GGGGSGGGGSGGGGSGGGGSGG 309
GGGGSGGGGSGGGGSGGGGSGGGGSGGG 310 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG
311 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 312
GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG 313 KGANKK 314
KGANQK 315 KGANKQ 316 KGANQQ 317 QGANKK 318 QGANQK 319 QGANKQ 320
QGANQQ 321 GGGGSGGGGSKGANKK 322 GGGGSGGGGSKGANQK 323
GGGGSGGGGSKGANKQ 324 GGGGSGGGGSKGANQQ 325 GGGGSGGGGSQGANKK 326
GGGGSGGGGSQGANQK 327 GGGGSGGGGSQGANKQ 328 GGGGSGGGGSQGANQQ 329
GGGDLQVDTQSQAAWAQLLQEHPNAQQYKGANKK 330
GGGGSGGGGSGGGGSGGGGSGGGGSGGGKGANKK 331
GGGGSGGGGSGGGGSGGGGSGGGGSGGGKGANQK 332
GGGGSGGGGSGGGGSGGGGSGGGGSGGGQGANQQ 333 QEHPNARKYKGANKK 334
GQEHPNARKYKGANKK 335 PGQEHPNARKYKGANKK 336 SGGGGSGGGGSGGGG 337
ASTSPANPQPAASSP 338 PSSAAPQPNAPSTSA 339 SGGGGSGGGKGANKK 340
SGGGGSGGGQGANQQ 341 SGGGGSGGGKGANKQ 342 SGGGGSGGGKGANQK 343
SGGGGSGGGQGANKK 344 SGGGGSGGGKGANQQ 345 SGGGGSGGGQGANQK 346
SGGGGSGGGQGANKQ 347 ASTSPANPQPAASSG 348 GSSAAPQPNAPSTSA 349
GSSAAPRPNAPSTSAGLSKG 350 ASTSPANPRPAASSG 351 HGPQGQEHPNARKYKGANKK
352 HKLRGQEHPNARKYKGANKK 353 GHGPQGQEHPNARKYKGANKK 354
GHKLRGQEHPNARKYKGANKK 355 GGHGPQGQEHPNARKYKGANKK 356
GGHKLRGQEHPNARKYKGANKK 357 GGGHGPQGQEHPNARKYKGANKK 358
GGGHKLRGQEHPNARKYKGANKK 359 GGGGHGPQGQEHPNARKYKGANKK 360
GGGGHKLRGQEHPNARKYKGANKK 361 GGGGGHGPQGQEHPNARKYKGANKK 362
GGGGGHKLRGQEHPNARKYKGANKK 363 HGPQGSGGGGSGGGKGANKK 364
HKLRGSGGGGSGGGKGANKK 365 GGGHGPQGSGGGGSGGGKGANKK 366
GGGHKLRGSGGGGSGGGKGANKK 367 SGGGGQEHPNARKYKGANKK 368
GGGSGGGGQEHPNARKYKGANKK 369 SGGGGSGGGGSGGGKGANKK 370
SGGGGSGGGGSGGGGSGGGKGANKK 371 GGGSGGGGSGGGGSGGGKGANKK 372
GGGSGGGGSGGGGSGGGGSGGGKGANKK 373 HGPQG 374 HKLRG 375 GHGPQG 376
GGHGPQG 377 GGGHGPQG 378 GGGGHGPQG 379 GGGGGHGPQG 380 GHKLRG 381
GGHKLRG 382 GGGHKLRG 383 GGGGHKLRG 384 GGGGGHKLRG 385
GGQEHPNARKYKGANKK 386 GGGQEHPNARKYKGANKK 387 GGGGQEHPNARKYKGANKK
388 GGGGGQEHPNARKYKGANKK 389 LK 390 DI 391
[0263] In some embodiments, the linker may include or consist of a
[(Gly).sub.m(Ser)].sub.n(Gly).sub.p or
(Gly).sub.p[(Ser)(Gly).sub.m].sub.n linker as described above,
followed by one of SEQ ID NOs: 314-321, e.g., one of SEQ ID NOs:
314, 315, or 321.
[0264] In other embodiments of polypeptides including an sALP, the
linker may include or consist of all or a fragment of an sALP. For
example, the 17-amino acid portion of human TNALP that is an
N-terminal signal sequence, or homologs or variants or fragments
thereof (e.g., residues 1-17 of SEQ ID NOs: 1202 or 1208), may be
used as a linker. Homologs of this 17-amino acid region may be
identified, e.g., by consulting a sequence alignment such as FIG. 8
(residues 1-17 of SEQ ID NO: 1216, where X can be any amino acid)
or in FIG. 11 (residues 1-17 of SEQ ID NO: 1219, where X can be any
amino acid but not a pathogenic mutation provided in Table 1). In
another example, the C-terminal GPI anchor portion of human TNALP,
or homologs or variants or fragments thereof (e.g., residues
503-524 of SEQ ID NO: 1208) may be used as a linker. Homologs of
this C-terminal region may be identified, e.g., by consulting a
sequence alignment such as FIG. 8 (residues 503-524 of SEQ ID NO:
1216, where X can be any amino acid) or in FIG. 11 (residues
503-524 of SEQ ID NO: 1219, where X can be any amino acid but not a
pathogenic mutation provided in Table 1).
[0265] In other embodiments of polypeptides including an NP, the
linker may include or consist of all or a fragment of an NP. For
example, the 31-amino acid portion of human CNP53 that is
N-terminal to CNP22, or homologs or variants thereof (e.g.,
residues 4-34 of SEQ ID NO: 320), may be used as a linker. Homologs
of this 31-amino acid region may be identified, e.g., by consulting
a sequence alignment such as FIGS. 15A-15G and identifying the
regions corresponding to the N-terminal 31 amino acid residues of
human CNP53. Other suitable linkers may also be identified, e.g.,
by choosing any portion of an NP, optionally excluding a ring
domain, as shown in FIGS. 15A-15G, or in any other NP or region of
an NP not shown in FIGS. 15A-15G. For example, the C-terminal
extension of DNP (SEQ ID NO: 117), or fragments or variants
thereof, may be used as a linker.
[0266] A linker may optionally be glycosylated at any appropriate
one or more amino acid residues.
[0267] In addition, a linker may have at least 50% (e.g., 55%, 60%,
65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence
identity to any of the linkers described herein. In addition, a
linker may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additions,
deletions, or substitutions relative to any of the linkers
described herein.
[0268] A linker as described herein may include any other sequence
or moiety, attached covalently or non-covalently.
[0269] In some embodiments, the linker is absent, meaning that the
Fc fragment and the sALP are fused together directly or that the Fc
fragment and the NP are fused together directly, with no
intervening residues.
[0270] It should be noted that certain Fc-sALP or sALP-Fc fusion
proteins may be viewed, according to the present disclosure, either
as 1) having no linker, or as 2) having a linker which corresponds
to a portion of the sALP. For example, Fc fused directly to hsTNALP
(1-502) may be viewed, e.g., either as having no linker, wherein
the NP is hsTNALP (1-502), or as having a 17-amino acid linker,
wherein the NP is hsTNALP (18-502).
[0271] Further, it should be noted that certain Fc-NP or NP-Fc
fusion proteins may be viewed, according to the present disclosure,
either as 1) having no linker, or as 2) having a linker which
corresponds to a portion of the NP. For example, Fc fused directly
to CNP53 may be viewed, e.g., either as having no linker, wherein
the NP is CNP53, or as having a 31-amino acid linker, wherein the
NP is CNP22.
sALP Polypeptides
[0272] Any of the sALPs and linkers described herein may be
combined in an sALP polypeptide, e.g., an sALP polypeptide of
A-sALP-B, wherein each of A and B is absent or is an amino acid
sequence of at least one amino acid. When present, A and/or B can
be any linker described herein (e.g., the amino acid sequence of
any one of SEQ ID NOs: 301-391). In some embodiments, A is absent,
B is absent, or A and B are both absent.
[0273] The sALP polypeptides of the invention can optionally
include an Fc region to provide an sALP fusion polypeptide, as
described herein.
[0274] The sALP polypeptide can optionally include a bone-targeting
moiety (e.g., any described herein). In some embodiments, a linker,
e.g., a flexible linker, may be included between the bone-targeting
moiety and the sALP. For example, FIG. 5B provide polypeptides
having both a bone-targeting moiety and a linker (shown in bold)
between the Fc region and the bone-targeting moiety. In some
embodiments, the linker is a dipeptide sequence (e.g.,
leucine-lysine or aspartic acid-isoleucine) or the amino acid
sequence of any one of SEQ ID NOs: 301-391.
[0275] The sALP polypeptide can include any ALPs, mutations,
N-terminal signal sequence, C-terminal GPI sequence, and/or
linkers, or fragments thereof, described herein. For example, the
italicized regions in FIGS. 5B-5C and sequences provided in FIG. 6
or 8-11 may be used as for any of the sALPs disclosed herein (e.g.,
SEQ ID NOs: 1201, 1202, 1204-1216, 1218, and 1219).
[0276] sALP Fusion Polypeptides
[0277] Any of the sALPs, linkers, and Fc regions described herein
may be combined in a fusion polypeptide, e.g., a recombinant fusion
polypeptide, that includes the structure C-sALP-D-Fc-E,
C-Fc-D-sALP-E, C-sALP-D-Fc-G-I.sub.n-H, or C-Fc-D-sALP-G-I.sub.n-H,
where each of C, D (the linker region), E, G (linker region), and H
is absent or is an amino acid sequence of at least one amino acid;
I represents an aspartic acid or a glutamic acid; and n=10 to 16. D
and G can be absent or can optionally include any linker described
herein (e.g., the amino acid sequence of any one of SEQ ID NOs:
301-391).
[0278] The polypeptides of the invention optionally include one or
more additional amino acid residues 1) at the N-terminus of the
polypeptide, 2) between the sALP and Fc regions of the polypeptide,
and 3) at the C-terminus of the polypeptide. Thus, the invention
includes, for example, polypeptides of the form C-sALP-D-Fc-E or
the structure C-Fc-D-sALP-E, where C is one or more (e.g., 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45,
50, or more) additional amino acid residues at the N-terminus of
the polypeptide, D is one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35, 40, 45, 50, or more)
additional amino acid residues (i.e., a linker) between the sALP
and Fc regions of the polypeptide, and E is one or more (e.g., 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 20, 25, 30, 35,
40, 45, 50, or more) additional amino acid residues at the
C-terminus of the polypeptide. In a particular example, D is the
dipeptide leucine-lysine. Alternatively, any combination of C, D,
and E may be present or absent. For example, in some embodiments, C
and E are both absent, and D is absent or is an amino acid sequence
of at least one amino acid. For example, the polypeptide may
consist of the structure sALP-D-Fc or the structure Fc-D-sALP. In
some embodiments of polypeptides consisting of the structure
sALP-D-Fc or Fc-D-sALP, D may consist of two amino acid residues,
e.g., leucine-lysine. For example, the polypeptide may consist of
the structure sALP-D-Fc. Optionally, the amino acid sequence of
sALP is the amino acid sequence of SEQ ID NO: 1205, the amino acid
sequence of D is leucine-lysine, and/or the amino acid sequence of
Fc is the amino acid sequence of SEQ ID NO: 401. In some
embodiments, the amino acid sequence of the polypeptide consists of
the amino acid sequence of SEQ ID NO: 1204. In some embodiments,
polypeptide includes an amino acid sequence of at least 50% (e.g.,
55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or
more) sequence identity to the amino acid sequence of SEQ ID NOs:
1204 or 1221.
