U.S. patent application number 10/074152 was filed with the patent office on 2003-04-03 for immunological methods to modulate myostatin in vertebrate subjects.
This patent application is currently assigned to MetaMorphix International, Inc.. Invention is credited to Barker, Christopher A., Morsey, Mohamad.
Application Number | 20030065137 10/074152 |
Document ID | / |
Family ID | 22124285 |
Filed Date | 2003-04-03 |
United States Patent
Application |
20030065137 |
Kind Code |
A1 |
Barker, Christopher A. ; et
al. |
April 3, 2003 |
Immunological methods to modulate myostatin in vertebrate
subjects
Abstract
Immunological compositions and methods for reducing myostatin
activity in vertebrate subjects are disclosed. The compositions
include myostatin peptide immunogens, myostatin multimers and or
myostatin immunoconjugates capable of eliciting an immune response
in a vertebrate subject to which the compositions are administered.
The methods are useful for the treatment of a wide variety of
disorders.
Inventors: |
Barker, Christopher A.;
(Saskatoon, CA) ; Morsey, Mohamad; (Niantic,
CT) |
Correspondence
Address: |
Roberta L. Robins
ROBINS & PASTERNAK LLP
Suite 180
545 Middlefield Road
Menlo Park
CA
94025
US
|
Assignee: |
MetaMorphix International,
Inc.
|
Family ID: |
22124285 |
Appl. No.: |
10/074152 |
Filed: |
February 11, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10074152 |
Feb 11, 2002 |
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09252149 |
Feb 18, 1999 |
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6369201 |
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60075212 |
Feb 19, 1998 |
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60075213 |
Feb 19, 1998 |
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Current U.S.
Class: |
530/324 ;
530/325; 530/326; 530/327; 530/328; 530/329; 530/330 |
Current CPC
Class: |
A61P 21/00 20180101;
C07K 2319/00 20130101; A61P 3/04 20180101; A61P 37/06 20180101;
A61P 43/00 20180101; A61K 2039/51 20130101; A61P 5/00 20180101;
A61K 38/00 20130101; C07K 14/475 20130101; A61P 3/00 20180101; A61K
39/00 20130101 |
Class at
Publication: |
530/324 ;
530/325; 530/326; 530/327; 530/328; 530/329; 530/330 |
International
Class: |
C07K 014/575; C07K
007/08; C07K 007/06 |
Claims
We claim:
1. A myostatin peptide consisting of about 3 to about 100 amino
acids, said peptide comprising at least one epitope of
myostatin.
2. The myostatin peptide of claim 1, wherein said myostatin peptide
consists of about 3 to about 30 amino acids.
3. The myostatin peptide of claim 1, wherein said myostatin peptide
consists of about 3 to about 15 amino acids.
4. The myostatin peptide of claim 1, wherein said myostatin peptide
is derived from the region of myostatin spanning amino acids 45
through 376, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36).
5. The myostatin peptide of claim 2, wherein said myostatin peptide
is derived from the region of myostatin spanning amino acids 45
through 376, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36).
6. The myostatin peptide of claim 4, wherein said myostatin peptide
is derived from the region of myostatin spanning amino acids 235
through 376, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36).
7. The myostatin peptide of claim 4, wherein said myostatin peptide
has at least about 75% amino acid identity to a peptide comprising
an amino acid sequence selected from the group consisting of amino
acids 3-18, inclusive of SEQ ID NO:4; amino acids 3-15, inclusive
of SEQ ID NO:6; amino acids 3-17, inclusive, of SEQ ID NO:8; amino
acids 3-16, inclusive of SEQ ID NO:10; amino acids 3-22, inclusive
of SEQ ID NO:12; amino acids 3-25, inclusive of SEQ ID NO:14; amino
acids 3-22, inclusive of SEQ ID NO:16; amino acids 3-18, inclusive
of SEQ ID NO:20; and amino acids 3-18, inclusive, of SEQ ID
NO:22.
8. The myostatin peptide of claim 7, wherein said myostatin peptide
comprises the amino acid sequence of amino acids 3-18, inclusive,
of SEQ ID NO:4.
9. The myostatin peptide of claim 7, wherein said myostatin peptide
comprises the amino acid sequence of amino acids 3-15, inclusive,
of SEQ ID NO:6.
10. The myostatin peptide of claim 7, wherein said myostatin
peptide comprises the amino acid sequence of amino acids 3-17,
inclusive, of SEQ ID NO:8.
11. The myostatin peptide of claim 7, wherein said myostatin
peptide comprises the amino acid sequence of amino acids 3-16,
inclusive, of SEQ ID NO:10.
12. The myostatin peptide of claim 7, wherein said myostatin
peptide comprises the amino acid sequence of amino acids 3-22,
inclusive, of SEQ ID NO:12.
13. The myostatin peptide of claim 7, wherein said myostatin
peptide comprises the amino acid sequence of amino acids 3-25,
inclusive, of SEQ ID NO:14.
14. The myostatin peptide of claim 7, wherein said myostatin
peptide comprises the amino acid sequence of amino acids 3-22,
inclusive, of SEQ ID NO:16.
15. The myostatin peptide of claim 7, wherein said myostatin
peptide comprises the amino acid sequence of amino acids 3-18,
inclusive, of SEQ ID NO:20.
16. The myostatin peptide of claim 7, wherein said myostatin
peptide comprises the amino acid sequence of amino acids 3-18,
inclusive of SEQ ID NO:22.
17. The myostatin peptide of claim 1, wherein said myostatin
peptide comprises the amino acid sequence Lys-Arg-Ser-Arg-Arg-Asp
(SEQ ID NO:37).
18. The myostatin peptide of claim 2, wherein said myostatin
peptide comprises the amino acid sequence Lys-Arg-Ser-Arg-Arg-Asp
(SEQ ID NO:37).
19. The myostatin peptide of claim 1, wherein said myostatin
peptide comprises the amino acid sequence
Lys-Glu-Asn-Val-Glu-Lys-Glu (SEQ ID NO:38).
20. The myostatin peptide of claim 2, wherein said myostatin
peptide comprises the amino acid sequence
Lys-Glu-Asn-Val-Glu-Lys-Glu (SEQ ID NO:38).
21. The myostatin peptide of claim 1, wherein said myostatin
peptide comprises the amino acid sequence Ser-Leu-Lys-Asp-Asp-Asp
(SEQ ID NO:39).
22. The myostatin peptide of claim 2, wherein said myostatin
peptide comprises the amino acid sequence Ser-Leu-Lys-Asp-Asp-Asp
(SEQ ID NO:39).
23. A myostatin peptide consisting of about 3 to about 200 amino
acids, said peptide comprising at least one epitope of myostatin,
wherein said peptide is derived from a region of myostatin selected
from the group consisting of the region of myostatin spanning amino
acids 1 through 350, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36);
the region of myostatin spanning amino acids 1 through 275,
inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); the region of
myostatin spanning amino acids 25 through 300, inclusive, of FIGS.
1A-1D (SEQ ID NOS:27-36); the region of myostatin spanning amino
acids 50 through 325, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36);
and the region of myostatin spanning amino acids 75 through 350,
inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36).
24. The myostatin peptide of claim 23, wherein said myostatin
peptide consists of about 3 to about 30 amino acids.
25. The myostatin peptide of claim 23, wherein said myostatin
peptide consists of about 3 to about 15 amino acids.
26. The myostatin peptide of claim 23, wherein said myostatin
peptide comprises the amino acid sequence of amino acids 3-19,
inclusive, of SEQ ID NO:18.
27. The myostatin peptide of claim 23, wherein said myostatin
peptide comprises the amino acid sequence Lys-Arg-Ser-Arg-Arg-Asp
(SEQ ID NO:37).
28. The myostatin peptide of claim 24, wherein said myostatin
peptide comprises the amino acid sequence Lys-Arg-Ser-Arg-Arg-Asp
(SEQ ID NO:37).
29. The myostatin peptide of claim 23, wherein said myostatin
peptide comprises the amino acid sequence
Lys-Glu-Asn-Val-Glu-Lys-Glu (SEQ ID NO:38).
30. The myostatin peptide of claim 24, wherein said myostatin
peptide comprises the amino acid sequence
Lys-Glu-Asn-Val-Glu-Lys-Glu (SEQ ID NO:38).
31. The myostatin peptide of claim 23, wherein said myostatin
peptide comprises the amino acid sequence Ser-Leu-Lys-Asp-Asp-Asp
(SEQ ID NO:39).
32. The myostatin peptide of claim 24, wherein said myostatin
peptide comprises the amino acid sequence Ser-Leu-Lys-Asp-Asp-Asp
(SEQ ID NO:39).
33. A myostatin multimer comprising two or more selected myostatin
immunogens, wherein each of said myostatin immunogens independently
comprises at least 3 amino acids defining at least one epitope of
myostatin.
34. The myostatin multimer of claim 33, wherein each of said
selected myostatin immunogens independently consists of about 3 to
about 200 amino acids and comprises at least one epitope of
myostatin.
35. The myostatin multimer of claim 33, wherein each of said
selected myostatin immunogens independently consists of about 3 to
about 100 amino acids and comprises at least one epitope of
myostatin.
36. The myostatin multimer of claim 33, wherein each of said
selected myostatin immunogens independently consists of about 3 to
about 30 amino acids and comprises at least one epitope of
myostatin.
37. The myostatin multimer of claim 33, wherein each of said
selected myostatin immunogens independently consists of about 3 to
about 15 amino acids and comprises at least one epitope of
myostatin.
38. The myostatin multimer of claim 33, wherein each of said
selected myostatin immunogens is independently derived from a
region of myostatin selected from the group consisting of the
region of myostatin spanning amino acids 100 through 376,
inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); the region of
myostatin spanning amino acids 235 through 376, inclusive, of FIGS.
1A-1D (SEQ ID NOS:27-36); the region of myostatin spanning amino
acids 1 through 376, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36);
the region of myostatin spanning amino acids 1 through 350,
inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); the region of
myostatin spanning amino acids 1 through 275, inclusive, of FIGS.
1A-1D (SEQ ID NOS:27-36); the region of myostatin spanning amino
acids 25 through 300, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36);
the region of myostatin spanning amino acids 50 through 325,
inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); and the region of
myostatin spanning amino acids 75 through 350, inclusive, of FIGS.
1A-1D (SEQ ID NOS:27-36).
39. The myostatin multimer of claim 33, wherein each of said
selected myostatin immunogens independently has at least about 75%
amino acid identity to a peptide comprising an amino acid sequence
selected from the group consisting of amino acids 3-18, inclusive
of SEQ ID NO:4; amino acids 3-15, inclusive of SEQ ID NO:6; amino
acids 3-17, inclusive, of SEQ ID NO:8; amino acids 3-16, inclusive
of SEQ ID NO:10; amino acids 3-22, inclusive of SEQ ID NO:12; amino
acids 3-25, inclusive of SEQ ID NO:14; amino acids 3-22, inclusive
of SEQ ID NO:16; amino acids 3-19, inclusive, of SEQ ID NO:18;
amino acids 3-18, inclusive of SEQ ID NO:20; and amino acids 3-18,
inclusive, of SEQ ID NO:22.
40. The myostatin multimer of claim 33, wherein at least one of
said selected myostatin immunogens comprises the amino acid
sequence Lys-Arg-Ser-Arg-Arg-Asp (SEQ ID NO:37).
41. The myostatin multimer of claim 34, wherein at least one of
said selected myostatin immunogens comprises the amino acid
sequence Lys-Arg-Ser-Arg-Arg-Asp (SEQ ID NO:37).
42. The myostatin multimer of claim 33, wherein at least one of
said selected myostatin immunogens comprises the amino acid
sequence Lys-Glu-Asn-Val-Glu-Lys-Glu (SEQ ID NO:38).
43. The myostatin multimer of claim 34, wherein at least one of
said selected myostatin immunogens comprises the amino acid
sequence Lys-Glu-Asn-Val-Glu-Lys-Glu (SEQ ID NO:38).
44. The myostatin multimer of claim 33, wherein at least one of
said selected myostatin immunogens comprises the amino acid
sequence Ser-Leu-Lys-Asp-Asp-Asp (SEQ ID NO:39).
45. The myostatin multimer of claim 34, wherein at least one of
said selected myostatin immunogens comprises the amino acid
sequence Ser-Leu-Lys-Asp-Asp-Asp (SEQ ID NO:39).
46. The myostatin multimer of claim 33, wherein said multimer
comprises a molecule according to the general formula (MP-X-MP)y,
wherein MP is a myostatin peptide, X is selected from the group
consisting of a peptide linkage, an amino acid spacer group, a
leukotoxin polypeptide and [MP].sub.n, where n is greater than or
equal to 1, and y is greater than or equal to 1.
47. The myostatin multimer of claim 46, wherein X comprises an
amino acid spacer group including at least one helper T-cell
epitope.
48. The myostatin multimer of claim 46, wherein the myostatin
peptides present in the multimer are the same.
49. The myostatin multimer of claim 46, wherein the myostatin
peptides present in the multimer are different.
50. A myostatin immunoconjugate comprising at least one myostatin
peptide according to claim 1, linked to an immunological
carrier.
51. A myostatin immunoconjugate comprising at least one myostatin
peptide according to claim 7, linked to an immunological
carrier.
52. A myostatin immunoconjugate comprising at least one myostatin
peptide according to claim 23, linked to an immunological
carrier.
53. A myostatin immunoconjugate comprising at least one myostatin
multimer according to claim 33, linked to an immunological
carrier.
54. The myostatin immunoconjugate of claim 50, wherein the
immunological carrier is a leukotoxin polypeptide.
55. The myostatin immunoconjugate of claim 51, wherein the
immunological carrier is a leukotoxin polypeptide.
56. The myostatin immunoconjugate of claim 52, wherein the
immunological carrier is a leukotoxin polypeptide.
57. The myostatin immunoconjugate of claim 53, wherein the
immunological carrier is a leukotoxin polypeptide.
58. A vaccine composition comprising a myostatin peptide according
to of claim 1 and a pharmaceutically acceptable excipient.
59. A vaccine composition comprising a myostatin peptide according
to of claim 7 and a pharmaceutically acceptable excipient.
60. A vaccine composition comprising a myostatin peptide according
to of claim 23 and a pharmaceutically acceptable excipient.
61. A vaccine composition comprising a myostatin multimer according
to claim 33 and a pharmaceutically acceptable excipient.
62. A vaccine composition comprising a myostatin immunoconjugate
according to claim 50 and a pharmaceutically acceptable
excipient.
63. A vaccine composition comprising a myostatin immunoconjugate
according to claim 51 and a pharmaceutically acceptable
excipient.
64. A vaccine composition comprising a myostatin immunoconjugate
according to claim 52 and a pharmaceutically acceptable
excipient.
65. A vaccine composition comprising a myostatin immunoconjugate
according to claim 53 and a pharmaceutically acceptable
excipient.
66. The vaccine composition of claim 58, further comprising an
adjuvant.
67. The vaccine composition of claim 59, further comprising an
adjuvant.
68. The vaccine composition of claim 60, further comprising an
adjuvant.
69. The vaccine composition of claim 61, further comprising an
adjuvant.
70. The vaccine composition of claim 62, further comprising an
adjuvant.
71. The vaccine composition of claim 63, further comprising an
adjuvant.
72. The vaccine composition of claim 64, further comprising an
adjuvant.
73. The vaccine composition of claim 65, further comprising an
adjuvant.
74. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
vaccine composition of claim 58 to said vertebrate subject.
75. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
vaccine composition of claim 59 to said vertebrate subject.
76. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
vaccine composition of claim 60 to said vertebrate subject.
77. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
vaccine composition of claim 61 to said vertebrate subject.
78. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
vaccine composition of claim 62 to said vertebrate subject.
79. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
vaccine composition of claim 63 to said vertebrate subject.
80. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
vaccine composition of claim 64 to said vertebrate subject.
81. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
vaccine composition of claim 65 to said vertebrate subject.
82. The method of claim 74, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
83. The method of claim 75, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
84. The method of claim 76, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
85. The method of claim 77, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
86. The method of claim 78, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
87. The method of claim 79, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
88. The method of claim 80, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
89. The method of claim 81, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
90. A method of treating a disorder which comprises degeneration or
wasting of muscle in a vertebrate subject, said method comprising
administering the vaccine composition of claim 58 to said
subject.
91. A method of treating a disorder which comprises degeneration or
wasting of muscle in a vertebrate subject, said method comprising
administering the vaccine composition of claim 59 to said
subject.
92. A method of treating a disorder which comprises degeneration or
wasting of muscle in a vertebrate subject, said method comprising
administering the vaccine composition of claim 60 to said
subject.
93. A method of treating a disorder which comprises degeneration or
wasting of muscle in a vertebrate subject, said method comprising
administering the vaccine composition of claim 61 to said
subject.
94. A method of treating a disorder which comprises degeneration or
wasting of muscle in a vertebrate subject, said method comprising
administering the vaccine composition of claim 62 to said
subject.
95. A method of treating a disorder which comprises degeneration or
wasting of muscle in a vertebrate subject, said method comprising
administering the vaccine composition of claim 63 to said
subject.
96. A method of treating a disorder which comprises degeneration or
wasting of muscle in a vertebrate subject, said method comprising
administering the vaccine composition of claim 64 to said
subject.
97. A method of treating a disorder which comprises degeneration or
wasting of muscle in a vertebrate subject, said method comprising
administering the vaccine composition of claim 65 to said
subject.
98. A method of modulating GDF11 activity in a vertebrate subject
comprising administering the vaccine composition of claim 58 to
said vertebrate subject.
99. A method of modulating GDF11 activity in a vertebrate subject
comprising administering the vaccine composition of claim 60 to
said vertebrate subject.
100. A method of modulating GDF11 activity in a vertebrate subject
comprising administering the vaccine composition of claim 61 to
said vertebrate subject.
101. A method of modulating GDF11 activity in a vertebrate subject
comprising administering the vaccine composition of claim 62 to
said vertebrate subject.
102. A method of modulating GDF11 activity in a vertebrate subject
comprising administering the vaccine composition of claim 64 to
said vertebrate subject.
103. A method of modulating GDF11activity in a vertebrate subject
comprising administering the vaccine composition of claim 65 to
said vertebrate subject.
104. A polynucleotide encoding a myostatin peptide according to
claim 1.
105. A polynucleotide encoding a myostatin peptide according to
claim 7.
106. A polynucleotide encoding a myostatin peptide according to
claim 23.
107. A polynucleotide encoding a myostatin multimer according to
claim 33.
108. A polynucleotide encoding a myostatin immunoconjugate
according to claim 50.
109. A polynucleotide encoding a myostatin immunoconjugate
according to claim 51.
110. A polynucleotide encoding a myostatin immunoconjugate
according to claim 52.
111. A recombinant vector comprising: (a) a polynucleotide
according to claim 104; and (b) control elements that are operably
linked to said polynucleotide whereby a coding sequence within said
polynucleotide can be transcribed and translated in a host cell,
and at least one of said control elements is heterologous to said
coding sequence.
112. A recombinant vector comprising: (a) a polynucleotide
according to claim 105; and (b) control elements that are operably
linked to said polynucleotide whereby a coding sequence within said
polynucleotide can be transcribed and translated in a host cell,
and at least one of said control elements is heterologous to said
coding sequence.
113. A recombinant vector comprising: (a) a polynucleotide
according to claim 106; and (b) control elements that are operably
linked to said polynucleotide whereby a coding sequence within said
polynucleotide can be transcribed and translated in a host cell,
and at least one of said control elements is heterologous to said
coding sequence.
114. A recombinant vector comprising: (a) a polynucleotide
according to claim 107; and (b) control elements that are operably
linked to said polynucleotide whereby a coding sequence within said
polynucleotide can be transcribed and translated in a host cell,
and at least one of said control elements is heterologous to said
coding sequence.
115. A recombinant vector comprising: (a) a polynucleotide
according to claim 108; and (b) control elements that are operably
linked to said polynucleotide whereby a coding sequence within said
polynucleotide can be transcribed and translated in a host cell,
and at least one of said control elements is heterologous to said
coding sequence.
116. A recombinant vector comprising: (a) a polynucleotide
according to claim 109; and (b) control elements that are operably
linked to said polynucleotide whereby a coding sequence within said
polynucleotide can be transcribed and translated in a host cell,
and at least one of said control elements is heterologous to said
coding sequence.
117. A recombinant vector comprising: (a) a polynucleotide
according to claim 110; and (b) control elements that are operably
linked to said polynucleotide whereby a coding sequence within said
polynucleotide can be transcribed and translated in a host cell,
and at least one of said control elements is heterologous to said
coding sequence.
118. A host cell transformed with the recombinant vector of claim
111.
119. A host cell transformed with the recombinant vector of claim
112.
120. A host cell transformed with the recombinant vector of claim
113.
121. A host cell transformed with the recombinant vector of claim
114.
122. A host cell transformed with the recombinant vector of claim
115.
123. A host cell transformed with the recombinant vector of claim
116.
124. A host cell transformed with the recombinant vector of claim
117.
125. A method of producing a recombinant myostatin peptide
comprising: (a) providing a population of host cells according to
claim 118; and (b) culturing said population of cells under
conditions whereby the myostatin peptide encoded by the coding
sequence present in said recombinant vector is expressed.
126. A method of producing a recombinant myostatin multimer
comprising: (a) providing a population of host cells according to
claim 119; and (b) culturing said population of cells under
conditions whereby the myostatin multimer encoded by the coding
sequence present in said recombinant vector is expressed.
127. A method of producing a recombinant myostatin immunoconjugate
comprising: (a) providing a population of host cells according to
claim 120; and (b) culturing said population of cells under
conditions whereby the myostatin multimer encoded by the coding
sequence present in said recombinant vector is expressed.
128. A method of producing a recombinant myostatin peptide
comprising: (a) providing a population of host cells according to
claim 121; and (b) culturing said population of cells under
conditions whereby the myostatin peptide encoded by the coding
sequence present in said recombinant vector is expressed.
129. A method of producing a recombinant myostatin multimer
comprising: (a) providing a population of host cells according to
claim 122; and (b) culturing said population of cells under
conditions whereby the myostatin multimer encoded by the coding
sequence present in said recombinant vector is expressed.
130. A method of producing a recombinant myostatin immunoconjugate
comprising: (a) providing a population of host cells according to
claim 123; and (b) culturing said population of cells under
conditions whereby the myostatin multimer encoded by the coding
sequence present in said recombinant vector is expressed.
131. A method of producing a recombinant myostatin immunoconjugate
comprising: (a) providing a population of host cells according to
claim 124; and (b) culturing said population of cells under
conditions whereby the myostatin multimer encoded by the coding
sequence present in said recombinant vector is expressed.
132. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
polynucleotide of claim 104 to said vertebrate subject.
133. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
polynucleotide of claim 106 to said vertebrate subject.
134. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
polynucleotide of claim 107 to said vertebrate subject.
135. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
polynucleotide of claim 108 to said vertebrate subject.
136. A method of eliciting an immune response against a myostatin
immunogen in a vertebrate subject, comprising administering the
polynucleotide of claim 110 to said vertebrate subject.
137. The method of claim 132, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
138. The method of claim 133, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
139. The method of claim 134, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
140. The method of claim 135, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
141. The method of claim 136, wherein the immune response elicited
reduces endogenous myostatin activity in said vertebrate subject
and results in at least one of the following biological effects:
(a) an increase in body weight; (b) an increase in muscle mass; (c)
an increase in the number of muscle cells; (d) an increase in the
size of muscle cells; (e) a reduction in body fat content; (f) an
increase in muscle strength; (g) an increase in mammary gland
tissue; (h) an increase in lactation; (i) an increase in appetite
or feed uptake; or (j) an increase in the life span of the
vertebrate subject.
142. A method of treating a disorder which comprises degeneration
or wasting of muscle in a vertebrate subject, said method
comprising administering the polynucleotide of claim 104 to said
subject.
143. A method of treating a disorder which comprises degeneration
or wasting of muscle in a vertebrate subject, said method
comprising administering the polynucleotide of claim 106 to said
subject.
144. A method of treating a disorder which comprises degeneration
or wasting of muscle in a vertebrate subject, said method
comprising administering the polynucleotide of claim 107 to said
subject.
145. A method of treating a disorder which comprises degeneration
or wasting of muscle in a vertebrate subject, said method
comprising administering the polynucleotide of claim 108 to said
subject.
146. A method of treating a disorder which comprises degeneration
or wasting of muscle in a vertebrate subject, said method
comprising administering the polynucleotide of claim 110 to said
subject.
147. An isolated antibody reactive with a myostatin peptide
according to claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is related to provisional patent
application serial No. 60/075,213, filed Feb. 19, 1998, from which
priority is claimed under 35 USC .sctn.119(e)(1) and which is
incorporated herein by reference in its entirety.
TECHNICAL FIELD
[0002] The present invention relates generally to compositions and
methods for increasing muscle synthesis and treating disease in
vertebrate subjects. More particularly, the invention is directed
to immunological compositions and methods for reducing myostatin
activity in vertebrate subjects.