[0279] In some embodiments, the polypeptide includes a
bone-targeting moiety, e.g., having a series of consecutive Asp or
Glu residues, e.g., E.sub.6, E.sub.7, E.sub.8, E.sub.9, E.sub.10,
E.sub.11, E.sub.12, E.sub.13, E.sub.14, E.sub.15, E.sub.16,
D.sub.6, D.sub.7, D.sub.8, D.sub.9, D.sub.10, D.sub.11, D.sub.12,
D.sub.13, D.sub.14, D.sub.15, or D.sub.16. The bone-targeting
moiety, if present, may be positioned anywhere in the fusion
polypeptide, e.g., at or near the N-terminal or C-terminal end,
and/or in the linker region. For example, any one of C, D, and/or E
may include a bone-targeting moiety. In some embodiments, the
bone-targeting moiety is at the C-terminal end. For example, the
polypeptide may comprise or consist of the structure
C-sALP-D-Fc-G-I.sub.n-H or the structure C-Fc-D-sALP-G-I.sub.n-H,
where each of C, D (the linker region), G (linker region), and H is
absent or is an amino acid sequence of at least one amino acid, I
represents an aspartic acid or a glutamic acid, and n=10 to 16. In
some embodiments, C and H are both absent, and D and G can be
absent or can optionally include any linker described herein (e.g.,
the amino acid sequence of any one of SEQ ID NOs: 301-391). For
example, the polypeptide may comprise or consist of the structure
sALP-D-Fc-G-I.sub.n or the structure Fc-D-sALP-G-I.sub.n.
[0280] In some embodiments, polypeptide does not include a
bone-targeting moiety.
[0281] A fusion polypeptide as described herein may have at least
50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or more) sequence identity to any of the fusion polypeptides
described herein, e.g., SEQ ID NOs: 1201, 1204, 1220, or 1221. In
addition, a fusion polypeptide as described herein may have 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
25, 30, 35, 40, 50, or more additions, deletions, or substitutions
relative to any of the fusion polypeptides described herein.
Furthermore, in some embodiments, a fusion polypeptide as described
herein may be encoded by a nucleic acid molecule that hybridizes
under high stringency conditions to at least a portion, e.g., to
20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,
98%, 99%, or even 100%, of a nucleic acid molecule that encodes any
of the polypeptides, e.g., fusion polypeptides, described
herein.
[0282] In some embodiments, additional amino acid residues can be
introduced into the polypeptide according to the cloning strategy
used to produce the fusion polypeptides. In some embodiments, the
additional amino acid residues do not provide an additional GPI
anchoring signal so as to maintain the polypeptide in a soluble
form. Furthermore, in some embodiments, any such additional amino
acid residues, when incorporated into the polypeptide of the
invention, do not provide a cleavage site for endoproteases of the
host cell. The likelihood that a designed sequence would be cleaved
by the endoproteases of the host cell can be predicted as
described, e.g., by Ikezawa (Biol. Pharm. Bull. 25:409-417,
2002).
[0283] In certain embodiments, the polypeptides of the invention
are associated into dimers or tetramers. For example, two sALP-Fc
monomers can covalently be linked through two disulfide bonds
located in the hinge regions of the Fc fragments.
NP Polypeptides
[0284] Any of the NPs and linkers described herein may be combined
in an NP polypeptide, e.g., an NP polypeptide of V-NP-W, wherein
each of V and W is absent or is an amino acid sequence of at least
one amino acid.
[0285] The NP polypeptides of the invention can optionally include
an Fc region to provide an NP fusion polypeptide, as described
herein.
[0286] The NP polypeptide can optionally include a bone-targeting
moiety. In some embodiments, a linker, e.g., a flexible linker, may
be included between the bone-targeting moiety and the NP. For
example, FIG. 27 provides polypeptides having a linker or both a
bone-targeting moiety and a linker (SEQ ID NOs: 128-150); FIGS.
31A-31B provide polypeptides having a linker or both a
bone-targeting moiety and a linker, where X in any one of SEQ ID
NOs: 173-220 can be any amino acid, e.g., F, L, I, T, E, R, Y, C,
P, or D; and FIG. 32 provides amino acid sequences for particular
variants, where X in SEQ ID NOs: 186-198 is a leucine to provide
the sequences in SEQ ID NOs: 221-233.
[0287] The NP polypeptide can include any NPs, N-terminal
extensions, C-terminal extensions, and/or linkers described herein.
For example, the italicized regions in FIGS. 30, 31A-31B, and 32
may be used as for any of the NPs disclosed herein (see, e.g., SEQ
ID NOs: 511-516 and 553-558).
[0288] NP Fusion Polypeptides
[0289] Any of the NPs, linkers, and Fc regions described herein may
be combined in a fusion polypeptide, e.g., a recombinant fusion
polypeptide, that includes the structure X-Fc-Y--NP-Z or the
structure X-NP-Y-Fc-Z, wherein each of X, Y (the linker region),
and Z is absent or is an amino acid sequence of at least one amino
acid.
[0290] FIGS. 17A-17E depict several possible schematic structures
of fusion polypeptides as described herein. Fc-NP or NP-Fc
homodimers may be formed, e.g., due to disulfide bonds formed by Fc
(FIGS. 17A and 17B, respectively). Alternative, monomer-dimer
hybrids are possible in which an NP-Fc or Fc-NP fusion polypeptide
is joined to a free Fc domain (FIGS. 17C and 17D, respectively).
Furthermore, an NP-Fc monomer may be joined to an Fc-NP monomer, as
shown in FIG. 17E. These configurations not intended to be
exhaustive but are merely exemplary.
[0291] Exemplary fusion polypeptides having an N-terminal NP domain
and a C-terminal Fc domain include those shown in FIG. 25A and
include CNP-16AAlinker-Fc-His.sub.10 (NC1) (SEQ ID NO: 521);
CNP-6AAlinker-Fc-His.sub.10 (NC3) (SEQ ID NO: 522);
CNP-6AAlinker-Fc (SEQ ID NO: 523); CDNP-Fc (SEQ ID NO: 524), which
has no linker between the CDNP and Fc moieties; CDNP-A17saa-Fc (SEQ
ID NO: 525), which has a mutation to alanine at position 17 of the
CNP22 region and mutations S3, A4, and A5 in the DNP tail region;
and CDNP-A17sra-Fc (SEQ ID NO: 526), which has a mutation to
alanine at position 17 of the CNP22 region and mutations S3 and A5
in the DNP tail region. FIG. 25B is a listing of the nucleic acid
sequence (SEQ ID NO: 806) of NC1.
[0292] The NP domain in the fusion polypeptide can be any NP
described herein. For example, any of the molecules shown in FIGS.
30, 31A-31B, and 32, with or without the bone-targeting moiety, may
be fused to an Fc domain and may optionally further include a
linker region between the Fc and NP, as disclosed herein. For
example, a CNP variant with M17X mutation can be fused to an Fc
domain, e.g., as shown in FIGS. 33A-33E, where X can be any amino
acid, e.g., F, L, I, T, E, R, Y, C, P, D, G, A, S, V, W, N, Q, H,
or K, e.g., F, L, I, T, E, R, Y, C, P, or D, e.g., F or L. In some
embodiments, the sequence is SEQ ID NO: 530, and X is any amino
acid described herein, e.g., F, L, I, T, E, R, Y, C, P, D, G, A, S,
V, W, N, Q, H, or K, e.g., F, L, I, T, E, R, Y, C, P, or D, e.g., F
or L.
[0293] In the structure X-Fc-Y-NP-Z or the structure X-NP-Y-Fc-Z, X
may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more)
additional amino acid residues at the N-terminus of the
polypeptide, and Z may independently include one or more (e.g., 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
25, 30, 35, 40, 45, 50, or more) additional amino acid residues at
the C-terminus of the polypeptide.
[0294] In some embodiments, the polypeptide includes a
bone-targeting moiety, e.g., having a series of consecutive Asp or
Glu residues, e.g., E.sub.6, E.sub.7, E.sub.8, E.sub.9, E.sub.10,
E.sub.11, E.sub.12, E.sub.13, E.sub.14, E.sub.15, E.sub.16,
D.sub.6, D.sub.7, D.sub.8, D.sub.9, D.sub.10, D.sub.11, D.sub.12,
D.sub.13, D.sub.14, D.sub.15, or D.sub.16. The bone-targeting
moiety, if present, may be positioned anywhere in the fusion
polypeptide, e.g., at or near the N-terminal or C-terminal end,
and/or in the linker region. For example, any one of X, Y, and/or Z
may include a bone-targeting moiety.
[0295] In some instances, one or more amino acid residues are
introduced into the fusion polypeptide, e.g., within X, Y, or Z, as
a result of the cloning strategy used. In some embodiments, any
such additional amino acid residues, when incorporated into the
polypeptide of the invention, do not provide a cleavage site for
endoproteases of the host cell. The likelihood that a designed
sequence would be cleaved by the endoproteases of the host cell can
be predicted as described, e.g., by Ikezawa (Biol. Pharm. Bull.
25:409-417, 2002), hereby incorporated by reference.
[0296] In certain embodiments, the fusion polypeptides of the
invention are associated into dimers, e.g., through two disulfide
bonds located in the hinge regions of the Fc fragments.
[0297] In some embodiments, the fusion polypeptides of the
invention have at least, e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7,
8, 9, 10, 12.5, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150,
175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000,
1,500, 2,000, 3,000, 4,000, 5,000, 7,500, 10,000, 15,000, 20,000,
25,000, 30,000, 40,000, or 50,000 times the half-life of CNP22 in
vivo.
[0298] Any NP fusion protein may be expressed with an N-terminal
signal sequence to facilitate secretion, e.g., amino acid residues
1-25 of SEQ ID NO: 501, or any other signal sequence known in the
art. Such sequences are generally cleaved co-translationally,
resulting in secretion of the mature version of the protein.