BACKGROUND OF THE INVENTION
[0003] Livestock producers have traditionally used breeding
programs to select animals that yield maximum amounts of protein
with acceptable performance as measured by feed efficiency,
reproductive function and general health. Cattle which exhibit
increased muscle mass due to both hypertrophy and hyperplasia of
muscle cells have been observed in a number of breeds. The
incidence of this condition, which is referred to as
double-muscling, is most pronounced in Belgian Blue cattle. Muscle
mass is increased by approximately 20% with a decrease in bone and
fat mass in these animals (Shahin and Berg, Can. J. Anim. Sci.
(1985) 65:279293). Belgian Blue cattle also utilize feed
efficiently and give rise to a higher percentage of desirable cuts
of meat (Casas et al., J. Anim. Sci. (1997) 75(Supp 1):149).
Double-muscling in Belgian Blue cattle is inherited and is believed
to be recessive since heterozygotes may be normal or have only a
modest increase in muscle mass.
[0004] Despite the advantages of this condition, double-muscled
cattle often have undesirable traits. For example, because calves
are generally 10-38% heavier than normal, dystocias are prevalent,
requiring cesarean deliveries. Animals also exhibit abnormal
reproduction due to poorly developed reproductive tracts and have
other anatomical abnormalities such as macroglossia. Other breeds
of cattle, such as the Piemontese from northern Italy, have varying
degrees of double-muscling and also display many of these
undesirable traits.
[0005] The double-muscling characteristic identified in some cattle
breeds has now been traced to mutations in the myostatin gene
(Grobet et al., Nature Genetics (1997) 17:71-74; Kambadur et al.,
Genome Research (1997) 7:910-915; McPherron and Lee, Proc. Natl.
Acad. Sci. USA (1997) 94:12457-12461). This mutation appears to
result mainly in an increase in the number of muscle cells
(hyperplasia) rather than an increase in the size of individual
muscle fibers (hypertrophy). A condition referred to as muscular
hypertrophy has also been identified in the Pietrain breed of pig.
This condition is not related to the myostatin gene and has been
identified as a mutation in a gene responsible for calcium
transport.
[0006] McPherron et al., Nature (1997) 387:83-90, have identified a
member of the transforming growth factor-.beta. (TGF-.beta.)
superfamily of proteins in mice, referred to as
growth/differentiating factor-8 (GDF-8). GDF-8 acts as a negative
regulator for skeletal muscle growth and is expressed in developing
and adult skeletal muscles. Gene knockout experiments in mice have
resulted in homozygous mutants which are 30% larger than wild-type
mice. This increase in size is due primarily to an increase in
muscle mass with individual muscles from the mutants weighing 2-3
times more than those from wild-type mice (McPherron et al., Nature
(1997) 387:83-90). McPherron and Lee, Proc. Natl. Acad. Sci. USA
(1997) 94:12457-12461 and Grobet et al., Nature Genetics (1997)
17:71-74 evaluated similar genomic sequences in a number of
species, including cattle, and reported that double-muscled cattle
had defects in the gene coding for a protein highly homologous to
GDF-8. This protein is now called myostatin.
[0007] Thus, it appears that myostatin is produced by muscle cells
and regulates the proliferation and differentiation of myoblasts.
In Belgian Blue and Piemontese cattle, natural defects in the gene
are believed to result either in production of an abnormal protein
or a reduced amount of myostatin, either of which has the effect of
increasing muscle growth.
[0008] The myostatin gene from a number of vertebrate species,
including mouse, rat, human, baboon, cattle, pig, sheep, chicken,
turkey, and zebrafish has been identified and the proteins
sequenced (McPherron and Lee, Proc. Natl. Acad. Sci. USA (1997)
94:12457-12461). The myostatin protein sequence is highly conserved
across all of these species. Similarly, the nucleotide sequence for
myostatin from mouse, rat, human, baboon, cattle, pig, sheep,
chicken and turkey has been determined. See, e.g., U.S. Pat. No.
5,827,733 for the nucleotide sequences of murine and human
myostatin; International Publication No. WO 99/02667 for the
nucleotide sequence of bovine myostatin; International Publication
No. WO 98/33887, for the nucleotide sequences of rat, human,
baboon, bovine, porcine, ovine, chicken and turkey myostatin.
[0009] The nucleotide sequence of the myostatin gene predicts a
protein of about 376 amino acids with a molecular weight of
approximately 43 kDa. This protein contains a secretion leader
sequence and a proteolytic processing site which releases a 13 kDa
peptide, containing 9 cysteine residues. Cloned myostatin expressed
in Chinese hamster ovary cells yields two proteins. The first has
an apparent molecular weight of about 52 kDa and the second about
15 kDa. Under nonreducing conditions, these proteins appear to be
dimers with molecular weights of about 101 kDa and 25 kDa
(McPherron et al., Nature (1997) 387:83-90).
[0010] Researchers have proposed delivery of mutated myostatin
genes to animal subjects for the production of transgenic species
having increased muscle tissue. See, e.g., International
Publication No. WO 98/33887. However, such approaches pose several
drawbacks. For example, because the myostatin gene becomes active
during the embryonic stage, reduced myostatin production causes
excessive muscle development in utero. Thus, transgenic animals
which include mutated genes would likely require cesarean delivery,
a serious burden to large animal producers. Additionally, public
opposition to genetically engineered animals for human consumption
exists and other methods of producing such animals would be
desirable.
DISCLOSURE OF THE INVENTION
[0011] The present invention is directed to immunological
compositions and methods for modulating endogenous myostatin
activity in a vertebrate subject. The invention is also useful for
treating a number of conditions in vertebrates, including humans
and other animals, such as a variety of disorders that cause
degeneration or wasting of muscle. Due to the ubiquitous nature of
myostatin, the compositions and methods described herein find use
in a wide variety of vertebrate subjects, as described further
below.
[0012] Surprisingly, the invention achieves these results by
immunological techniques. It is readily known in the art that
immunization against endogenous molecules, such as myostatin, is
problematic because the immune system does not recognize such
"self" molecules. Thus, the present invention provides a solution
to a problem which would normally be encountered when immunizing
against an endogenous substance.
[0013] Accordingly, in one embodiment, the invention is directed to
a myostatin peptide consisting of about 3 to about 100 amino acids.
The peptide comprises at least one epitope of myostatin. In
preferred embodiments, the myostatin peptide is derived from the
region of myostatin spanning amino acids 45 through 376, inclusive,
of FIGS. 1A-1D (SEQ ID NOS:27-36) or amino acids 235 through 376,
inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36).
[0014] In other embodiments, the myostatin peptide has at least
about 75% amino acid identity to a peptide comprising an amino acid
sequence selected from the group consisting of amino acids 3-18,
inclusive of SEQ ID NO:4; amino acids 3-15, inclusive of SEQ ID
NO:6; amino acids 3-17, inclusive, of SEQ ID NO:8; amino acids
3-16, inclusive of SEQ ID NO:10; amino acids 3-22, inclusive of SEQ
ID NO:12; amino acids 3-25, inclusive of SEQ ID NO:14; amino acids
3-22, inclusive of SEQ ID NO:16; amino acids 3-18, inclusive of SEQ
ID NO:20; and amino acids 3-18, inclusive, of SEQ ID NO:22.
[0015] In still further embodiments, the invention is directed to a
myostatin peptide consisting of about 3 to about 200 amino acids.
The peptide comprises at least one epitope of myostatin and is
derived from a region of myostatin selected from the group
consisting of the region of myostatin spanning amino acids 1
through 350, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); the
region of myostatin spanning amino acids 1 through 275, inclusive,
of FIGS. 1A-1D (SEQ ID NOS:27-36); the region of myostatin spanning
amino acids 25 through 300, inclusive, of FIGS. 1A-1D (SEQ ID
NOS:27-36); the region of myostatin spanning amino acids 50 through
325, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); and the region
of myostatin spanning amino acids 75 through 350, inclusive, of
FIGS. 1A-1D (SEQ ID NOS:27-36).
[0016] In yet further embodiments, the myostatin peptide comprises
the amino acid sequence Lys-Arg-Ser-Arg-Arg-Asp (SEQ ID NO:37), the
amino acid sequence Lys-Glu-Asn-Val-Glu-Lys-Glu (SEQ ID NO:38) or
the amino acid sequence Ser-Leu-Lys-Asp-Asp-Asp (SEQ ID NO:39).
[0017] In yet another embodiment, the invention is directed to a
myostatin multimer comprising two or more selected myostatin
immunogens, wherein each of the immunogens independently comprises
at least 3 amino acids defining at least one epitope of myostatin.
In particularly preferred embodiments, each of the selected
myostatin immunogens comprises at least one epitope of myostatin
and independently consists of about 3 to about 200 amino acids, or
about 3 to about 100 amino acids, or about 3 to about 30 amino
acids, or about 3 to about 15 amino acids.
[0018] In other embodiments, each of the selected myostatin
immunogens in the multimer independently comprise a selected
myostatin peptide as described above. In particularly preferred
embodiments, the multimer comprises a molecule with repeating units
according to the general formula (MP-X-MP)y, wherein MP is a
myostatin peptide, X is selected from the group consisting of a
peptide linkage, an amino acid spacer group, a leukotoxin
polypeptide and [MP].sub.n, where n is greater than or equal to 1,
and y is greater than or equal to 1.
[0019] In another embodiment, the invention is directed to a
myostatin immunoconjugate comprising at least one myostatin peptide
or multimer, as described above, linked to an immunological
carrier.
[0020] In still further embodiments, the invention is directed to
vaccine compositions comprising the myostatin peptide, the
myostatin multimer and/or the myostatin immunoconjugate, and a
pharmaceutically acceptable excipient.
[0021] In yet other embodiments, the invention is directed to
polynucleotides encoding the myostatin peptides, the myostatin
multimers and the myostatin immunoconjugates above, as well as
recombinant vectors comprising the polynucleotides, host cells
transformed with the recombinant vectors, and methods of
recombinantly producing the myostatin peptides, myostatin multimers
and myostatin immunoconjugates.
[0022] In other embodiments, the invention is directed to methods
of eliciting an immune response against a myostatin immunogen in a
vertebrate subject comprising administering the vaccine
compositions or polynucleotides above to the vertebrate subject. In
particularly preferred embodiments, the immune response elicited
reduces endogenous myostatin activity in the vertebrate subject and
results in at least one of the following biological effects:
[0023] (a) an increase in body weight;
[0024] (b) an increase in muscle mass;
[0025] (c) an increase in the number of muscle cells;
[0026] (d) an increase in the size of muscle cells;
[0027] (e) a reduction in body fat content;
[0028] (f) an increase in muscle strength;
[0029] (g) an increase in mammary gland tissue;
[0030] (h) an increase in lactation;
[0031] (i) an increase in appetite or feed uptake; or
[0032] (j) an increase in the life span of the vertebrate
subject.
[0033] In other embodiments, the invention is directed to methods
of treating a disorder which comprises degeneration or wasting of
muscle in a vertebrate subject, the method comprising administering
the vaccine compositions or polynucleotides above to the subject.
The invention is also directed to methods of modulating GDF11
activity in a vertebrate subject comprising administering the
vaccine compositions above.
[0034] These and other embodiments of the present invention will
readily occur to those of ordinary skill in the art in view of the
disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIGS. 1A-1D show a comparison of myostatin derived from
various species as follows: Mouse (SEQ ID NO:27); Rat (SEQ ID
NO:28); Human (SEQ ID NO:29); Baboon (SEQ ID NO:30); Bovine (SEQ ID
NO:31); Porcine (SEQ ID NO:32); Ovine (SEQ ID NO:33); Chicken (SEQ
ID NO:34); Turkey (SEQ ID NO:35); and Zebrafish (SEQ ID NO:36).
Amino acids are numbered to the right and left of the
sequences.
[0036] FIG. 2 shows the nucleotide sequence (SEQ ID NO:3) and
corresponding amino acid sequence (SEQ ID NO:4) of the MYOS 1
peptide. MYOS 1 includes the proteolytic cleavage site,
Arg-Ser-Arg-Arg, and the N-terminus of the active protein.
[0037] FIG. 3 depicts the nucleotide sequence (SEQ ID NO:5) and
corresponding amino acid sequence (SEQ ID NO:6) of the MYOS 3
peptide.
[0038] FIG. 4 depicts the nucleotide sequence (SEQ ID NO:7) and
corresponding amino acid sequence (SEQ ID NO:8) of the MYOS 5
peptide.
[0039] FIG. 5 shows the nucleotide sequence (SEQ ID NO:9) and
corresponding amino acid sequence (SEQ ID NO:10) of the MYOS 7
peptide.
[0040] FIG. 6 shows the nucleotide sequence (SEQ ID NO:ll) and
corresponding amino acid sequence (SEQ ID NO:12) of the MYOS 9
peptide.
[0041] FIG. 7 shows the nucleotide sequence (SEQ ID NO:13) and
corresponding amino acid sequence (SEQ ID NO:14) of the MYOS 11
peptide.
[0042] FIG. 8 shows the nucleotide sequence (SEQ ID NO:15) and
corresponding amino acid sequence (SEQ ID NO:16) of the MYOS 13
peptide.
[0043] FIG. 9 shows the nucleotide sequence (SEQ ID NO:17) and
corresponding amino acid sequence (SEQ ID NO:18) of the MYOS 15
peptide.
[0044] FIG. 10 shows the nucleotide sequence (SEQ ID NO:19) and
corresponding amino acid sequence (SEQ ID NO:20) of the MYOS 17
peptide.
[0045] FIG. 11 shows the nucleotide sequence (SEQ ID NO:21) and
corresponding amino acid sequence (SEQ ID NO:22) of the MYOS 19
peptide. MYOS 19 includes the proteolytic cleavage site,
Arg-Ser-Arg-Arg.
[0046] FIG. 12 shows the approximate position of MYOS peptides 1,
3, 5, 7, 9, 11, 13, 15, 17 and 19 within the myostatin
sequence.
[0047] FIG. 13 shows the nucleotide sequence (SEQ ID NO:23) and
corresponding amino acid sequence (SEQ ID NO:24) for a
reconstructed myostatin active region containing three sets of two
amino acid linkers (Arg-Ser) inserted in the sequence at nucleotide
positions 55-60, 139-144 and 241-246 and at the C-terminus.
[0048] FIG. 14 is a diagram of plasmid pCB150, encoding a
leukotoxin polypeptide carrier and used to create myostatin
expression vectors as described in the examples.
[0049] FIGS. 15A-15D show the nucleotide sequence (SEQ ID NO:25)
and corresponding amino acid sequence (SEQ ID NO:26) of the
leukotoxin carrier polypeptide present in plasmid pCB150. Myostatin
oligo repeats are inserted into the BamH1 site present at
nucleotide position 3334.
[0050] FIG. 16A shows the nucleotide sequence (SEQ ID NO:1) and
[0051] FIG. 16B shows the predicted amino acid sequence (SEQ ID
NO:2) of a representative myostatin for use with the present
invention. The proteolytic cleavage site is found at positions
263-266 of FIG. 16B. The myostatin active region of the polypeptide
spans amino acids 264-375.
[0052] FIG. 17 shows a hydrophilicity profile of the myostatin
protein. The profile was computed using an average group length of
six amino acids. The three highest points of hydrophilicity are
found at amino acid positions 263-268, which span the proteolytic
cleavage site; positions 31-37; and positions 106-111.
[0053] FIG. 18 shows the amount of weight gain in animals treated
with myostatin peptide immunogens, as described in the
examples.
DETAILED DESCRIPTION
[0054] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
microbiology, virology, recombinant DNA technology, and immunology,
which are within the skill of the art. Such techniques are
explained fully in the literature. See, e.g., Sambrook, Fritsch
& Maniatis, Molecular Cloning: A Laboratory Manual; DNA
Cloning, Vols. I and II (D. N. Glover ed.); Oligonucleotide
Synthesis (M. J. Gait ed.); Nucleic Acid Hybridization (B. D. Hames
& S. J. Higgins eds.); B. Perbal, A Practical Guide to
Molecular Cloning; the series, Methods In Enzymology (S. Colowick
and N. Kaplan eds., Academic Press, Inc.); and Handbook of
Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell
eds., Blackwell Scientific Publications).
[0055] All patents, patent applications, and publications mentioned
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
[0056] A. Definitions
[0057] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0058] By "myostatin immunogen" is meant a polypeptide derived from
a myostatin molecule which elicits an immunological response as
defined below. The term includes molecules that elicit an
immunological response without an associated immunological carrier,
adjuvant or immunostimulant, as well as myostatin polypeptides
capable of being rendered immunogenic, or more immunogenic, by way
of association with a carrier molecule, adjuvant or
immunostimulant, or by mutation of a native sequence, and/or by
incorporation into a molecule containing multiple repeating units
of at least one epitope of a myostatin molecule. The term may be
used to refer to an individual macromolecule or to a homogeneous or
heterogeneous population of antigenic macromolecules derived from
myostatin.
[0059] For purposes of the present invention, a myostatin immunogen
may be derived from any of the various known myostatin sequences,
including without limitation, myostatin polypeptides derived from
mouse, rat, human, baboon, cattle, pig, sheep, chicken, turkey, and
zebrafish (see, McPherron and Lee, Proc. Natl. Acad. Sci. USA
(1997) 94:12457-12461). The myostatin protein sequence is highly
conserved across all of these species (see FIGS. 1A-1D).
[0060] Additionally, the term "myostatin immunogen" includes a
myostatin polypeptide molecule differing from the reference
sequence by having one or more amino acid substitutions, deletions
and/or additions and which has at least about 50% amino acid
identity to the reference molecule, more preferably about 75-85%
identity and most preferably about 90-95% identity or more, to the
relevant portion of the native peptide sequence in question. The
amino acid sequence will have not more than about 10-20 amino acid
substitutions, or not more than about 5-10 amino acid
substitutions, or even only 1, 2, 3 or up to 5 substitutions.
Particularly preferred substitutions will generally be conservative
in nature, i.e., those substitutions that take place within a
family of amino acids. In this regard, amino acids are generally
divided into four families: (1) acidic--aspartate and glutamate;
(2) basic--lysine, arginine, histidine; (3) non-polar--alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar--glycine, asparagine,
glutamine, cystine, serine threonine, tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified as aromatic amino
acids. For example, it is reasonably predictable that an isolated
replacement of leucine with isoleucine or valine, or vice versa; an
aspartate with a glutamate or vice versa; a threonine with a serine
or vice versa; or a similar conservative replacement of an amino
acid with a structurally related amino acid, will not have a major
effect on the activity. Proteins having substantially the same
amino acid sequence as the reference molecule, but possessing minor
amino acid substitutions that do not substantially affect the
immunogenicity of the protein, are therefore within the definition
of a myostatin immunogen.
[0061] As used herein a "myostatin immunogen" also includes a
molecule derived from a native myostatin sequence, as well as
recombinantly produced or chemically synthesized myostatin
polypeptides including the full-length myostatin reference
sequence, as well as myostatin peptides which remain immunogenic,
as described below.
[0062] A "myostatin immunogen" thus includes molecules having the
native sequence, molecules with single or multiple amino acid
additions, substitutions and/or deletions, as well as peptide
fragments of the reference myostatin molecule, so long as the
molecule retains the ability to elicit formation of antibodies that
cross-react with the naturally occurring myostatin of the
vertebrate species to which such an immunogen is delivered.
Epitopes of myostatin are also captured by the definition.
[0063] A "myostatin peptide" is a myostatin immunogen, as described
herein, which includes less than the full-length of the reference
myostatin molecule in question and which includes at least one
epitope as defined below. Thus, a vaccine composition comprising a
myostatin peptide would include a portion of the full-length
molecule but not the entire myostatin molecule in question.
[0064] By "myostatin multimer" is meant a molecule having more than
one copy of a selected myostatin immunogen, myostatin peptide or
epitope, or multiple tandem repeats of a selected myostatin
immunogen, myostatin peptide or epitope. The myostatin multimer may
correspond to a molecule with repeating units of the general
formula (MP-X-MP)y wherein MP is a myostatin peptide, X is selected
from the group consisting of a peptide linkage, an amino acid
spacer group and [MP].sub.n, where n is greater than or equal to 1,
y is greater than or equal to 1, and further wherein "MP" may
comprise any MP peptide. Y may therefore define 1-40 or more
repeating units, more preferably, 1-30 repeating units and most
preferably, 1-20 repeating units. Further, the selected myostatin
peptide sequences may all be the same, or may correspond to
different derivatives, analogs, variants or epitopes of myostatin
so long as they retain the ability to elicit an immune response.
Additionally, if the myostatin peptides are linked either
chemically or recombinantly to a carrier, myostatin peptides may be
linked to either the 5'-end, the 3'-end, or may flank the carrier
in question. Further, the myostatin multimer may be located at
sites internal to the carrier. Myostatin multimers are discussed in
further detail below.
[0065] "Homology" refers to the percent identity between two
polynucleotide or two polypeptide moieties. Two DNA, or two
polypeptide sequences are "substantially homologous" to each other
when the sequences exhibit at least about 75%-85%, preferably at
least about 90%, and most preferably at least about 95%-98%
sequence identity over a defined length of the molecules. As used
herein, substantially homologous also refers to sequences showing
complete identity to the specified DNA or polypeptide sequence.
[0066] Percent "identity" between two amino acid or polynucleotide
sequences can be determined by a direct comparison of the sequence
information between two molecules by aligning the sequences,
counting the exact number of matches between the two aligned
sequences, dividing by the length of the shorter sequence, and
multiplying the result by 100. Readily available computer programs
can be used to aid in the analysis, such as ALIGN, Dayhoff, M. O.
in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5
Suppl. 3:353-358, National biomedical Research Foundation,
Washington, D.C., which adapts the local homology algorithm of
Smith and Waterman (1981) Advances in Appl. Math. 2:482-489 for
peptide analysis. Programs for determining nucleotide sequence
identity are available in the Wisconsin Sequence Analysis Package,
Version 8 (available from Genetics Computer Group, Madison, Wis.)
for example, the BESTFIT, FASTA and GAP programs, which also rely
on the Smith and Waterman algorithm. These programs are readily
utilized with the default parameters recommended by the
manufacturer and described in the Wisconsin Sequence Analysis
Package referred to above. For example, percent identity of a
particular nucleotide sequence to a reference sequence can be
determined using the homology algorithm of Smith and Waterman with
a default scoring table and a gap penalty of six nucleotide
positions.
[0067] Alternatively, identity can be determined by hybridization
of polynucleotides under conditions which form stable duplexes
between homologous regions, followed by digestion with
single-stranded-specific nuclease(s), and size determination of the
digested fragments. DNA sequences that are substantially homologous
can be identified in a Southern hybridization experiment under, for
example, stringent conditions, as defined for that particular
system. Defining appropriate hybridization conditions is within the
skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning,
supra; Nucleic Acid Hybridization, supra.
[0068] By the term "degenerate variant" is intended a
polynucleotide containing changes in the nucleic acid sequence
thereof, that encodes a polypeptide having the same amino acid
sequence as the polypeptide encoded by the polynucleotide from
which the degenerate variant is derived.
[0069] An "immunological response" to an immunogen or vaccine is
the development in the host of a cellular and/or antibody-mediated
immune response to the immunogen or vaccine of interest. Usually,
such a response includes but is not limited to one or more of the
following effects; the production of antibodies, B cells, helper T
cells, suppressor T cells, and/or cytotoxic T cells and/or
.gamma..delta. T cells, directed specifically to an immunogen or
immunogens included in a composition or vaccine of interest. An
immunological response can be detected using any of several assays
well known in the art, such as standard immunoassays and
neutralization assays, including Western blots, dot blots and
immunoaffinity assays. The presence of a cell-mediated
immunological responses may be determined using CTL cytotoxic cell
assays, well known in the art, such as the assay described in
Erickson et al. J. Immunol. (1993) 151:4189-4199; and Doe et al.
Eur. J. Immunol. (1994) 24:2369-2376.
[0070] An "epitope" refers to any portion or region of a molecule
with the ability or potential to elicit, and combine with, a
myostatin-specific antibody. For the purpose of the present
invention, a polypeptide epitope will usually include at least
about 3 amino acids, preferably at least about 5 amino acids, and
most preferably at least about 10-15 amino acids to 20-30 or more
amino acids, of the reference molecule. There is no critical upper
limit to the length of the fragment, which could comprise nearly
the full-length of a protein sequence, or even a fusion protein
comprising two or more epitopes of a protein in question.
[0071] Epitopes in polypeptide molecules can be identified using
any number of epitope mapping techniques, well known in the art.
See, e.g., Epitope Mapping Protocols in Methods in Molecular
Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press, Totowa,
N.J. For example, linear epitopes may be determined by e.g.,
concurrently synthesizing large numbers of peptides on solid
supports, the peptides corresponding to portions of the protein
molecule, and reacting the peptides with antibodies while the
peptides are still attached to the supports. Such techniques are
known in the art and described in, e.g., U.S. Pat. No. 4,708,871;
Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002;
Geysen et al. (1986) Molec. Immunol. 23:709-715, all incorporated
herein by reference in their entireties. Similarly, conformational
epitopes are readily identified by determining spatial conformation
of amino acids such as by, e.g., x-ray crystallography and
2-dimensional nuclear magnetic resonance. See, e.g., Epitope
Mapping Protocols, supra. Computer programs that formulate
hydropathy scales from the amino acid sequence of the protein,
utilizing the hydrophobic and hydrophilic properties of each of the
20 amino acids, as described in, e.g., Kyte et al., J. Mol. Biol.