[0299] A fusion polypeptide as described herein may have at least
50% (e.g., 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or more) sequence identity to any of the fusion polypeptides
described herein, e.g., SEQ ID NOs: 501-608. In addition, a fusion
polypeptide as described herein may have 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, or
more additions, deletions, or substitutions relative to any of the
fusion polypeptides described herein. Furthermore, in some
embodiments, a fusion polypeptide as described herein may be
encoded by a nucleic acid molecule that hybridizes under high
stringency conditions to at least a portion, e.g., to 20%, 30%,
40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or
even 100%, of a nucleic acid molecule that encodes any of the
polypeptides, e.g., fusion polypeptides, described herein.
[0300] Fusion proteins include those having one or more
modifications that are cleaved during expression. For example, an
Fc-CNP fusion protein was designed as shown in FIGS. 18A and 18B.
This protein, termed "NC2 Streptag" or "NC2st," has a 25-amino acid
N-terminal signal sequence that is cleaved during expression. The
mature protein has the following domain structure, from N terminus
to C terminus: Strep-tag II sequence (to facilitate purification)
flanked by short linker sequences; TEV protease cleavage sequence,
followed by a short linker; Fc domain of human IgG-1; 16-amino acid
glycine-rich linker; and CNP22. This protein can be produced by
chemically synthesizing the coding sequence (FIG. 18C, SEQ ID NO:
801) and inserting the coding sequence into a small cloning plasmid
using standard techniques known in the art.
[0301] Fusion proteins may be varied in several respects. For
example, NC2st can be varied by eliminating the sequence that is
N-terminal to the Fc domain (resulting, e.g., in NC2B, as shown in
FIGS. 19A-19B), adding a bone-targeting moiety (resulting, e.g., in
D10-NC2, as shown in FIG. 19A), and/or altering of the length of
the linker between Fc and CNP22 (e.g., NC2B-22 (also referred to as
NC2-22), NC2B-28 (also referred to as NC2-28), and NC2B-34 (also
referred to as NC2-34), as shown in FIGS. 20A-20D). Other exemplary
NC2st variants are shown in FIG. 21 (NC2-KGANKK and NC2-KGANQK) and
FIG. 22 (NC2-CNP53mut2).
[0302] NC2B may be varied in several respects, including having a
point mutation, e.g., at position 17 relative to CNP22, having a
bone-targeting moiety, and/or having modified or altered linker
regions. Exemplary fusion protein include any sequences having a
modified or altered linker region (e.g., SEQ ID NOs: 511-516, as
shown in FIG. 34A), as compared to NC2B (as shown in FIGS.
16A-16B); any sequences having a bone-targeting moiety, e.g., a
D.sub.10 moiety (e.g., SEQ ID NOs: 553-558, as shown in FIG. 34B);
any N-terminal extensions, C-terminal extensions, and/or linkers
for any of the NPs disclosed herein (e.g., the italicized regions
in SEQ ID NOs: 511-516 and 553-558, as shown in FIGS. 30, 31A-31B,
and 32); any sequences having a phenylalanine (Phe, F) substitution
at position 17 relative to CNP22 (e.g., SEQ ID NOs: 559-564, as
shown in FIG. 34C), including those sequences having a further
modification of a D.sub.10 bone-targeting moiety at the N-terminus
(e.g., SEQ ID NOs: 565-570, as shown in FIG. 34D); any sequences
having a leucine (Leu, L) substitution at position 17 relative to
CNP22 (e.g., SEQ ID NOs: 571-576, as shown in FIG. 34E), including
those sequences having a further modification of a D.sub.10
bone-targeting moiety at the N-terminus (e.g., SEQ ID NOs: 577-582,
as shown in FIG. 34F); any sequences having an arginine (Arg, R)
substitution at position 17 relative to CNP22 (e.g., SEQ ID NOs:
583-588, as shown in FIG. 34G), including those sequences having a
further modification of a D.sub.10 bone-targeting moiety at the
N-terminus (e.g., SEQ ID NOs: 589-594, as shown in FIG. 34H); and
any sequences having a tyrosine (Tyr, Y) substitution at position
17 relative to CNP22 (e.g., SEQ ID NOs: 595-600, as shown in FIG.
34I), including those sequences having a further modification of a
D.sub.10 bone-targeting moiety at the N-terminus (e.g., SEQ ID NOs:
601-606, as shown in FIG. 34J).
[0303] Additional constructs include fusion proteins in which an
N-terminal Fc domain is fused to a variant of CNP53 with a short
Gly.sub.3 linker region. An alternative way to analyze these fusion
polypeptides is that the linker region is Gly.sub.3 followed by
amino acid residues 1-31 of CNP53 (or variants thereof); viewed in
this way, the linker region connects the Fc domain to CNP22 and is
34 amino acid residues in length. For Fc-CNP53-A (also referred to
as "Fc-CNP53 wt") (SEQ ID NOs: 517 (with signal sequence) and 518
(without signal sequence); FIG. 23), position 48 of CNP53,
corresponding to position 17 of CNP22, was mutated to alanine.
Fc-CNP53-AAA (also referred to as "Fc-CNP53mut") (SEQ ID NOs: 519
(with signal sequence) and 520 (without signal sequence); FIG. 23)
has the same sequence as Fc-CNP53-A with the exception that
residues 30 and 31 of CNP53, the two residues immediately before
CNP22, are mutated to alanine in order to reduce the likelihood of
proteolytic cleavage. In some cases, modifying the linker region of
an Fc-CNP22 fusion to include the first 31 amino acid residues of
CNP53 could result in constructs having even greater potency and
efficacy than NC2st in in vitro membrane and whole cell assays.
Additional Polypeptide Features
[0304] The polypeptides of the invention also include any
polypeptide having one or more post-translational modifications
such as glycosylation (e.g., mannosylation and other forms of
glycosylation discussed herein), acetylation, amidation, blockage,
formylation, gamma-carboxyglutamic acid hydroxylation, methylation,
ubiquitination, phosphorylation, pyrrolidone carboxylic acid
modification, and sulfation. Artificial modifications, e.g.,
pegylation, may also be made.
Production of Nucleic Acids and Polypeptides
[0305] The nucleic acids and polypeptides of the invention can be
produced by any method known in the art. Typically, a nucleic acid
encoding the desired fusion protein is generated using molecular
cloning methods, and is generally placed within a vector, such as a
plasmid or virus. The vector is used to transform the nucleic acid
into a host cell appropriate for the expression of the fusion
protein. Representative methods are disclosed, for example, in
Maniatis et al. (Cold Springs Harbor Laboratory, 1989). Many cell
types can be used as appropriate host cells, although mammalian
cells are preferable because they are able to confer appropriate
post-translational modifications. For example, Human Embryonic
Kidney 293 (HEK293) cells have been used as a host for expressing
the fusion proteins of the present invention, as described in more
detail in the Examples below.
[0306] The polypeptides of the invention can be produced under any
conditions suitable to effect expression of the polypeptide in the
host cell. Such conditions include appropriate selection of a media
prepared with components such as a buffer, bicarbonate and/or
HEPES, ions like chloride, phosphate, calcium, sodium, potassium,
magnesium, iron, carbon sources like simple sugars, amino acids,
potentially lipids, nucleotides, vitamins and growth factors like
insulin; regular commercially available media like alpha-MEM, DMEM,
Ham's-F12, and IMDM supplemented with 2-4 mM L-glutamine and 5%
Fetal bovine serum; regular commercially available animal protein
free media like Hyclone.TM. SFM4CHO, Sigma CHO DHFR.sup.-, Cambrex
POWER.TM. CHO CD supplemented with 2-4 mM L-glutamine. These media
are desirably prepared without thymidine, hypoxanthine and
L-glycine to maintain selective pressure, allowing stable
protein-product expression.
[0307] Additional details of the production of the polypeptides and
nucleic acids of the invention are given in the Examples.
Therapeutic Applications
[0308] The polypeptides and nucleic acid molecules described herein
can have a wide variety of therapeutic applications, e.g., in the
fields of neurocutaneous syndromes (e.g., neurofibromatosis) or
disorders associated with overactivation of FGFR3 (e.g., bone and
cartilage disorders, e.g., achondroplasia, or cancers, e.g.,
multiple myeloma) or bone or cartilage disorders (e.g., that are
not associated with overactivation of FGFR3) or vascular smooth
muscle disorders. In addition, the polypeptides and nucleic acid
molecules described herein can be used for any condition or
disorder that would benefit from elongation of bone.
[0309] Neurocutaneous Syndromes
[0310] The polypeptides and nucleic acid molecules described herein
can be used to treat any disorder, disease, or other abnormality
associated with elevated blood and/or urine levels of inorganic
pyrophosphate (PPi) and/or overactivation of MAP kinase, such as
neurocutaneous syndromes. In particular embodiments, the
polypeptide and nucleic acid molecules are used to treat a
neurocutaneous syndrome either with or without a bone
manifestation. Exemplary neurocutaneous syndromes include
neurofibromatosis (e.g., any type described herein, such as type I
or type II); Noonan syndrome-like disorder with loose anagen hair;
Noonan syndrome-like disorder with juvenile myelomonocytic leukemia
(JMML); tuberous sclerosis; Sturge-Weber disease; ataxia
telangiectasia; von Hippel-Lindau disease; incontinentia pigmenti;
epidermal nevus syndromes, such as linear sebaceous nevus of
Jadassohn; nevoid basal cell carcinoma syndrome; hypomelanosis of
Ito; neurocutaneous melanosis; Klippel-Ternaunay syndrome; and
Waardenburg syndrome, including types I, II, III, and IV.
[0311] Neurofibromatosis
[0312] Neurofibromatosis is an autosomal dominant neurocutaneous
syndrome characterized by abnormal growth or proliferation of nerve
tissue, such as to produce tumors (or neurofibromas) or abnormal
pigmentation (e.g., cafe au lait spots). In particular,
neurofibromatosis can be accompanied by bone manifestations (e.g.,
manifestations arising from hypophosphatasia), such as short
stature, scoliosis, osteomalacia, osseous fibrous dysplasia (e.g.,
one or more lesions at the ends of or within one or more bones,
such as the femur, tibia, or fibula), pseudarthrosis (e.g., tibial
pseudarthrosis), and/or skeletal dysplasia (e.g., tibial dysplasia,
orbital dysplasia, and/or sphenoid wing dysplasia).