(1982) 157:105-132; and Hopp and Woods, Proc. Natl. Acad. Sci. USA
(1981) 78:3824-3828, can also be used to determine antigenic
portions of a given molecule. For example, the technique of Hopp
and Woods assigns each amino acid a numerical hydrophilicity value
and then repetitively averages these values along the peptide
chain. The points of highest local average hydrophilicities are
indicative of antigenic portions of the molecule.
[0072] By "immunological carrier" is meant any molecule which, when
associated with a myostatin immunogen of interest, imparts
immunogenicity to that molecule, or enhances the immunogenicity of
the molecule. Examples of suitable carriers include large, slowly
metabolized macromolecules such as: proteins; polysaccharides, such
as sepharose, agarose, cellulose, cellulose beads and the like;
polymeric amino acids such as polyglutamic acid, polylysine, and
the like; amino acid copolymers; inactive virus particles;
bacterial toxins such as toxoid from diphtheria, tetanus, cholera,
leukotoxin molecules, and the like. Carriers are described in
further detail below.
[0073] A myostatin immunogen is "linked" to a specified carrier
molecule when the immunogen is chemically coupled to, or associated
with the carrier, or when the immunogen is expressed from a
chimeric DNA molecule which encodes the immunogen and the carrier
of interest.
[0074] An "immunoconjugate" is a myostatin immunogen such as a
myostatin peptide or multimer which is linked to a carrier
molecule, as defined above.
[0075] The term "leukotoxin polypeptide" or "LKT polypeptide"
intends a polypeptide which is derived from a protein belonging to
the family of molecules characterized by the carboxy-terminus
consensus amino acid sequence Gly-Gly-X-Gly-X-Asp (Highlander et
al. (1989) DNA 8:15-28), wherein X is Lys, Asp, Val or Asn. Such
proteins include, among others, leukotoxins derived from P.
haemolytica and Actinobacillus pleuropneumoniae, as well as E. coli
alpha hemolysin (Strathdee et al. (1987) Infect. Immun.
55:3233-3236; Lo (1990) Can. J. Vet. Res. 54:S33-S35; Welch (1991)
Mol. Microbiol. 5:521-528). This family of toxins is known as the
"RTX" family of toxins (Lo (1990) Can. J. Vet. Res. 54:S33-S35). In
addition, the term "leukotoxin polypeptide" refers to a leukotoxin
polypeptide which is chemically synthesized, isolated from an
organism expressing the same, or recombinantly produced.
Furthermore, the term intends an immunogenic protein having an
amino acid sequence substantially homologous to a contiguous amino
acid sequence found in the particular native leukotoxin molecule.
Thus, the term includes both full-length and partial sequences, as
well as analogues. Although native full-length leukotoxins display
cytotoxic activity, the term "leukotoxin" also intends molecules
which remain immunogenic yet lack the cytotoxic character of native
leukotoxins. The nucleotide sequences and corresponding amino acid
sequences for several leukotoxins are known. See, e.g., U.S. Pat.
Nos. 4,957,739 and 5,055,400; Lo et al. (1985) Infect. Immun.
50:667-67; Lo et al. (1987) Infect. Immun. 55:1987-1996; Strathdee
et al. (1987) Infect. Immun. 55:3233-3236; Highlander et al. (1989)
DNA 8:15-28; and Welch (1991) Mol. Microbiol. 5:521-528. In
preferred embodiments of the invention, leukotoxin chimeras are
provided having a selected leukotoxin polypeptide sequence that
imparts enhanced immunogenicity to one or more myostatin multimers
fused thereto.
[0076] Particular examples of immunogenic leukotoxin polypeptides
for use in the present invention are truncated leukotoxin molecules
described in U.S. Pat. Nos. 5,476,657 and 5,837,268, incorporated
herein by reference in their entireties. These truncated molecules
include LKT 352, LKT 111 and LKT 114. LKT 352 is derived from the
lktA gene present in plasmid pAA352 (ATCC Accession No. 68283). The
nucleotide sequence and corresponding amino acid sequence of this
gene are described in U.S. Pat. No. 5,476,657. The gene encodes a
truncated leukotoxin, having 914 amino acids and an estimated
molecular weight of around 99 kDa. LKT 111 is a leukotoxin
polypeptide derived from the lktA gene present in plasmid pCB11
(ATCC Accession No. 69748). The nucleotide sequence of this gene
and the corresponding amino acid sequence are disclosed in U.S.
Pat. No. 5,837,268. The gene encodes a shortened version of
leukotoxin which was developed from the recombinant leukotoxin gene
present in plasmid pAA352 (ATCC Accession No. 68283) by removal of
an internal DNA fragment of approximately 1300 bp in length. The
LKT 111 polypeptide has an estimated molecular weight of 52 kDa (as
compared to the 99 kDa LKT 352 polypeptide), but retains portions
of the LKT 352 N-terminus containing T-cell epitopes which are
necessary for sufficient T-cell immunogenicity, and portions of the
LKT 352 C-terminus containing convenient restriction sites for use
in producing fusion proteins for use in the present invention. LKT
114 is derived from the gene present in plasmid pAA114 (described
in U.S. Pat. No. 5,837,268) and is shown in FIGS. 15A-15D herein.
LKT 114 differs from LKT ill by virtue of an additional amino acid
deletion from the internal portion of the molecule.
[0077] "Adjuvants" refer to agents which act in a nonspecific
manner to increase an immune response to a particular antigen, thus
reducing the quantity of antigen necessary in any given vaccine,
and/or the frequency of injection necessary in order to generate an
adequate immune response to the antigen of interest. See, e.g., A.
C. Allison J. Reticuloendothel. Soc. (1979) 26:619-630.
[0078] "Native" proteins, polypeptides or peptides are proteins,
polypeptides or peptides isolated from the source in which the
proteins naturally occur. "Recombinant" polypeptides refer to
polypeptides produced by recombinant DNA techniques; i.e., produced
from cells transformed by an exogenous DNA construct encoding the
desired polypeptide. "Synthetic" polypeptides are those prepared by
chemical synthesis.
[0079] By "polynucleotide" is meant a sequence of nucleotides
including, but is not limited to, RNA such as mRNA, cDNA, genomic
DNA sequences and even synthetic DNA sequences. The term also
captures sequences that include any of the known base analogs of
DNA and RNA.
[0080] A "vector" is a replicon, such as a plasmid, phage, or
cosmid, to which another DNA segment may be attached so as to bring
about the replication of the attached segment.
[0081] A DNA "coding sequence" or a "sequence encoding" a
particular protein, is a DNA sequence which is transcribed and
translated into a polypeptide in vitro or in vivo when placed under
the control of appropriate regulatory elements. The boundaries of
the coding sequence are determined by a start codon at the
5'-terminus and a translation stop codon at the 3'-terminus. A
coding sequence can include, but is not limited to, procaryotic
sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from
eucaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
A transcription termination sequence will usually be located 3' to
the coding sequence.
[0082] The term DNA "control elements" refers collectively to
promoters, ribosome binding sites, polyadenylation signals,
transcription termination sequences, upstream regulatory domains,
enhancers, and the like, which collectively provide for the
transcription and translation of a coding sequence in a host cell.
Not all of these control sequences need always be present in a
recombinant vector so long as the desired gene is capable of being
transcribed and translated.
[0083] "Operably linked" refers to an arrangement of elements
wherein the components so described are configured so as to perform
their usual function. Thus, control elements operably linked to a
coding sequence are capable of effecting the expression of the
coding sequence. The control elements need not be contiguous with
the coding sequence, so long as they function to direct the
expression thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter and the
coding sequence and the promoter can still be considered "operably
linked" to the coding sequence.
[0084] A control element, such as a promoter, "directs the
transcription" of a coding sequence in a cell when RNA polymerase
will bind the promoter and transcribe the coding sequence into
mRNA, which is then translated into the polypeptide encoded by the
coding sequence.
[0085] A "host cell" is a cell which has been transformed, or is
capable of transformation, by an exogenous nucleic acid
molecule.
[0086] A cell has been "transformed" by exogenous DNA when such
exogenous DNA has been introduced inside the cell membrane.
Exogenous DNA may or may not be integrated (covalently linked) into
chromosomal DNA making up the genome of the cell. In procaryotes
and yeasts, for example, the exogenous DNA may be maintained on an
episomal element, such as a plasmid. With respect to eucaryotic
cells, a stably transformed cell is one in which the exogenous DNA
has become integrated into the chromosome so that it is inherited
by daughter cells through chromosome replication. This stability is
demonstrated by the ability of the eucaryotic cell to establish
cell lines or clones comprised of a population of daughter cells
containing the exogenous DNA.
[0087] The term "derived from," as it is used herein, denotes an
actual or theoretical source or origin of the subject molecule or
immunogen. For example, an immunogen that is "derived from" a
particular myostatin molecule will bear close sequence similarity
with a relevant portion of the reference molecule. Thus, an
immunogen that is "derived from" a particular myostatin molecule
may include all of the wild-type myostatin sequence, or may be
altered by insertion, deletion or substitution of amino acid
residues, so long as the derived sequence provides for an immunogen
that corresponds to the targeted myostatin molecule. Immunogens
derived from a denoted molecule will contain at least one epitope
specific to the denoted molecule.
[0088] By "vertebrate subject" is meant any member of the subphylum
cordata, including, without limitation, mammals such as cattle,
sheep, pigs, goats, horses, and humans; domestic animals such as
dogs and cats; and birds, including domestic, wild and game birds
such as cocks and hens including chickens, turkeys and other
gallinaceous birds; and fish. The term does not denote a particular
age or gender. Thus, both male and female adult and newborn
animals, as well as fetuses and eggs, are intended to be
covered.
[0089] The compositions and methods of the present invention will
serve to "reduce myostatin activity." This reduction in activity
may be a reduction of circulating levels of myostatin normally
found in a vertebrate subject, or a reduction of circulating levels
of myostatin in subjects with disorders that result in elevated
circulating levels of myostatin. A reduction in myostatin activity
generally results from inactivation of circulating myostatin by
antibodies generated against the myostatin peptide immunogen
delivered to the subject in question. However, the reduction of
activity is not limited to a particular mode of inactivation, but
may be the result of decreased production or secretion of myostatin
into the circulation. While not being bound by a particular theory,
the myostatin peptide immunogens may elicit the production of
antibodies which prevent myostatin from being cleaved to release
the active portion of the protein, or prevent the protein from
binding to its receptor. Alternatively, the antibodies may remove
secreted myostatin from circulation or other body fluids before it
reaches the active site.
[0090] The reduction in myostatin activity may manifest itself in a
variety of ways. For example, reduction in myostatin activity may
result in an increase in body weight, enhanced muscle mass,
increased muscle strength, an alteration in the ratio of muscle to
fat, an increase in fat-free muscle mass, an increase in the size
and/or number of muscle cells, a reduction in body fat content, an
increase in life span in a normal or diseased vertebrate, an
increase in appetite or feed uptake, an enhanced quality of life,
and in mammals, an increase in mammary gland tissue and
lactation.
[0091] By "enhancing muscle mass" is meant that the animal
administered a composition of the present invention displays an
increase in muscle cell size (hypertrophy) or muscle cell numbers
(hyperplasia). The increase can be in type 1 and/or type 2 muscle
fibers. The term "muscle" as used herein is intended to capture
analogous tissue types in fish. Methods for determining "enhanced
muscle mass" are well known in the art. For example, muscle content
can be measured before and after administration of a myostatin
peptide of the invention using standard techniques, such as under
water weighing (see, e.g., Bhasin et al. New Eng. J. Med. (1996)
335:1-7) and dual-energy x-ray absorptiometry (see, e.g., Bhasin et
al. Mol. Endocrinol. (1998) 83:3155-3162). An increase in muscle
size may be evidenced by weight gain of at least about 5-10%,
preferably at least about 10-20% or more.
[0092] B. General Methods
[0093] Before describing the present invention in detail, it is to
be understood that this invention is not limited to particular
formulations or process parameters as such may, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments of the invention
only, and is not intended to be limiting.
[0094] Although a number of compositions and methods similar or
equivalent to those described herein can be used in the practice of
the present invention, the preferred materials and methods are
described herein.
[0095] Central to the instant invention is the development of
immunological compositions and methods for modulating endogenous
myostatin production in a vertebrate subject. Although myostatin is
generally recognized as "self" and hence nonimmunogenic, the
compositions described herein surprising provide a means for
producing an immunological response in a subject immunized
therewith.
[0096] Accordingly, the invention is directed to immunogenic
myostatin peptides, myostatin multimers and myostatin
immunoconjugates for use in generating an immune response in a
vertebrate subject. Since the myostatin protein is secreted, active
or passive immunization of young animals serves to increase muscle
mass but avoids the problems associated with other abnormalities
which arise from changes induced during the embryonic period. Thus,
for example, vaccination schedules can be initiated shortly after
birth to achieve both hypertrophy and/or hyperplasia.
Alternatively, immunization can be done at a later stage of
development, (e.g., to cattle in feedlots) to improve muscle
protein yield. Additionally, immunization can be done prenatally or
to animals in utero, to achieve the desired results.
[0097] The compositions and techniques described herein are equally
applicable to egg-laying vertebrates, such as birds and fish. In
this regard, McPherron and Lee, Proc. Natl. Acad. Sci. USA (1997)
94:12457-12461, have identified myostatin genes in birds and fish
which are highly homologous to mammalian myostatin genes.
Therefore, the gene is conserved among species and is believed to
serve a similar function in all species. Thus, for example,
egg-laying birds and fish are immunized to create high antibody
titers in maternal plasma. Since antibodies are transferred to the
yolk sac of the egg, these antibodies are able to reduce myostatin
during the embryonic period and cause the desired increase in size
and/or numbers of muscle cells. Alternatively, immunization may be
done in ovo.
[0098] Furthermore, the methods and vaccines described herein will
find use for the treatment of various disorders in humans and other
animals. For example, modulation of myostatin production is useful
for the treatment of individuals with disorders that either
primarily or incidentally cause muscle wasting such as for the
treatment of paraplegics and quadriplegics, where muscle atrophy is
a serious concern. Elderly subjects may also benefit from the
methods and vaccines described herein where lack of muscle strength
is often a serious limitation to an active, healthy lifestyle.
Additionally, the compositions of the present invention can be used
to treat or prevent muscle wasting due to various cancers,
anorexia, cachexia, AIDS, and like disorders.
[0099] The methods and vaccines of the present invention will find
use for treating various dystrophies, such as pseudohypertrophic
muscular dystrophies, facioscapulohumeral dystrophies, limb-girdle
muscular dystrophies, distal muscular dystrophies, ocular
myopathies, and myotonic dystrophies. These diseases include the
disorders known as Becker's type muscular dystrophy,
Dejerine-Landouzy muscular dystrophy, Duchenne's type muscular
dystrophy, Landouzy muscular dystrophy, Emery-Dreifuss muscular
dystrophy, Erb's muscular dystrophy, Fukuyama type muscular
dystrophy, Gowers' muscular dystrophy, infantile neuroaxonal
muscular dystrophy, Leyden-Moblus muscular dystrophy,
oculopharyngeal muscular dystrophy, pelvifemoral muscular
dystrophy, progressive muscular dystrophy, scapulohumeral muscular
dystrophy and Simmerl in's muscular dystrophy.
[0100] Additionally, since myostatin is highly homologous to GDF11,
the myostatin peptides of the present invention will also find use
in modulating GDF11 activity. See, e.g., NCBI Accession No.
AF092734 for the sequence of GDF11.
[0101] Immunization can be achieved by any of the methods known in
the art including, but not limited to, use of peptide vaccines or
DNA immunization. Such methods are described in detail below.
[0102] 1. Myostatin Peptides
[0103] Myostatin peptides for use with the present invention will
generally include at least about 3 amino acids to about 200 amino
acids, preferably at least about 3 amino acids to about 100 amino
acids, more preferably at least about 3 to about 50 amino acids,
even more preferably at least about 3 amino acids to about 30 amino
acids, preferably about 3 to about 15 amino acids, and most
preferably at least about 5 amino acids to about 25 amino acids or
5 to about 15 amino acids, from a selected myostatin protein.
[0104] Representative myostatin proteins from 10 species from which
the myostatin peptides of the present invention can be derived are
shown in FIGS. 1A-1D. The amino acid sequence of bovine myostatin
is also shown in FIG. 16B. The peptide will include at least one
epitope which imparts immunogenicity to the myostatin molecule.
[0105] In preferred embodiments, the myostatin peptide is derived
from the region of myostatin including but not limited to the
region spanning amino acids 1 through 350, inclusive, of FIGS.
1A-1D (SEQ ID NOS:27-36); the region of myostatin spanning amino
acids 1 through 275, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36);
the region of myostatin spanning amino acids 25 through 300,
inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); the region of
myostatin spanning amino acids 50 through 325, inclusive, of FIGS.
1A-1D (SEQ ID NOS:27-36); the region of myostatin spanning amino
acids 75 through 350, inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36);
the region of myostatin spanning amino acids 45 through 376,
inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); 100 through 376,
inclusive, of FIGS. 1A-1D (SEQ ID NOS:27-36); the region of
myostatin spanning amino acids 235 through 376, inclusive, of FIGS.
1A-1D (SEQ ID NOS:27-36); or from any region believed to include an
epitope of myostatin capable of eliciting an immune response in a
subject to which the peptide is delivered.
[0106] In certain embodiments, myostatin peptides are derived from
one of three regions of myostatin which display the highest points
of hydrophilicity in the hydrophilicity profile shown in FIG. 17.
The three highest points of hydrophilicity are found at amino acid
positions 263-268, which spans the proteolytic cleavage site;
positions 31-37; and positions 106-111. Thus, in these embodiments,
the myostatin peptide comprises the amino acid sequence
Lys-Arg-Ser-Arg-Arg-Asp (SEQ ID NO:37) which spans the proteolytic
cleavage site; the amino acid sequence Lys-Glu-Asn-Val-Glu-Lys-Glu
(SEQ ID NO:38) which corresponds to amino acids 31-37 of myostatin;
or the amino acid sequence Ser-Leu-Lys-Asp-Asp-Asp (SEQ ID NO:39)
which corresponds to amino acids 106 to 111 of myostatin.
[0107] In other embodiments, the myostatin peptide has at least
about 75% amino acid identity to a peptide comprising the amino
acid sequence of amino acids 3-18, inclusive of SEQ ID NO:4 (MYOS
1, shown in FIG. 2); amino acids 3-15, inclusive of SEQ ID NO:6
(MYOS 3, shown in FIG. 3); amino acids 3-17, inclusive, of SEQ ID
NO:8 (MYOS 5, shown in FIG. 4); amino acids 3-16, inclusive of SEQ
ID NO:10 (MYOS 7, shown in FIG. 5); amino acids 3-22, inclusive of
SEQ ID NO:12 (MYOS 9, shown in FIG. 6); amino acids 3-25, inclusive
of SEQ ID NO:14 (MYOS 11, shown in FIG. 7); amino acids 3-22,
inclusive of SEQ ID NO:16 (MYOS 13, shown in FIG. 8); amino acids
3-19, inclusive, of SEQ ID NO:18 (MYOS 15, shown in FIG. 9); amino
acids 3-18, inclusive, of SEQ ID NO:20 (MYOS 17, shown in FIG. 10);
or amino acids 3-18, inclusive of SEQ ID NO:22 (MYOS 19, shown in
FIG. 11). The positions of the various MYOS peptides above relative
to full-length myostatin are shown in FIG. 12.
[0108] The myostatin peptide is optionally linked to an
immunological carrier molecule in order to form a myostatin
immunoconjugate, as described further below.
[0109] 2. Myostatin Immunoconjugates
[0110] As explained above, myostatin is an endogenous molecule and,
as such, it may be desirable to further increase the immunogenicity
of the myostatin peptide (or multimers described below) by linking
it to a carrier to form a myostatin immunoconjugate. This is
especially necessary if the myostatin immunogen will be
administered to the same species from which it is derived.
[0111] Suitable carriers are generally polypeptides which include
antigenic regions of a protein derived from an infectious material
such as a viral surface protein, or a carrier peptide sequence.
These carriers serve to non-specifically stimulate T-helper cell
activity and to help direct an immunogen of interest to antigen
presenting cells (APCs) for processing and presentation at the cell
surface in association with molecules of the major
histocompatibility complex (MHC).
[0112] Several carrier systems have been developed for this
purpose. For example, small peptide haptens are often coupled to
protein carriers such as keyhole limpet hemocyanin (Bittle et al.
(1982) Nature 298:30-33), bacterial toxins such as tetanus toxoid
(Muller et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79:569-573),
ovalbumin, leukotoxin polypeptides, and sperm whale myoglobin, to
produce an immune response. These coupling reactions typically
result in the incorporation of several moles of peptide hapten per
mole of carrier protein.
[0113] Other suitable carriers for use with the present invention
include VP6 polypeptides of rotaviruses, or functional fragments
thereof, as disclosed in U.S. Pat. No. 5,071,651. Also useful is a
fusion product of a viral protein and one or more epitopes from
myostatin, which fusion products are made by the methods disclosed
in U.S. Pat. No. 4,722,840. Still other suitable carriers include
cells, such as lymphocytes, since presentation in this form mimics
the natural mode of presentation in the subject, which gives rise
to the immunized state. Alternatively, the myostatin immunogens may
be coupled to erythrocytes, preferably the subject's own
erythrocytes. Methods of coupling peptides to proteins or cells are
known to those of skill in the art.
[0114] Delivery systems useful in the practice of the present
invention may also utilize particulate carriers. For example,
pre-formed particles have been used as platforms onto which
immunogens can be coupled and incorporated. Systems based on
proteosomes (Lowell et al. (1988) Science 240:800-802) and immune
stimulatory complexes (Morein et al. (1984) Nature 308:457-460) are
also known in the art.
[0115] Carrier systems using recombinantly produced chimeric
proteins that self-assemble into particles may also be used with
the present invention. For example, the yeast retrotransposon, Ty,
encodes a series of proteins that assemble into virus like
particles (Ty-VLPs; Kingsman et al. (1988) Vaccines 6:304-306).
Thus, a gene, or fragment thereof, encoding the myostatin immunogen
of interest may be inserted into the TyA gene and expressed in
yeast as a fusion protein. The fusion protein retains the capacity
to self assemble into particles of uniform size. Other useful
virus-like carrier systems are based on HBsAg, (Valenzuela et al.
(1985) Bio/Technol. 3:323-326; U.S. Pat. No. 4,722,840; Delpeyroux
et al. (1986) Science 233:472-475), Hepatitis B core antigen
(Clarke et al. (1988) Vaccines 88 (Ed. H. Ginsberg, et al.) pp.
127-131), Poliovirus (Burke et al. (1988) Nature 332:81-82), and
Tobacco Mosaic Virus (Haynes et al. (1986) Bio/Technol.
4:637-641).
[0116] Especially preferred carriers include serum albumins,
keyhole limpet hemocyanin, ovalbumin, sperm whale myoglobin,
leukotoxin molecules as described above, and other proteins well
known to those skilled in the art. One particular leukotoxin
polypeptide, for use as a carrier herein, is shown in FIGS.
15A-15D. Myostatin is conveniently inserted into the BamH1 site
present at nucleotide position 3334, as described further in the
examples.
[0117] Protein carriers may be used in their native form or their
functional group content may be modified by, for example,
succinylation of lysine residues or reaction with Cys-thiolactone.
A sulfhydryl group may also be incorporated into the carrier (or
antigen) by, for example, reaction of amino functions with
2-iminothiolane or the N-hydroxysuccinimide ester of
3-(4-dithiopyridyl propionate. Suitable carriers may also be
modified to incorporate spacer arms (such as hexamethylene diamine
or other bifunctional molecules of similar size) for attachment of
peptide immunogens.
[0118] Carriers can be physically conjugated to the myostatin
immunogen of interest, using standard coupling reactions.
Alternatively, chimeric molecules can be prepared recombinantly for
use in the present invention, such as by fusing a gene encoding a
suitable polypeptide carrier to one or more copies of a gene, or
fragment thereof, encoding for a selected myostatin immunogen.
[0119] The myostatin immunogens can also be administered via a
carrier virus which expresses the same. Carrier viruses which will
find use herein include, but are not limited to, the vaccinia and
other pox viruses, adenovirus, and herpes virus. By way of example,
vaccinia virus recombinants expressing the proteins can be
constructed as follows. The DNA encoding a particular protein is
first inserted into an appropriate vector so that it is adjacent to
a vaccinia promoter and flanking vaccinia DNA sequences, such as
the sequence encoding thymidine kinase (TK). This vector is then
used to transfect cells which are simultaneously infected with
vaccinia. Homologous recombination serves to insert the vaccinia
promoter plus the gene encoding the desired immunogen into the
viral genome. The resulting TK-recombinant can be selected by
culturing the cells in the presence of 5-bromodeoxyuridine and
picking viral plaques resistant thereto.
[0120] 3. Myostatin Multimers
[0121] Immunogenicity of the myostatin immunogens may also be
significantly increased by producing immunogenic forms of the
molecules that comprise multiple copies of selected epitopes. In
this way, endogenous myostatin may be rendered an effective
autoantigen.