[0313] Neurofibromatosis, or any of its manifestations or
phenotypes, can be treated using the compositions and methods
described herein. Examples of such disorders, manifestations, and
phenotypes include the classic von Recklinghausen type (type I),
either with gastrointestinal stromal tumors (i.e., as in intestinal
neurofibromatosis (type 3B)) or without such tumors; an acoustic
neuroma type (type II); a mixed type that combines the features of
types I and II with predominant features, such as bilateral
acoustic neuromas, posterior fossa and upper cervical meningiomas,
and spinal/paraspinal neurofibromas (type III, Riccardi type or
type 3A); an atypical type that is distinguished from by the lack
of iris Lisch nodules that are characteristic of type I (type VI);
segmental neurofibromatosis, which is a variant of type I having
lesions affecting a specific area of the body, such as a single
segment of the body or an area that crosses the midline (type V); a
type having only the symptoms of cafe au lait spots without other
manifestations of neurofibromatosis (type VI); familial spinal
neurofibromatosis, which is caused by mutation in the neurofibromin
gene NF1 and considered a distinguishable variant of type I; other
variants of type I, such as
neurofibromatosis-pheochromocytoma-duodenal carcinoid syndrome;
neurofibromatosis with manifestations of Noonan syndrome, such as
short stature, ptosis, midface hypoplasia, webbed neck, learning
disabilities, and muscle weakness; and schwannomatosis, where any
of these disorders can include or exclude one or more bone
manifestations.
[0314] Disorders Associated with Overactivation of RAS and/or
ERK
[0315] Any disorder, disease, or other abnormality that is caused
by, or is associated with, overactivation of RAS and/or ERK may be
treated using the compositions and methods described herein. These
disorders, diseases, and other abnormalities include, without
limitation, Noonan syndrome, Costello syndrome, Noonan syndrome
with multiple lentigines/LEOPARD syndrome, neurofibromatosis type
1, NF1-Noonan syndrome, hereditary gingival fibromatosis type 1,
capillary malformation-AV malformation syndrome, Legius syndrome,
Noonan syndrome-like disorder with loose anagen hair, Noonan
syndrome-like disorder with juvenile myelomonocytic leukemia
(JMML), cardio-facio-cutaneous syndrome, or autoimmune
lymphoproliferative syndrome, where any of these disorders can
include or exclude one or more bone manifestations.
[0316] Disorders Associated with Overactivation of FGFR3
[0317] Any disorder, disease, or other abnormality that is caused
by, or is associated with, overactivation of FGFR3, e.g., stemming
from a gain-of-function FGFR3 mutation, may be treated using the
compositions and methods described herein. These disorders,
diseases, and other abnormalities include, without limitation, bone
or cartilage disorders and cancers, each of which is described in
more detail below.
[0318] Bone or Cartilage Disorders Associated with Overactivation
of FGFR3
[0319] Any disorder, disease, or other abnormality, e.g., skeletal
dysplasia, that affects the function, structure, or growth of bone
or cartilage, may be treated using the compositions and methods
described herein. In particular, the disorder may be a skeletal
dysplasia that is associated with overactivation of FGFR3, such as
achondroplasia, including severe achondroplasia with developmental
delay and acanthosis; Muenke syndrome (Muenke coronal
craniosynostosis); Crouzonodermoskeletal syndrome;
hypochondroplasia; thanatophoric dysplasia type I; and
thanatophoric dysplasia type II. The compositions and methods of
the invention can also be used to treat bone or cartilage disorders
not associated with overactivation of FGFR3, and these disorders
are described in more detail below.
[0320] Cancers
[0321] Any cancer that is caused by, or is associated with,
overactivation of FGFR3, may be treated using the compositions and
methods described herein. These cancers include, e.g., multiple
myeloma, myeloproliferative syndromes, leukemia (e.g., plasma cell
leukemia), lymphomas, glioblastoma, prostate cancer, bladder
cancer, and mammary cancer.
[0322] Bone or Cartilage Disorders
[0323] The polypeptides and nucleic acid molecules described herein
can be used to treat any disorder, disease, phenotype, or other
abnormality that affects the function, structure, or growth of bone
or cartilage. These bone or cartilage disorders may be, but do not
necessarily have to be, associated with overactivation of
FGFR3.
[0324] Exemplary bone or cartilage disorders include skeletal
dysplasia and any other disorders, diseases, phenotypes, or other
abnormalities related to the bone or cartilage, including
achondroplasia (e.g., homozygous or heterozygous achondroplasia),
achondrogenesis, acrodysostosis, acromesomelic dysplasia,
atelosteogenesis, bone pain, calcium pyrophosphate dihydrate (CPPD)
crystal deposition, camptomelic dysplasia, chondrodysplasia
punctata (e.g., rhizomelic type of chondrodysplasia punctata),
cleidocranial dysostosis, congenital short femur, craniosynostosis
(e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome,
Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal
syndrome), dactyly (e.g., brachydactyly, camptodactyly,
polydactyly, or syndactyly), diastrophic dysplasia, dental
disorders (e.g., decrease in teeth mineralization and premature
loss of deciduous teeth, such as through aplasia, hypoplasia or
dysplasia of the dental cementum), dwarfism, dyssegmental
dysplasia, enchondromatosis, fibrochondrogenesis, fibrous
dysplasia, hereditary multiple exostoses, hypochondroplasia,
hypophosphatasia (HPP) (e.g., infantile HPP, childhood HPP,
perinatal HPP, adult HPP, or odontohypophosphatasia), HPP-related
seizure, hypophosphatemic rickets, incomplete bone mineralization,
Jaffe-Lichtenstein syndrome, Kniest dysplasia, Kniest syndrome,
Langer-type mesomelic dysplasia, Marfan syndrome, McCune-Albright
syndrome, micromelia, metaphyseal dysplasia (e.g., Jansen-type
metaphyseal dysplasia), metatrophic dysplasia, Morquio syndrome,
Nievergelt-type mesomelic dysplasia, neurofibromatosis (e.g., type
1, e.g., with bone manifestations or without bone manifestations;
type 2; or schwannomatosis), osteoarthritis, osteochondrodysplasia,
osteogenesis imperfecta (e.g., perinatal lethal type of
osteogenesis imperfecta), osteomalacia, osteopetrosis,
osteopoikilosis, osteoporosis, peripheral dysostosis, Reinhardt
syndrome, Roberts syndrome, Robinow syndrome, short-rib polydactyly
syndromes, short stature, spondyloepiphyseal dysplasia congenita,
spondyloepimetaphyseal dysplasia, or thanatophoric dysplasia. Bone
or cartilage disorders also include those that can be diagnosed,
for example, by elevated blood and/or urine levels of one or more
clinical markers related to hypophosphatasia (e.g., elevated levels
of inorganic pyrophosphate (PPi), phosphoethanolamine (PEA), and/or
pyridoxal 5'-phosphate (PLP)), growth retardation with a decrease
of long bone length (such as femur, tibia, humerus, radius, ulna),
a decrease of the mean density of total bone, or a decrease of bone
mineralization in bones such as femur, tibia, ribs and metatarsi,
and phalange. Without being so limited, treatment of bone or
cartilage disorders may be observed by one or more of the
following: an increase of long bone length, an increase of
mineralization in bone and/or teeth, a correction of bowing of the
legs, a reduction of bone pain, and a reduction of CPPD crystal
deposition in joints.
[0325] Skeletal Dysplasia
[0326] Skeletal dysplasias are bone or cartilage disorders
characterized by short stature or dwarfism. Skeletal dysplasias are
typically congenital and may include numerous abnormalities in
addition to short stature, e.g., short limbs and trunk; bowlegs; a
waddling gait; skull malformations, e.g., a large head, cloverleaf
skull, craniosynostosis (premature fusion of the bones in the
skull), or wormian bones (abnormal thread-like connections between
the bones in the skull); anomalies of the hands and feet, e.g.,
polydactyly (extra fingers), "hitchhiker" thumbs, and abnormal
fingernails and toenails; or chest anomalies, e.g., pear-shaped
chest or narrow thorax. Non-skeletal abnormalities may also be
present in individuals having skeletal dysplasia, e.g., anomalies
of the eyes, mouth, and ears, such as congenital cataracts, myopia,
cleft palate, or deafness; brain malformations, such as
hydrocephaly, porencephaly, hydranencephaly, or agenesis of the
corpus callosum; heart defects, such as atrial septal defect,
patent ductus arteriosus, or transposition of the great vessels;
developmental delays; or mental retardation. Skeletal dysplasias
associated with overactivation of FGFR3 include achondroplasia.
[0327] Skeletal dysplasias include achondroplasia (e.g., homozygous
or heterozygous achondroplasia), achondrogenesis, acrodysostosis,
acromesomelic dysplasia, atelosteogenesis, camptomelic dysplasia,
chondrodysplasia punctata (e.g., rhizomelic type of
chondrodysplasia punctata), cleidocranial dysostosis, congenital
short femur, craniosynostosis (e.g., Muenke syndrome, Crouzon
syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer
syndrome, or Crouzonodermoskeletal syndrome), dactyly (e.g.,
brachydactyly, camptodactyly, polydactyly, or syndactyly),
diastrophic dysplasia, dwarfism, dyssegmental dysplasia,
enchondromatosis, fibrochondrogenesis, fibrous dysplasia,
hereditary multiple exostoses, hypochondroplasia, hypophosphatasia
(HPP) (e.g., infantile HPP, childhood HPP, perinatal HPP, adult
HPP, or odontohypophosphatasia), hypophosphatemic rickets,
Jaffe-Lichtenstein syndrome, Kniest dysplasia, Kniest syndrome,
Langer-type mesomelic dysplasia, Marfan syndrome, McCune-Albright
syndrome, micromelia, metaphyseal dysplasia (e.g., Jansen-type
metaphyseal dysplasia), metatrophic dysplasia, Morquio syndrome,
Nievergelt-type mesomelic dysplasia, neurofibromatosis (e.g., type
1, e.g., with bone manifestations or without bone manifestations;
type 2; or schwannomatosis), osteoarthritis, osteochondrodysplasia,
osteogenesis imperfecta (e.g., perinatal lethal type of
osteogenesis imperfecta), osteopetrosis, osteopoikilosis,
peripheral dysostosis, Reinhardt syndrome, Roberts syndrome,
Robinow syndrome, short-rib polydactyly syndromes, short stature,
spondyloepiphyseal dysplasia congenita, spondyloepimetaphyseal
dysplasia, or thanatophoric dysplasia.
[0328] In particular, some forms of craniosynostosis are the result
of mutations in one of the fibroblast growth factor receptors
(e.g., one or more of FGFR1, FGFR2, or FGFR3) that cause the
activation of the MAPK pathway. This is the case for Muenke (Muenke
coronal craniosynostosis), Crouzon, Apert, Jackson-Weiss, Pfeiffer,
and Crouzonodermoskeletal syndromes, for example. There is genetic
and biochemical evidence in the scientific literature that agents
that can prevent activation of the MAP-kinase (ERK 1/2) can prevent
craniosynostosis in animal models. In particular, use of a MEK1/2
inhibitor (e.g., U0126), which prevents activation of ERK1/2 can
prevent craniosynostosis in an animal model of Apert syndrome
(Shukla et al., Nat. Genet. 39:1145, 2007). Accordingly, the
compounds of the present invention, which can prevent activation of
the MAP-kinase pathway, could be used to treat these forms of
craniosynostosis.
[0329] Achondroplasia
[0330] Achondroplasia is an autosomal dominant skeletal dysplasia
that is the most common cause of dwarfism in humans. Its incidence
is approximately 1 in 20,000 live births. Skeletal manifestations
include growth retardation (with an average adult height of 123-131
cm (4 feet 1/2 in.-4 feet 31/2 in.)), skull deformities, and
orthodontic defects. Extraskeletal manifestations include cervical
cord compression (with risk of death, e.g., from central apnea or
seizures); spinal stenosis (e.g., leg and lower back pain);
hydrocephalus (e.g., requiring cerebral shunt surgery); hearing
loss due to chronic otitis; cardiovascular disease; neurological
disease; higher frequency of accidents; and obesity.
[0331] Babies are often diagnosed at birth. While the homozygous
form is usually lethal, individuals diagnosed with the heterozygous
form have a life expectancy, on average, of 15 years less than the
normal population.
[0332] Heterozygous or homozygous achondroplasia, or any of its
manifestations or phenotypes, can be treated using the compositions
and methods described herein. Treatment of either form may be
started as early as possible in the patient's life, e.g., shortly
after birth, or even in utero; this is particularly important for
treatment of the homozygous form, which is typically much more
severe and is often lethal if untreated.
[0333] Hypophosphatasia (HPP)
[0334] HPP is a matrix mineralization disorder that is historically
classified according to age at diagnosis and includes (in order
from most severe to least severe) perinatal, infantile, childhood,
adult, and odontohypophosphatasia forms. The most severe form,
perinatal (lethal) HPP, is manifest as an almost complete absence
of bone mineralization in utero and can cause stillbirth. Some
neonates with perinatal HPP may survive for several days, but
suffer increased respiratory compromise due to hypoplastic and
rachitic disease of the chest. In infantile HPP, diagnosed before 6
months-of-age, postnatal development seems normal until the onset
of poor feeding, inadequate weight gain, and appearance of rickets.
Infantile HPP has characteristic radiological features showing
impaired skeletal mineralization, sometimes with progressive
skeletal demineralization leading to rib fractures and chest
deformity. Childhood HPP has highly variable clinical expression.
One symptom of childhood HPP is the premature loss of deciduous
teeth resulting from aplasia, hypoplasia or dysplasia of dental
cementum that connects the tooth root with the periodontal
ligament. Another symptom of childhood HPP is rickets, which causes
short stature and skeletal deformities such as bowed legs and
enlarged wrists, knees and ankles as a result of flared metaphysis.
Adult HPP usually presents during middle age, but is frequently
preceded by a history of rickets and/or early loss of teeth
followed by good health during adolescence and young adult life. In
adult HPP, recurrent metatarsal stress fractures are common, and
calcium pyrophosphate dihydrate deposition can cause attacks of
arthritis and pyrophosphate arthropathy. Finally,
odontohypophosphatasia is diagnosed when the only clinical
abnormality is dental disease, and radiological studies and even
bone biopsies reveal no signs of rickets or osteomalacia.
[0335] The more severe clinical forms of HPP are usually inherited
as autosomal recessive traits, with parents of such patients
showing subnormal levels of serum AP activity. For the milder forms
of HPP, i.e., adult HPP and odontohypophosphatasia, an autosomal
dominant pattern of inheritance has also been documented.
[0336] In the healthy skeleton, TNALP is an ectoenzyme present on
the surface of the plasma membrane of osteoblasts and chondrocytes,
and on the membranes of their shed matrix vesicles (MVs), where the
enzyme is particularly enriched. Deposition of hydroxyapatite
during bone mineralization normally initiates within the lumen of
these MVs. Electron microscopy has shown that TNALP-deficient MVs
from severely affected HPP patients and Akp2.sup.-/- mice (a TNALP
null mouse model, see below) contain hydroxyapatite crystals, but
that extravesicular crystal propagation appears retarded. This
defect is attributed to the extracellular accumulation of PP.sub.i,
a potent inhibitor of calcification, due to a deficiency of TNALP
activity.
[0337] At physiological concentrations (0.01-0.1 mM), PP.sub.i has
the ability to stimulate mineralization. This has been demonstrated
in organ-cultured chick femurs and in isolated rat MVs. However, at
concentrations above 1 mM, PP.sub.i inhibits calcium phosphate
mineral formation by coating hydroxyapatite crystals, thus
preventing mineral crystal growth and proliferative
self-nucleation. Thus, PP.sub.i has a dual physiological role: it
functions as a promoter of mineralization at low concentrations but
as an inhibitor of mineralization at higher concentrations. TNALP
has been shown to hydrolyze the mineralization inhibitor PP.sub.i
to facilitate mineral precipitation and growth. Recent studies
using the Akp2.sup.-/- mice have indicated that the primary role of
TNALP in vivo is to restrict the size of the extracellular PP.sub.i
pool to allow proper skeletal mineralization.
[0338] The severity of hypophosphatasia depends on the nature of
the TNALP mutation. Missense mutations at a variety of positions in
TNALP, including the enzyme's active site vicinity, homodimer
interface, crown domain, amino-terminal arm, and calcium-binding
site, have all been found to affect its catalytic activity. In
addition, missense, nonsense, frame-shift, and splice site
mutations have also been shown to lead to aberrant mutant proteins
or intracellular trafficking defects that lead to subnormal
activity on the cell surface. The multitude of mutations that cause
HPP, and the fact that compound heterozygosity is a common
occurrence in HPP, explain the variable expressivity and incomplete
penetrance often observed in this disease.
[0339] Progress on the human form of HPP has benefited greatly from
the existence of the TNALP null mice (Akp2.sup.-/-), an animal
model of HPP. Akp2.sup.-/- mice phenocopy infantile HPP remarkably
well: they are born with a normally mineralized skeleton but
develop radiographically apparent rickets at about 6 days of age,
and die between days 12-16 suffering severe skeletal
hypomineralization and episodes of apnea and epileptic seizures
attributable to disturbances in PLP (vitamin B.sub.6)
metabolism.
[0340] Both PP.sub.i and PLP are confirmed natural substrates of
TNALP, and some TNALP active site mutations have been shown to have
different effects on the ability of the enzyme to metabolize
PP.sub.i and PLP. Abnormalities in PLP metabolism explain the
epileptic seizures observed in Akp2.sup.-/- mice, while
abnormalities in PP.sub.i metabolism explain the skeletal phenotype
in this mouse model of HPP.
[0341] In any of the methods of the invention, the pharmaceutical
compositions described herein are optionally administered in an
amount that is therapeutically effective to treat a HPP phenotype
selected from the group consisting of HPP-related seizure,
premature loss of deciduous teeth, incomplete bone mineralization,
elevated blood and/or urine levels of PP.sub.i, elevated blood
and/or urine levels of PEA, elevated blood and/or urine levels of
PLP, inadequate weight gain, rickets, bone pain, calcium
pyrophosphate dihydrate crystal deposition, aplasia, hypoplasia,
and dysplasia of the dental cementum. In some embodiments, the
incomplete bone mineralization is incomplete femoral bone
mineralization, incomplete tibial bone mineralization, incomplete
metatarsal bone mineralization, or incomplete rib bone
mineralization.
[0342] Vascular Smooth Muscle Disorders
[0343] The polypeptides and nucleic acid molecules described herein
can be used to treat any disorder, disease, or other abnormality
that affects the function, structure, or growth of vascular smooth
muscle. For example, natriuretic peptides modulate salt and water
homeostasis in the body and in this way act as regulators of blood
pressure. The peptides belonging to this family have varying amino
acid sequences and are secreted through different mechanisms by
various tissues in the body For example, ANP is released by muscle
cells in the upper chambers (atria) of the heart (atrial myocytes)
and acts as a vasodilator; BNP is secreted by the lower chambers
(ventricles) of the heart in response to cardiac stress; and CNP
exerts natruretic and natriuretic effect and regulates vessel tone,
inhibits migration and proliferation of vascular smooth muscle
cell. Accordingly, the polypeptides and compositions of the
invention can be used to treat vascular smooth muscle disorders.
Exemplary vascular smooth muscle disorders are hypertension,
restenosis, arteriosclerosis, acute decompensated heart failure,
congestive heart failure, cardiac edema, nephredema, hepatic edema,
acute renal insufficiency, and chronic renal insufficiency.
[0344] Conditions for Elongation of Bone
[0345] Any condition, disorder, disease, or other abnormality that
would benefit from elongation of bone may be treated using the
compositions and methods described herein. These conditions,
disorders, diseases, and other abnormalities include, without
limitation, insufficient or impaired bone growth arising from
fractures, renal failure or insufficiency, poor diet, vitamin
deficiency, or hormone deficiency. Healthy subjects, e.g., those
without any conditions, disorders, diseases, or other abnormalities
related to bone or cartilage, may also be treated using the
compositions and methods described herein, e.g., for cosmetic
purposes.
[0346] Skeletal dysplasias are also associated with shortened
segments of long bones. Exemplary skeletal dysplasias include those
associated with rhizomelia (or shortening in a proximal segment of
a limb, e.g., in the humerus or femur), such as achondroplasia,
atelosteogenesis, congenital short femur, diastrophic dysplasia,
hypochondroplasia, Jansen type of metaphyseal dysplasia, rhizomelic
type of chondrodysplasia punctata, spondyloepiphyseal dysplasia
congenita, and thanatophoric dysplasia; mesomelia (or shortening in
a middle segment of a limb, e.g., in the radius, ulna, tibia, or
fibula), such as Langer and Nievergelt types of mesomelic
dysplasias, Robinow syndrome, and Reinhardt syndrome; acromelia (or
shortening in a distal segment of a limb, e.g., in the metacarpals
or phalanges), such as acrodysostosis and peripheral dysostosis;
acromesomelia (or shortening in the middle and distal segments of
limbs, e.g., in the forearms and hands), such as acromesomelic
dysplasia; micromelia (or shortening in the entire limb), such as
achondrogenesis, fibrochondrogenesis, dyssegmental dysplasia,
Kniest dysplasia, and Roberts syndrome; or short-trunk, such as
Dyggve-Melchior-Clausen disease, Kniest syndrome, metatrophic
dysplasia, Morquio syndrome, spondyloepimetaphyseal dysplasia, and
spondyloepiphyseal dysplasia congenita.