[0122] Accordingly, in one aspect of the invention, vaccine
compositions containing myostatin multimers are provided in either
nucleic acid or peptide form for delivery to a subject. The
myostatin multimer will have more than one copy of selected
myostatin immunogens, peptides or epitopes, as described above, or
multiple tandem repeats of a selected myostatin immunogen, peptide
or epitope. Thus, the myostatin multimers may comprise either
multiple or tandem repeats of selected myostatin sequences,
multiple or tandem repeats of selected myostatin epitopes, or any
conceivable combination thereof. Myostatin epitopes may be
identified using techniques as described in detail above.
[0123] For example, the myostatin multimer may correspond to a
molecule with repeating units of the general formula (MP-X-MP)y
wherein MP is a myostatin peptide, X is selected from the group
consisting of a peptide linkage, an amino acid spacer group and
[MP].sub.n, where n is greater than or equal to 1, y is greater
than or equal to 1, and further wherein "MP" may comprise any MP
peptide. Thus, the myostatin multimer may contain from 2-64 or more
myostatin peptides, more preferably 2-32 or 2-16 myostatin
peptides.
[0124] Further, the selected myostatin immunogen sequences may all
be the same, or may correspond to different derivatives, analogs,
variants or epitopes of myostatin so long as they retain the
ability to elicit an immune response. Additionally, if the
myostatin immunogens are linked either chemically or recombinantly
to a carrier, myostatin peptides may be linked to either the
5'-end, the 3'-end, or may flank the carrier in question. Further,
the myostatin multimer may be located at sites internal to the
carrier.
[0125] One particular carrier for use with the present myostatin
multimers is a leukotoxin polypeptide as described above. For
example, myostatin oligo repeats can be conveniently inserted into
the BamH1 site present at nucleotide position 3334 of the
leukotoxin polypeptide shown in FIGS. 15A-15D.
[0126] As explained above, spacer sequences may be present between
the myostatin moieties. For example, Arg-Ser and Gly-Ser dimers are
present in the MYOS peptides exemplified herein which provide
spacers between repeating sequences of the myostatin peptides. The
strategic placement of various spacer sequences between selected
myostatin immunogens can be used to confer increased immunogenicity
on the subject constructs. Accordingly, under the invention, a
selected spacer sequence may encode a wide variety of moieties such
as a single amino acid linker or a sequence of two to several amino
acids. Selected spacer groups may preferably provide enzyme
cleavage sites so that the expressed multimer can be processed by
proteolytic enzymes in vivo (by APCs, or the like) to yield a
number of peptides, each of which contain at least one T-cell
epitope derived from the carrier portion, and which are preferably
fused to a substantially complete myostatin peptide sequence.
[0127] The spacer groups may be constructed so that the junction
region between selected myostatin moieties comprises a clearly
foreign sequence to the immunized subject, thereby conferring
enhanced immunogenicity upon the associated myostatin peptides.
Additionally, spacer sequences may be constructed so as to provide
T-cell antigenicity, such as those sequences which encode
amphipathic and/or .alpha.-helical peptide sequences which are
generally recognized in the art as providing immunogenic helper
T-cell epitopes. The choice of particular T-cell epitopes to be
provided by such spacer sequences may vary depending on the
particular vertebrate species to be vaccinated. Although particular
myostatin portions are exemplified which include spacer sequences,
it is also an object of the invention to provide one or more
myostatin multimers comprising directly adjacent myostatin
sequences (without intervening spacer sequences).
[0128] The myostatin multimeric sequence thus produced renders a
highly immunogenic myostatin antigen for use in the compositions of
the invention.
[0129] The myostatin peptides, immunoconjugates and multimers can
be produced using the methods described below, and used for nucleic
acid immunization, gene therapy, protein-based immunization
methods, and the like.
[0130] 4. Nucleic Acid-Based Immunization Methods
[0131] Generally, nucleic acid-based vaccines for use with the
present invention will include relevant regions encoding a
myostatin immunogen, with suitable control sequences and,
optionally, ancillary therapeutic nucleotide sequences. The nucleic
acid molecules are prepared in the form of vectors which include
the necessary elements to direct transcription and translation in a
recipient cell.
[0132] In order to augment an immune response in an immunized
subject, the nucleic acid molecules can be administered in
conjunction with ancillary substances, such as pharmacological
agents, adjuvants, or in conjunction with delivery of vectors
encoding biological response modifiers such as cytokines and the
like. Other ancillary substances include, but are not limited to,
substances to increase weight gain, muscle mass or muscle strength,
such as growth hormones, growth promoting agents, beta antagonists,
partitioning agents and antibiotics.
[0133] Nucleotide sequences selected for use in the present
invention can be derived from known sources, for example, by
isolating the same from cells or tissue containing a desired gene
or nucleotide sequence using standard techniques, or by using
recombinant or synthetic techniques.
[0134] Once coding sequences for the myostatin immunogens have been
prepared or isolated, such sequences can be cloned into any
suitable vector or replicon. Numerous cloning vectors are known to
those of skill in the art, and the selection of an appropriate
cloning vector is a matter of choice. Ligations to other sequences,
e.g., ancillary molecules or carrier molecules, are performed using
standard procedures, known in the art. One or more myostatin
immunogen portions of the chimera can be fused 5' and/or 3' to a
desired ancillary sequence or carrier molecule. Alternatively, one
or more myostatin immunogen portions may be located at sites
internal to the carrier molecule, or such portions can be
positioned at both terminal and internal locations in the
chimera.
[0135] Alternatively, DNA sequences encoding the myostatin
immunogens of interest, optionally linked to carrier molecules, can
be prepared synthetically rather than cloned. The DNA sequences can
be designed with appropriate codons for the particular sequence.
The complete sequence of the immunogen is then assembled from
overlapping oligonucleotides prepared by standard methods and
assembled into a complete coding sequence. See, e.g., Edge (1981)
Nature 292:756; Nambair et al. (1984) Science 223:1299; and Jay et
al. (1984) J. Biol. Chem. 259:6311.
[0136] The coding sequence is then placed under the control of
suitable control elements for expression in suitable host tissue in
vivo. The choice of control elements will depend on the subject
being treated and the type of preparation used. Thus, if the
subject's endogenous transcription and translation machinery will
be used to express the immunogens, control elements compatible with
the particular subject will be utilized. In this regard, several
promoters for use in mammalian systems are known in the art. For
example, typical promoters for mammalian cell expression include
the SV40 early promoter, a CMV promoter such as the CMV immediate
early promoter, the mouse mammary tumor virus LTR promoter, the
adenovirus major late promoter (Ad MLP), and the herpes simplex
virus promoter, among others. Other nonviral promoters, such as a
promoter derived from the murine metallothionein gene, will also
find use for mammalian expression.
[0137] Typically, transcription termination and polyadenylation
sequences will also be present, located 3' to the translation stop
codon. Preferably, a sequence for optimization of initiation of
translation, located 5' to the coding sequence, is also present.
Examples of transcription terminator/polyadenylation signals
include those derived from SV40, as described in Sambrook et al.,
supra, as well as a bovine growth hormone terminator sequence.
Introns, containing splice donor and acceptor sites, may also be
designed into the constructs for use with the present
invention.
[0138] Enhancer elements may also be used herein to increase
expression levels of the constructs. Examples include the SV40
early gene enhancer (Dijkema et al. (1985) EMBO J. 4:761), the
enhancer/promoter derived from the long terminal repeat (LTR) of
the Rous Sarcoma Virus (Gorman et al. (1982) Proc. Natl. Acad. Sci.
USA 79:6777) and elements derived from human CMV (Boshart et al.
(1985) Cell 41:521), such as elements included in the CMV intron A
sequence.
[0139] Once prepared, the nucleic acid vaccine compositions can be
delivered to the subject using known methods. In this regard,
various techniques for immunization with antigen-encoding DNAs have
been described. See, e.g., U.S. Pat. No. 5,589,466 to Felgner et
al.; Tang et al. (1992) Nature 358:152; Davis et al. (1993) Hum.
Molec. Genet. 2:1847; Ulmer et al. (1993) Science 258:1745; Wang et
al. (1993) Proc. Natl. Acad. Sci. USA 90:4156; Eisenbraun et al.
(1993) DNA Cell Biol. 12:791; Fynan et al. (1993) Proc. Natl. Acad.
Sci. USA 90:12476; Fuller et al. (1994) AIDS Res. Human Retrovir.
10:1433; and Raz et al. (1994) Proc. Natl. Acad. Sci. USA 91:9519.
General methods for delivering nucleic acid molecules to cells in
vitro, for the subsequent reintroduction into the host, can also be
used, such as liposome-mediated gene transfer. See, e.g., Hazinski
et al. (1991) Am. J. Respir. Cell Mol. Biol. 4:206-209; Brigham et
al. (1989) Am. J. Med. Sci. 298:278-281; Canonico et al. (1991)
Clin. Res. 39:219A; and Nabel et al. (1990) Science 249:1285-1288.
Thus, the nucleic acid vaccine compositions can be delivered in
either liquid or particulate form using a variety of known
techniques. Typical vaccine compositions are described more fully
below.
[0140] 5. Protein-Based Immunization Methods
[0141] Peptide-based vaccine compositions can also be produced
using a variety of methods known to those skilled in the art. In
particular, myostatin immunogens can be isolated directly from
native sources, using standard purification techniques.
Alternatively, the immunogens can be recombinantly produced using
the nucleic acid expression systems described above, and purified
using known techniques. Peptide immunogens can also be synthesized,
based on described amino acid sequences or amino acid sequences
derived from the DNA sequence of a molecule of interest, using
chemical polymer syntheses such as solid phase peptide synthesis.
Such methods are known to those skilled in the art. See, e.g., J.
M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed.,
Pierce Chemical Co., Rockford, Ill. (1984) and G. Barany and R. B.
Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E.
Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980),
pp. 3-254, for solid phase peptide synthesis techniques; and M.
Bodansky, Principles of Peptide Synthesis, Springer-Verlag, Berlin
(1984) and E. Gross and J. Meienhofer, Eds., The Peptides:
Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution
synthesis.
[0142] Peptide immunogens may also be produced by cloning the
coding sequences therefor into any suitable expression vector or
replicon. Numerous cloning vectors are known to those of skill in
the art, and the selection of an appropriate cloning vector is a
matter of choice. Examples of recombinant DNA vectors for cloning,
and host cells which they can transform, include the bacteriophage
lambda (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230
(gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFR1
(gram-negative bacteria), pME290 (non-E. coli gram-negative
bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus),
pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces),
YCp19 (Saccharomyces) and bovine papilloma virus (mammalian cells).
See, generally, DNA Cloning: Vols. I & II, supra; Sambrook et
al., supra; B. Perbal, supra.
[0143] For example, the coding sequence for myostatin from a number
of vertebrate species, including mouse, rat, human, baboon, cattle,
pig, sheep, chicken and turkey has been determined. See, e.g., U.S.
Pat. No. 5,827,733 and NCBI Accession No. U84OC5 for the nucleotide
sequence of murine myostatin; U.S. Pat. No. 5,827,733,
International Publication No. WO 98/33887, and NCBI Accession No.
AF019627 for the nucleotide sequence of human myostatin; FIG. 16A
herein, as well as International Publication Nos. WO 99/02667 and
WO 98/33887, and NCBI Accession No. AF019620 for the nucleotide
sequence of bovine myostatin; NCBI Accession No. AF019626 for the
nucleotide sequence of zebrafish myostatin; International
Publication No. WO 98/33887, for the nucleotide sequences of rat
(see, also NCBI Accession No. AF019624), baboon (see, also NCBI
Accession No. AF019619), porcine (see, also NCBI Accession No.
AF019623), ovine (see, also NCBI Accession No. AF019622), chicken
(see, also NCBI Accession No. AF019621) and turkey (seem also NCBI
Accession No. AF019625) myostatin. The myostatin sequence is highly
conserved across all of these species.
[0144] Portions of these sequences encoding desired myostatin
peptides, and if desired, a sequence encoding a carrier protein,
can be cloned, isolated and ligated together using recombinant
techniques generally known in the art. See, e.g., Sambrook et al.,
supra.
[0145] The gene can be placed under the control of a promoter,
ribosome binding site (for bacterial expression) and, optionally,
an operator, so that the DNA sequence of interest is transcribed
into RNA by a suitable transformant. The coding sequence may or may
not contain a signal peptide or leader sequence. The peptide
immunogens can be expressed using, for example, the E. coli tac
promoter or the protein A gene (spa) promoter and signal sequence.
Leader sequences can be removed by the bacterial host in
post-translational processing. See, e.g., U.S. Pat. Nos. 4,431,739;
4,425,437; 4,338,397. Ancillary sequences, such as those described
above, may also be present.
[0146] In addition to control sequences, it may be desirable to add
regulatory sequences which allow for regulation of the expression
of the immunogen sequences relative to the growth of the host cell.