[0347] Combination Therapy
[0348] The combinations of the polypeptides of the invention (e.g.,
a combination of an sALP polypeptide and an NP polypeptide, such as
a combination of an sALP fusion protein and an NP fusion protein)
are useful for the treatment of any disease or condition described
herein. Therapy may be performed alone or in conjunction with
another therapy (e.g., surgery, radiation therapy, immunotherapy,
or gene therapy). Additionally, a person having a greater risk of
developing a disease described herein (e.g., one who is genetically
predisposed or one who previously had a neurocutaneous syndrome)
may receive prophylactic treatment to inhibit or delay disease
formation. The duration of the combination therapy depends on the
type of disease or disorder being treated, the age and condition of
the patient, the stage and type of the patient's disease, and how
the patient responds to the treatment. Therapy may be given in
on-and-off cycles that include rest periods so that the patient's
body has a chance to recovery from any as yet unforeseen
side-effects.
[0349] The administration of a combination of the polypeptides of
the present invention (e.g., a combination of an sALP polypeptide
and an NP polypeptide) allows for the administration of lower doses
of each polypeptide, providing similar efficacy and lower toxicity
compared to administration of either polypeptide alone.
Alternatively, such combinations result in improved efficacy in
treating a disease described herein (e.g., a neurocutaneous
syndrome, such as neurofibromatosis) with similar or reduced
adverse events over the single agent alone, at moderate or high
doses.
[0350] Formulation
[0351] Formulation will depend on the route of administration, as
well as on other therapeutic goals. The polypeptides and nucleic
acid molecules described herein can be administered by any route
known in the art, e.g., subcutaneous (e.g., by subcutaneous
injection), intravenously, orally, nasally, intramuscularly,
sublingually, intrathecally, or intradermally. By way of example,
pharmaceutical compositions of the invention can be in the form of
a liquid, solution, suspension, pill, capsule, tablet, gelcap,
powder, gel, ointment, cream, nebulae, mist, atomized vapor,
aerosol, or phytosome.
[0352] In some embodiments of the invention, the compositions of
the invention can be administered subcutaneously. Subcutaneous
administration is advantageous because it is relatively
non-invasive and offers desirable pharmacokinetic profiles.
Suitable volumes are known to those skilled in the art, and are
typically 5 mL or smaller (e.g., 4 mL, 3.5 mL, 3 mL, 2.7 mL, 2.5
mL, 2.3 mL, 2.2 mL, 2.1 mL, 2.0 mL, 1.9 mL, 1.8 mL, 1.7 mL, 1.5 mL,
1.3 mL, 1.0 mL, 0.7 mL, 0.5 mL, 0.3 mL, 0.1 mL, 0.05 mL, 0.01 mL,
or smaller). Typically, the compositions of the invention can be
formulated at a concentration between 1 mg/mL and 500 mg/mL (e.g.,
between 10 mg/mL and 300 mg/mL, 20 mg/mL and 120 mg/mL, 40 mg/mL
and 200 mg/mL, 30 mg/mL and 150 mg/mL, 40 mg/mL and 100 mg/mL, 50
mg/mL and 80 mg/mL, or 60 mg/mL and 70 mg/mL) for subcutaneous
administration.
[0353] For oral administration, tablets or capsules can be prepared
by conventional means with pharmaceutically acceptable excipients
such as binding agents, fillers, lubricants, disintegrants, or
wetting agents. The tablets can be coated by methods known in the
art. Liquid preparations for oral administration can take the form
of, for example, solutions, syrups, or suspension, or they can be
presented as a dry product for constitution with saline or other
suitable liquid vehicle before use. Compositions of the invention
for oral administration also can contain pharmaceutically
acceptable excipients such as suspending agents, emulsifying
agents, non-aqueous vehicles, preservatives, buffer salts,
flavoring, coloring, and sweetening agents as appropriate.
Preparations for oral administration also can be suitably
formulated to give controlled release of the active
ingredients.
[0354] Enteric coatings can further be used on tablets of the
present invention to resist prolonged contact with the strongly
acidic gastric fluid, but dissolve in the mildly acidic or neutral
intestinal environment. Without being so limited, cellulose acetate
phthalate, Eudragit.TM. and hydroxypropyl methylcellulose phthalate
(HPMCP) can be used in enteric coatings of pharmaceutical
compositions of the present invention. Cellulose acetate phthalate
concentrations generally used are 0.5-9.0% of the core weight. The
addition of plasticizers improves the water resistance of this
coating material, and formulations using such plasticizers are more
effective than when cellulose acetate phthalate is used alone.
Cellulose acetate phthalate is compatible with many plasticizers,
including acetylated monoglyceride; butyl phthalylbutyl glycolate;
dibutyl tartrate; diethyl phthalate; dimethyl phthalate; ethyl
phthalylethyl glycolate; glycerin; propylene glycol; triacetin;
triacetin citrate; and tripropionin. It is also used in combination
with other coating agents such as ethyl cellulose, in drug
controlled-release preparations.
[0355] The compounds of the invention may be administered in
combination with pharmaceutically acceptable, sterile, aqueous or
non-aqueous solvents, suspensions or emulsions. Examples of
nonaqueous solvents are propylene glycol, polyethylene glycol,
vegetable oil, fish oil, and injectable organic esters. Aqueous
carriers include water, water-alcohol solutions, emulsions or
suspensions, including saline and buffered medical parenteral
vehicles including sodium chloride solution, Ringer's dextrose
solution, dextrose plus sodium chloride solution, Ringer's solution
containing lactose, or fixed oils. Intravenous vehicles may include
fluid and nutrient replenishers, electrolyte replenishers, such as
those based upon Ringer's dextrose, and the like.
[0356] In some embodiments, the pharmaceutical compositions of the
present invention can be delivered in a controlled release system.
In some embodiments, polymeric materials including polylactic acid,
polyorthoesters, cross-linked amphipathic block copolymers and
hydrogels, polyhydroxy butyric acid and polydihydropyrans can be
used (see also Smolen and Ball, Controlled Drug Bioavailability,
Drug product design and performance, 1984, John Wiley & Sons;
Ranade and Hollinger, Drug Delivery Systems, pharmacology and
toxicology series, 2003, 2.sup.nd edition, CRC Press). In another
embodiment, a pump may be used (Saudek et al., 1989, N. Engl. J.
Med. 321: 574).
[0357] The compositions of the invention could be formulated in the
form of a lyophilized powder using appropriate excipient solutions
(e.g., sucrose) as diluents.
[0358] Furthermore, cells can be isolated from an individual having
a neurocutaneous syndrome, a disorder associated with
overactivation of FGFR3, e.g., achondroplasia, a bone or cartilage
disorder, or a vascular smooth muscle disorder or from an
individual that would benefit from bone elongation; transformed
with a nucleic acid of the invention; and reintroduced to the
afflicted individual (e.g., subcutaneous or intravenous injection).
Alternatively, the nucleic acid can be administered directly to the
afflicted individual, for example, by injection. The nucleic acid
can also be delivered through a vehicle such as a liposome, which
can be designed to be targeted to a specific cell type, and
engineered to be administered through different routes.
[0359] The polypeptides or compositions of the present invention
may also be used in combination with at least one other active
ingredient to correct, e.g., an achondroplasia phenotype,
neurofibromatosis, HPP, or any other disorder or condition
described herein.
[0360] For combination therapy, two or more of the polypeptides of
the present invention (e.g., an sALP polypeptide and an NP
polypeptide) are formulated in a variety of ways that are known in
the art. For example, the first and second polypeptides may be
formulated together or separately. In some embodiments, the first
and second polypeptides are formulated together for the
simultaneous or near simultaneous administration of the
polypeptides. Such co-formulated compositions can include the sALP
polypeptide and the NP polypeptide formulated together in the same
pill, capsule, liquid, etc.
[0361] Administration of each compound in controlled release
formulations is useful where the sALP polypeptide or the NP
polypeptide, has (i) a narrow therapeutic index (e.g., the
difference between the plasma concentration leading to harmful side
effects or toxic reactions and the plasma concentration leading to
a therapeutic effect is small; generally, the therapeutic index,
TI, is defined as the ratio of median lethal dose (LD.sub.50) to
median effective dose (ED.sub.50)); (ii) a narrow absorption window
in the gastro-intestinal tract; (iii) a short biological half-life,
such as by degradation in vivo by NEP and/or IDE; or (iv) the
pharmacokinetic profile of each component must be modified to
maximize the exposure of the neoplasm to an amount of each agent,
together, that is therapeutically effective. Accordingly, a
sustained release formulation may be used to avoid frequent dosing
that may be required in order to sustain the plasma levels of both
agents at a therapeutic level.
[0362] Many strategies can be pursued to obtain controlled release
in which the rate of release outweighs the rate of metabolism of
the therapeutic polypeptide. For example, controlled release can be
obtained by the appropriate selection of formulation parameters and
ingredients (e.g., appropriate controlled release compositions and
coatings). Examples include single or multiple unit tablet or
capsule compositions, oil solutions, suspensions, emulsions,
microcapsules, microspheres, nanoparticles, patches, and liposomes.
The control release mechanism can be such that the compound of the
sALP polypeptide is released first, followed by the NP polypeptide,
or vice versa. The release mechanism can also be controlled that
the two polypeptides are released at period intervals, the release
could be simultaneous or a delayed release of one, when release of
a particular drug is preferred over the other.
[0363] Controlled release formulations may include a degradable or
nondegradable polymer, hydrogel, organogel, or other physical
construct that modifies the bioabsorption, half life or
biodegradation of the agent. The controlled release formulation can
be a material that is painted or otherwise applied onto the
afflicted site, either internally or externally. In one example,
hydrogels, such as those described in U.S. Pat. No. 5,626,863 can
be used in controlled release formulations of compositions of the
invention. These biodegradable polymers can be tailored to degrade
at a desired rate and with a desired kinetics by selecting the
appropriate monomers, method of preparation and molecular weight.
Differences in crystallinity of the monomer can alter the polymeric
degradation rate. Due to the relatively hydrophobic nature of most
polymers, actual mass loss can begin with the oligomeric fragments
that are small enough to be water soluble; hence, even the initial
molecular weight can influence the degradation rate.
[0364] The individually or separately formulated polypeptides can
be packaged together as in a kit. Non-limiting examples include
kits that contain, e.g., two pills, a pill and a powder, a
suppository and a liquid in a vial, two topical creams, among
others. The kit can include optional components that aid in the
administration of the unit dose to patients, such as vials for
reconstituting powder forms, syringes for injection or subcutaneous
administration, customized IV delivery systems, inhalers, among
others. Additionally, the unit dose kit can contain instructions
for preparation and administration of the compositions. The kit may
be manufactured as a single use unit dose for one subject, multiple
uses for a particular subject (at a constant dose or in which the
individual polypeptides may vary in potency as therapy progresses);
or the kit may contain multiple doses suitable for administration
to multiple subjects ("bulk packaging"). The kit components may be
assembled in cartons, blister packs, bottles, tubes, and the
like.
[0365] Gene Therapy
[0366] The polypeptides described herein could also be
advantageously delivered through gene therapy, where an exogenous
nucleic acid encoding the proteins is delivered to tissues of
interest and expressed in vivo. Gene therapy methods are discussed,
e.g., in Verme et al. (Nature 389:239-242, 1997), Yamamoto et al.
(Molecular Therapy 17:S67-S68, 2009), and Yamamoto et al., (J. Bone
Miner. Res. 26:135-142, 2011), each of which is hereby incorporated
by reference. Both viral and non-viral vector systems can be used.
The vectors may be, for example, plasmids, artificial chromosomes
(e.g., bacterial, mammalian, or yeast artificial chromosomes),
virus or phage vectors provided with an origin of replication, and
optionally, a promoter for the expression of the nucleic acid
encoding the viral polypeptide and optionally, a regulator of the
promoter. The vectors may contain one or more selectable marker
genes, for example, an ampicillin or kanamycin resistance gene in
the case of a bacterial plasmid or a resistance gene for a fungal
vector. Vectors may be used in in vitro, for example, for the
production of DNA, RNA, or the viral polypeptide, or may be used to
transfect or transform a host cell, for example, a mammalian host
cell, e.g., for the production of the viral polypeptide encoded by
the vector. The vectors may also be adapted to be used in vivo, for
example, in a method of vaccination or gene therapy.
[0367] Examples of suitable viral vectors include, retroviral,
lentiviral, adenoviral, adeno-associated viral, herpes viral,
including herpes simplex viral, alpha-viral, pox viral, such as
Canarypox and vaccinia-viral based systems. Gene transfer
techniques using these viruses are known in the art. Retrovirus
vectors, for example, may be used to stably integrate the nucleic
acids of the invention into the host genome. Replication-defective
adenovirus vectors by contrast remain episomal and therefore allow
transient expression. Vectors capable of driving expression in
insect cells (e.g., baculovirus vectors), in human cells, yeast, or
in bacteria may be employed in order to produce quantities of the
viral polypeptide(s) encoded by the nucleic acids of the invention,
for example, for use in subunit vaccines or in immunoassays. Useful
gene therapy methods include those described in WO 06/060641, U.S.
Pat. No. 7,179,903 and WO 01/36620 (each of which is hereby
incorporated by reference), which use an adenovirus vector to
target a nucleic acid of interest to hepatocytes as protein
producing cells.
[0368] In an additional example, a replication-deficient simian
adenovirus vector may be used as a live vector. These viruses
contain an E1 deletion and can be grown on cell lines that are
transformed with an E1 gene. Examples of these
replication-deficient simian adenovirus vectors are described in
U.S. Pat. No. 6,083,716 and WO 03/046124 (each of which is hereby
incorporated by reference). These vectors can be manipulated to
insert a nucleic acid of the invention, such that the encoded viral
polypeptide(s) may be expressed.
[0369] Promoters and other expression regulatory signals may be
selected to be compatible with the host cell for which expression
is designed. For example, mammalian promoters include the
metallothionein promoter, which can be induced in response to heavy
metals such as cadmium, and the .beta.-actin promoter. Viral
promoters, such as the SV40 large T antigen promoter, human
cytomegalovirus (CMV) immediate early (1E) promoter, rous sarcoma
virus LTR promoter, adenovirus promoter, or a HPV promoter,
particularly the HPV upstream regulatory region (URR) may also be
used. All these promoters, as well as additional promoters, are
well-described in the art.
[0370] The nucleic acid molecules described herein may also be
administered using non-viral based systems. For example, these
administration systems include microsphere encapsulation,
poly(lactide-co-glycolide), nanoparticle, and liposome-based
systems. Non-viral based systems also include techniques
facilitating the delivery of "naked" polynucleotides (such as
electroporation, "gene gun" delivery and various other techniques
used for the introduction of polynucleotides).
[0371] The introduced polynucleotide can be stably or transiently
maintained in the host cell. Stable maintenance typically requires
that the introduced polynucleotide either contains an origin of
replication compatible with the host cell or integrates into a
replicon of the host cell such as an extrachromosomal replicon
(e.g., a plasmid) or a nuclear or mitochondrial chromosome.
Dosage
[0372] Any amount of a polypeptide or a pharmaceutical composition
of the invention can be administered to a subject. The dosages will
depend on many factors, including the mode of administration and
the age of the subject. Typically, the amount of the composition of
the invention contained within a single dose will be an amount that
is effective to treat a neurocutaneous syndrome, a disorder
associated with overactivation of FGFR3, a bone or cartilage
disorder, or a vascular smooth muscle disorder, or to elongate
bone, without inducing significant toxicity. For example, the
polypeptides described herein can be administered to subjects in
individual doses ranging, e.g., from 0.01 mg/kg to 500 mg/kg (e.g.,
from 0.05 mg/kg to 500 mg/kg, from 0.2 mg/kg to 20 mg/kg, from 5
mg/kg to 500 mg/kg, from 0.1 mg/kg to 100 mg/kg, from 10 mg/kg to
100 mg/kg, from 0.1 mg/kg to 50 mg/kg, 0.5 mg/kg to 25 mg/kg, 1.0
mg/kg to 10 mg/kg, 1.5 mg/kg to 5 mg/kg, or 2.0 mg/kg to 3.0 mg/kg)
or from 1 .mu.g/kg to 1,000 .mu.g/kg (e.g., from 5 .mu.g/kg to
1,000 .mu.g/kg, from 1 .mu.g/kg to 750 .mu.g/kg, from 5 .mu.g/kg to
750 .mu.g/kg, from 10 .mu.g/kg to 750 .mu.g/kg, from 1 .mu.g/kg to
500 .mu.g/kg, from 5 .mu.g/kg to 500 .mu.g/kg, from 10 .mu.g/kg to
500 .mu.g/kg, from 1 .mu.g/kg to 100 .mu.g/kg, from 5 .mu.g/kg to
100 .mu.g/kg, from 10 .mu.g/kg to 100 .mu.g/kg, from 1 .mu.g/kg to
50 .mu.g/kg, from 5 .mu.g/kg to 50 .mu.g/kg, or from 10 .mu.g/kg to
50 .mu.g/kg). Exemplary doses include, e.g., 0.01, 0.05, 0.1, 0.5,
1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250, or 500
mg/kg; or 1, 2, 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 200, 250,
500, 750, 900, or 1,000 .mu.g/kg. For all dosages or ranges recited
herein, the term "about" may be used to modify these dosages by
.+-.10% of the recited values or range endpoints.
[0373] Doses can also be adjusted when two or more polypeptides or
compositions of the inventions are being administered. Exemplary
doses include an sALP polypeptide (e.g., an sALP fusion protein)
present in a dosage between about 0.2 mg/kg to about 20 mg/kg and
an NP polypeptide (e.g., an NP fusion protein) present in a dosage
of about 0.5 mg/kg to about 500 mg/kg. In particular embodiments,
the dose of each individual polypeptide or composition is lower
than the therapeutic dose of a single polypeptide or single
composition when administered alone.
[0374] Doses can be administered, e.g., hourly, bihourly, daily,
bidaily, twice a week, three times a week, four times a week, five
times a week, six times a week, weekly, biweekly, monthly,
bimonthly, or yearly. Alternatively, doses can be administered,
e.g., twice, three times, four times, five times, six times, seven
times, eight times, nine times, 10 times, 11 times, or 12 times per
day. In particular embodiments, the dosing regimen is once weekly.
The duration of the dosing regimen can be, e.g., 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, or 30 day(s), week(s), or month(s), or even
for the remaining lifespan of the subject. The amount, frequency,
and duration of dosage will be adapted by the clinician in
accordance with conventional factors such as the extent of the
disease and different parameters from the subject.
[0375] The nucleic acids of the invention can be administered
according the formulations described herein to a patient in dosages
suitable for gene therapy. The amount of the nucleic acids
administered will depend on a number of factors known to those
skilled in the art, including: the length and nature of the nucleic
acid, the vector (e.g., viral or non-viral) used, the activity of
the polypeptide encoded, the presence of excipients, the route and
method of administration, and the general condition and fitness of
the subject. Exemplary dosages and routes of administration are
described, e.g., in Melman et al. (Isr. Med. Assoc. J. 9:143-146,
2007; describing the intrapenile injection of 0.5 mg to 7.5 mg of a
human cDNA in a plasmid for treating erectile dysfunction), Powell
et al. (Circulation 118:58-65, 2008; describing the intramuscular
injection of 0.4 mg to 4.0 mg of a hepatocyte growth factor plasmid
to treat critical limb ischemia, Waddill et al. (AJR Am. J.
Roentgenol. 169:63-67, 1997; describing the CT-guided intratumoral
injection of 0.01 mg to 0.25 mg of plasmid DNA encoding an MHC
antigen to treat melanoma), Kastrup et al. (J. Am. Coll. Cardiol.
45:982-988, 2005; describing the intramyocardial injection of 0.5
mg of a VEGF plasmid to treat severe angina pectoris), and Romero
et al. (Hum. Gene. Ther. 15:1065-1076, 2004; describing the
intramuscular injection of 0.2 mg to 0.6 mg of a plasmid to treat
Duchenne/Becker muscular dystrophy), each of which is hereby
incorporated by reference.
[0376] In certain embodiments, the nucleic acids of the invention
can be administered to the subject at a dose in the range from,
e.g., 0.01 mg to 100 mg (e.g., from 0.05 mg to 50 mg, 0.1 mg to 10
mg, 0.3 mg to 3 mg, or about 1 mg) of nucleic acid. The total
volume at which the nucleic acid can be administered will depend on
its concentration, and can range from, e.g., 1 .mu.L to 10 mL (e.g.
from 10 .mu.L to 1 mL, 50 .mu.L to 500 .mu.L, 70 .mu.L to 200
.mu.L, 90 .mu.L to 150 .mu.L, or 100 .mu.L to 120 .mu.L).
[0377] The nucleic acids can be administered, e.g., hourly,
bihourly, daily, bidaily, twice a week, three times a week, four
times a week, five times a week, six times a week, weekly,
biweekly, monthly, bimonthly, or yearly. Alternatively, the nucleic
acids can be administered, e.g., twice, three times, four times,
five times, six times, seven times, eight times, nine times, 10
times, 11 times, or 12 times per day. The duration of the dosing
regimen can be, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or
30 day, weeks, or months, or even for the remaining lifespan of the
subject.
[0378] These are guidelines, since the actual dose should be
carefully selected and titrated by an attending physician or
nutritionist based upon clinical factors unique to each subject.
The optimal periodic dose will be determined by methods known in
the art and will be influenced by factors such as the age of the
subject, as indicated above, and other clinically relevant factors.
In addition, subjects may be taking medications for other diseases
or conditions. The other medications may be continued during the
time that a polypeptide or nucleic acid of the invention is given
to the subject, but it is advisable in such cases to begin with low
doses to determine if adverse side effects are experienced.
EXAMPLES
[0379] The following examples are provided for the purpose of
illustrating the invention and are not meant to limit the invention
in any way.
Example 1. Characterization of Osteoblasts in Mice Lacking NF1
[0380] To determine the effect of NF1 on bone matrix
mineralization, mice lacking NF1 in osteochondroprogenitor cells
were developed and characterized. These mice displayed skeletal
dysplasia defects similar to patients with neurofibromatosis type
I, where these defects included progressive scoliosis and kyphosis,
tibial bowing and abnormalities in skull and anterior chest wall
formation. In particular, NF1.sub.col2.sup.-/- osteoblasts secreted
increased levels of (PP.sub.i) as compared to osteoblasts from
wild-type mice (FIG. 1A). Bone marrow adherent stromal cells
(BMSCs) extracted from adult NF1.sub.col2.sup.-/- mice and grown in
vitro under osteogenic medium form less alkaline phosphatase
(AP)-positive and less mineralized (alizarin red-positive) nodules
compared to BMSCs extracted from wild-type mice, which is
accompanied by an increased amount of PP.sub.i released in the
medium over 24 hours. Accumulation of PP.sub.i prevents proper bone
matrix mineralization and likely contributes, at least in part, to
defects observed in NF1.sub.col2.sup.-/- mice. Thus, compounds that
reduce PP.sub.i accumulation, such as sALP or an sALP analog, could
be useful for treating NF1.
[0381] In addition, NF1.sub.col2.sup.-/- osteoblasts expressed
increased levels of mRNA for the progressive ankylosis gene (Ank)
as compared to osteoblasts from wild-type mice (FIG. 1B). Treatment
with a dual-specificity MEK1/MEK2 kinase inhibitor (U0126) provided
decreased levels of Ank mRNA expression in NF1.sub.col2.sup.-/-
osteoblasts as compared to vehicle, and these observed levels were
similar to that in wild-type osteoblasts (FIG. 1B, second and third
bars). Thus, compounds that inhibit a kinase pathway, such as NP or
an NP analog, could be useful for treating NF1.
[0382] Taken together, these data suggest that an sALP polypeptide
alone, an NP polypeptide alone, or the combination of both an sALP
polypeptide and an NP polypeptide could be useful for treating any
neurocutaneous syndrome with bone manifestations, such as
neurofibromatosis, or any other disorder described herein.
Example 2. Combination Therapy for the Treatment of
Neurofibromatosis
[0383] FIG. 2 provides a hypothetical working model for defective
bone matrix mineralization in NF1.sub.col2.sup.-/- mice, which can
include multiple imbalances (e.g., increase of PP.sub.i or
overactivation of one or more kinases, such as Ras or ERK) that
contribute to the disease. Without wishing to be limited by theory,
accumulation of PP.sub.i could be minimized by using any of the
compositions and methods described herein including a soluble
alkaline phosphatase (sALP) or sALP analog (see, e.g., the
polypeptide of SEQ ID NO: 1204). Furthermore, overactivation of one
or more kinases could be controlled by using any of the
compositions and methods described herein including an NP or NP
analog. As described herein, the intracellular production of cGMP
resulting from NPR-B activation is known to inhibit the MAP-kinase
pathway. Thus, an NP or NP analog that could activate the NPR-B
signaling pathway can be used for the treatment of neurocutaneous
syndromes, such as neurofibromatosis. Accordingly, the combination
of an sALP or sALP analog (e.g., an sALP polypeptide) with an NP or
NP analog (e.g., an NP polypeptide) could be particularly useful
for treating such diseases.
Example 3. In Vitro and In Vivo Effects of sTNALP-FcD.sub.10 on
NF1.sub.col2.sup.-/- Phenotype
[0384] To assess the effect of sALP polypeptides on NF1 phenotype,
cultures of osteoblasts from NF1.sub.col2.sup.-/- mice were treated
with either bone morphogenetic protein 2 (BMP2) or the sALP fusion
polypeptide of sTNALP-FcD.sub.10 (SEQ ID NO: 1204).
[0385] Both wild-type and NF1.sub.col2.sup.-/- osteoblasts were
treated with increasing concentrations of recombinant human BMP2
(rhBMP2, FIG. 3A). In NF1.sub.col2.sup.-/- osteoblasts, rhBMP2
rescued the differentiation defect, as evidenced by an increased
presence of alkaline phosphatase upon increasing doses of rhBMP2
(FIG. 3A, second row). However, increased mineralization was not
observed, as evidenced by the lack of calcium deposition (as
indicated by the lack of alizarin red S staining) (FIG. 3A, fourth
row).
[0386] In contrast, treatment with sTNALP-FcD.sub.10 rescued the
mineralization defect that is present in NF1.sub.col2.sup.-/-
osteoblasts (FIG. 3B). Increasing doses of sTNALP-FcD.sub.10
provided increased calcific deposition in a dose-dependent manner
(FIG. 3B, bottom, and FIG. 3C).
[0387] Furthermore, in vitro targeted deletion of the NF1 gene in
BMSCs resulted in significant reduction of mineralization, which is
at least partially rescued by treatment with 0.5 .mu.g/mL of
sTNALP-FcD.sub.10(FIG. 3D).
[0388] In vivo experiments were also conducted with
sTNALP-FcD.sub.10. NF1.sub.col2.sup.-/- mice were treated from day
1 to day 18 with 8.2 mg/kg of sTNALP-FcD.sub.10. Treatment of mice
with sTNALP-FcD.sub.10 increased bone mineral density deficit in
NF1.sub.col2.sup.-/- mice compared to vehicle (* p<0.5), as
determined by the ratio of mineralized bone volume (BV) to total
bone volume (TV) (FIG. 3E).
[0389] Accordingly, any sALP polypeptide described herein, either
alone or in combination with any NP polypeptide described herein,
could be particularly useful for treating neurofibromatosis or any
neurocutaneous syndrome with bone manifestations.
Example 4. In Vitro and In Vivo Effects of NC2-KGANKK on
NF1.sub.col2.sup.-/- Phenotype
[0390] To assess the effect of NP polypeptides on NF1 phenotype,
expression levels of the NPR-B gene were assessed in
NF1.sub.col2.sup.-/- mice (FIG. 4A). NPR-B was expressed in BMSCs
at all stages of differentiation, where lack of NF1 expression did
not affect NPR-B expression.
[0391] Additional experiments were conducted in which cultures of
chondrocytes from NF1.sub.col2.sup.-/- mice were treated with the
NP fusion polypeptide of NC2-KGANKK (SEQ ID NO: 512). Rib primary
chondrocytes from P0 mice were obtained from wild-type mice and
NF1.sub.col2.sup.-/- mice and treated for 30 minutes with
increasing concentrations of NC2-KGANKK. Without any treatment,
increased levels of phosphorylated ERK (p-ERK) were observed in
NF1.sub.col2.sup.-/- chondrocytes, as compared to levels in
wild-type chondrocytes (FIG. 4B). After treatment with NC2-KGANKK,
decreased levels of p-ERK were observed in NF1.sub.col2.sup.-/-
chondrocytes. These results support the use of NP polypeptides,
such as NC2-KGANKK, to inhibit the overactivation of one or more
kinases, such as ERK.
[0392] In vivo experiments were also conducted.
NF1.sub.col2.sup.-/- mice were treated with NC2B (SEQ ID NO: 504).
Treatment at least partially rescued the body length defect in
NF1.sub.col2.sup.-/- mice (FIG. 4C), the bone growth defect in
NF1.sub.col2.sup.-/- mice (FIG. 4D), and the proliferative and
hypertrophic chondrocyte zone defects in NF1.sub.col2.sup.-/- mice
(FIG. 4E).
[0393] Accordingly, any NP polypeptide described herein, either
alone or in combination with an sALP polypeptide described herein,
could be particularly useful for treating disorders associated with
overactivation of one or more kinases, such as neurofibromatosis or
any neurocutaneous syndrome with bone manifestations.
REFERENCES
[0394] The following documents are hereby incorporated by
reference. [0395] 1. Wang et al., "Mice lacking Nf1 in
osteochondroprogenitor cells display skeletal dysplasia similar to
patients with neurofibromatosis type I," Hum. Mol. Genet.
20:3910-24, 2011 (Epub 2011 Jul. 14). [0396] 2. Ali et al.,
"Isolation and characterization of calcifying matrix vesicles from
epiphyseal cartilage," Proc. Natl. Acad. Sci. USA 67:1513-20, 1970.
[0397] 3. Anderson et al., "Matrix vesicles in osteomalacic
hypophosphatasia bone contain apatite-like mineral crystals," Am.
J. Pathol. 151:1555-61, 1997. [0398] 4. Anderson et al., "The role
of matrix vesicles in growth plate development and
biomineralization," Frontiers in Bioscience 10:822-837, 2005.
[0399] 5. Anderson et al., "Sustained osteomalacia of long bones
despite major improvement in other hypophosphatasia-related mineral
deficits in tissue nonspecific alkaline phosphatase/nucleotide
pyrophosphatase phosphodiesterase 1 double-deficient mice," Am. J.
Pathol. 166:1711-1720, 2005. [0400] 6. Anderson et al.,
"Pyrophosphate stimulation of calcium uptake into cultured
embryonic bones. Fine structure of matrix vesicles and their role
in calcification," Dev. Biol. 34:211-227, 1973. [0401] 7. Anderson
et al., "Impaired Calcification Around Matrix Vesicles of Growth
Plate and Bone in Alkaline Phosphatase-Deficient Mice," Am. J.
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2009.
Other Embodiments
[0466] All publications, patents, and patent applications mentioned
in the above specification are hereby incorporated by reference.
Various modifications and variations of the described method and
system of the invention will be apparent to those skilled in the
art without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific embodiments, it should be understood that the invention as
claimed should not be unduly limited to such specific embodiments.
Indeed, various modifications of the described modes for carrying
out the invention that are obvious to those skilled in the art are
intended to be within the scope of the invention.
[0467] Other embodiments are in the claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180326019A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20180326019A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References