Regulatory sequences are known to those of skill in the art, and
examples include those which cause the expression of a gene to be
turned on or off in response to a chemical or physical stimulus,
including the presence of a regulatory compound. Other types of
regulatory elements may also be present in the vector, for example,
enhancer sequences.
[0147] An expression vector is constructed so that the particular
coding sequence is located in the vector with the appropriate
regulatory sequences, the positioning and orientation of the coding
sequence with respect to the control sequences being such that the
coding sequence is transcribed under the "control" of the control
sequences (i.e., RNA polymerase which binds to the DNA molecule at
the control sequences transcribes the coding sequence).
Modification of the sequences encoding the particular myostatin
immunogen may be desirable to achieve this end. For example, in
some cases it may be necessary to modify the sequence so that it
can be attached to the control sequences in the appropriate
orientation; i.e., to maintain the reading frame. The control
sequences and other regulatory sequences may be ligated to the
coding sequence prior to insertion into a vector, such as the
cloning vectors described above. Alternatively, the coding sequence
can be cloned directly into an expression vector which already
contains the control sequences and an appropriate restriction
site.
[0148] In some cases, it may be desirable to add sequences which
cause the secretion of the immunogen from the host organism, with
subsequent cleavage of the secretory signal. It may also be
desirable to produce mutants or analog of the immunogen. Mutants or
analogs may be prepared by the deletion of a portion of the
sequence encoding the immunogen, or if present, a portion of the
sequence encoding the desired carrier molecule, by insertion of a
sequence, and/or by substitution of one or more nucleotides within
the sequence. Techniques for modifying nucleotide sequences, such
as site-directed mutagenesis, and the like, are well known to those
skilled in the art. See, e.g., Sambrook et al., supra; DNA Cloning,
Vols. I and II, supra; Nucleic Acid Hybridization, supra; Kunkel,
T. A. Proc. Natl. Acad. Sci. USA (1985) 82:448; Geisselsoder et al.
BioTechniques (1987) 5:786; Zoller and Smith, Methods Enzymol.
(1983) 100:468; Dalbie-McFarland et al. Proc. Natl. Acad. Sci USA
(1982) 79:6409.
[0149] The myostatin immunogens can be expressed in a wide variety
of systems, including insect, mammalian, bacterial, viral and yeast
expression systems, all well known in the art. For example, insect
cell expression systems, such as baculovirus systems, are known to
those of skill in the art and described in, e.g., Summers and
Smith, Texas Agricultural Experiment Station Bulletin No. 1555
(1987). Materials and methods for baculovirus/insect cell
expression systems are commercially available in kit form from,
inter alia, Invitrogen, San Diego Calif. ("MaxBac" kit). Similarly,
bacterial and mammalian cell expression systems are well known in
the art and described in, e.g., Sambrook et al., supra. Yeast
expression systems are also known in the art and described in,
e.g., Yeast Genetic Engineering (Barr et al., eds., 1989)
Butterworths, London.
[0150] A number of appropriate host cells for use with the above
systems are also known. For example, mammalian cell lines are known
in the art and include immortalized cell lines available from the
American Type Culture Collection (ATCC), such as, but not limited
to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster
kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular
carcinoma cells (e.g., Hep G2), Madin-Darby bovine kidney ("MDBK")
cells, as well as others. Similarly, bacterial hosts such as E.
coli, Bacillus subtilis, and Streptococcus spp., will find use with
the present expression constructs. Yeast hosts useful in the
present invention include inter alia, Saccharomyces cerevisiae,
Candida albicans, Candida maltosa, Hansenula polymorpha,
Kluyveromyces fragilis, Kluyveromyces lactis, Pichia
guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and
Yarrowia lipolytica. Insect cells for use with baculovirus
expression vectors include, inter alia, Aedes aegypti, Autographa
californica, Bombyx mori, Drosophila melanogaster, Spodoptera
frugiperda, and Trichoplusia ni.
[0151] Depending on the expression system and host selected, the
myostatin immunogens are produced by growing host cells transformed
by an expression vector described above under conditions whereby
the immunogen is expressed. The expressed immunogen is then
isolated from the host cells and purified. If the expression system
secretes the immunogen into growth media, the product can be
purified directly from the media. If it is not secreted, it can be
isolated from cell lysates. The selection of the appropriate growth
conditions and recovery methods are within the skill of the
art.
[0152] Once obtained, the myostatin peptides, with or without
associated carrier, may be formulated into vaccine compositions,
such as vaccine compositions as described further below, in order
to elicit antibody production in a subject vertebrate.
[0153] 6. Antibody Production
[0154] The subject myostatin peptides can be used to generate
antibodies for use in passive immunization methods or for
immunopurification or immunodiagnostic purposes. Typically,
peptides useful for producing antibodies will usually be at least
about 3-5 amino acids in length, preferably 7-10 amino acids in
length, and most preferably at least about 10 to 15 amino acids in
length, or more.
[0155] Antibodies against the subject immunogens include polyclonal
and monoclonal antibody preparations, monospecific antisera, as
well as preparations including hybrid antibodies, altered
antibodies, F(ab').sub.2 fragments, F(ab) fragments, F.sub.v
fragments, single domain antibodies, chimeric antibodies, humanized
antibodies, and functional fragments thereof, which retain
specificity for the target molecule in question. For example, an
antibody can include variable regions, or fragments of variable
regions, which retain specificity for the molecule in question. The
remainder of the antibody can be derived from the species in which
the antibody will be used. Thus, if the antibody is to be used in a
human, the antibody can be "humanized" in order to reduce
immunogenicity yet retain activity. For a description of chimeric
antibodies, see, e.g., Winter, G. and Milstein, C. (1991) Nature
349:293-299; Jones, P. T. et al. (1986) Nature 321:522-525;
Riechmann, L. et al. (1988) 332:323-327; and Carter, P. et al.
(1992) Proc. Natl. Acad. Sci. USA 89:4285-4289. Such chimeric
antibodies may contain not only combining sites for the target
molecule, but also binding sites for other proteins. In this way,
bifunctional reagents can be generated with targeted specificity to
both external and internal antigens.
[0156] If polyclonal antibodies are desired, a selected mammal,
(e.g., mouse, rabbit, goat, horse, etc.) is immunized with the
desired antigen, or its fragment, or a mutated antigen, as
described above. Prior to immunization, it may be desirable to
further increase the immunogenicity of a particular immunogen. This
can be accomplished in any one of several ways known to those of
skill in the art.
[0157] For example, immunization for the production of antibodies
is generally performed by mixing or emulsifying the protein in a
suitable excipient, such as saline, preferably in an adjuvant such
as Freund's complete adjuvant, or any of the adjuvants described
below, and injecting the mixture or emulsion parenterally
(generally subcutaneously or intramuscularly). The animal is
generally boosted 2-6 weeks later with one or more injections of
the protein in saline, preferably using Freund's incomplete
adjuvant, or the like. Antibodies may also be generated by in vitro
immunization, using methods known in the art. Polyclonal antisera
is then obtained from the immunized animal and treated according to
known procedures. See, e.g., Jurgens et al. (1985) J. Chrom.
348:363-370. If serum containing polyclonal antibodies is used, the
polyclonal antibodies can be purified by immunoaffinity
chromatography, using known, procedures.
[0158] Monoclonal antibodies are generally prepared using the
method of Kohler and Milstein, Nature (1975) 256:495-96, or a
modification thereof. Typically, a mouse or rat is immunized as
described above. However, rather than bleeding the animal to
extract serum, the spleen (and optionally several large lymph
nodes) is removed and dissociated into single cells. If desired,
the spleen cells may be screened (after removal of non-specifically
adherent cells) by applying a cell suspension to a plate or well
coated with the protein antigen. B-cells, expressing membrane-bound
immunoglobulin specific for the antigen, will bind to the plate,
and are not rinsed away with the rest of the suspension. Resulting
B-cells, or all dissociated spleen cells, are then induced to fuse
with myeloma cells to form hybridomas, and are cultured in a
selective medium (e.g., hypo-xanthine, aminopterin, thymidine
medium, "HAT"). The resulting hybridomas are plated by limiting
dilution, and are assayed for the production of antibodies which
bind specifically to the immunizing antigen (and which do not bind
to unrelated antigens). The selected monoclonal antibody-secreting
hybridomas are then cultured either in vitro (e.g., in tissue
culture bottles or hollow fiber reactors), or in vivo (as ascites
in mice). See, e.g., M. Schreier et al., Hybridoma Techniques
(1980); Hammerling et al., Monoclonal Antibodies and T-cell
Hybridomas (1981); Kennett et al., Monoclonal Antibodies (1980);
see also U.S. Pat. Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887;
4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. Panels
of monoclonal antibodies produced against the myostatin peptide of
interest, or fragment thereof, can be screened for various
properties; i.e., for isotype, epitope, affinity, etc.
[0159] Functional fragments of the antibodies can also be made
against the myostatin peptide of interest and can be produced by
cleaving a constant region, not responsible for antigen binding,
from the antibody molecule, using e.g., pepsin, to produce
F(ab').sub.2 fragments. These fragments will contain two antigen
binding sites, but lack a portion of the constant region from each
of the heavy chains. Similarly, if desired, Fab fragments,
comprising a single antigen binding site, can be produced, e.g., by
digestion of polyclonal or monoclonal antibodies with papain.
Functional fragments, including only the variable regions of the
heavy and light chains, can also be produced, using standard
techniques. These fragments are known as F.sub.v.
[0160] Chimeric or humanized antibodies can also be produced using
the subject immunogens. These antibodies can be designed to
minimize unwanted immunological reactions attributable to
heterologous constant and species-specific framework variable
regions typically present in monoclonal and polyclonal antibodies.
For example, if the antibodies are to be used in human subjects,
chimeric antibodies can be created by replacing non-human constant
regions, in either the heavy and light chains, or both, with human
constant regions, using techniques generally known in the art. See,
e.g., Winter, G. and Milstein, C. (1991) Nature 349:293-299; Jones,
P. T. et al. (1986) Nature 321:522-525; Riechmann, L. et al. (1988)
332:323-327; and Carter, P. et al. (1992) Proc. Natl. Acad. Sci.
USA 89:4285-4289.
[0161] 7. Vaccine Compositions
[0162] Once the above molecules are produced, they are formulated
into vaccine compositions for delivery to a vertebrate subject. The
relevant myostatin molecule is administered alone, or mixed with a
pharmaceutically acceptable vehicle or excipient. Suitable vehicles
are, for example, water, saline, dextrose, glycerol, ethanol, or
the like, and combinations thereof. In addition, the vehicle may
contain minor amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, or adjuvants which enhance
the effectiveness of the vaccine. Suitable adjuvants are described
further below. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in the art. See, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., 18th edition, 1990. The composition or formulation to
be administered will contain a quantity of the myostatin immunogen
adequate to achieve the desired immunized state in the subject
being treated.
[0163] As explained above, the vaccine compositions of the present
invention may include adjuvants to further increase the
immunogenicity of the myostatin immunogen. Adjuvants may include
for example, emulsifiers, muramyl dipeptides, avridine, aluminum
hydroxide, oils, saponins and other substances known in the art.
For example, compounds which may serve as emulsifiers herein
include natural and synthetic emulsifying agents, as well as
anionic, cationic and nonionic compounds. Among the synthetic
compounds, anionic emulsifying agents include, for example, the
potassium, sodium and ammonium salts of lauric and oleic acid, the
calcium, magnesium and aluminum salts of fatty acids (i.e.,
metallic soaps), and organic sulfonates such as sodium lauryl
sulfate. Synthetic cationic agents include, for example,
cetyltrimethylammonium bromide, while synthetic nonionic agents are
exemplified by glyceryl esters (e.g., glyceryl monostearate),
polyoxyethylene glycol esters and ethers, and the sorbitan fatty
acid esters (e.g., sorbitan monopalmitate) and their
polyoxyethylene derivatives (e.g., polyoxyethylene sorbitan
monopalmitate). Natural emulsifying agents include acacia, gelatin,
lecithin and cholesterol.
[0164] Other suitable adjuvants can be formed with an oil
component, such as a single oil, a mixture of oils, a water-in-oil
emulsion, or an oil-in-water emulsion. The oil may be a mineral
oil, a vegetable oil, or an animal oil. Mineral oil, or
oil-in-water emulsions in which the oil component is mineral oil
are preferred. In this regard, a "mineral oil" is defined herein as
a mixture of liquid hydrocarbons obtained from petrolatum via a
distillation technique; the term is synonymous with "liquid
paraffin," "liquid petrolatum" and "white mineral oil." The term is
also intended to include "light mineral oil," i.e., an oil which is
similarly obtained by distillation of petrolatum, but which has a
slightly lower specific gravity than white mineral oil. See, e.g.,
Remington's Pharmaceutical Sciences, supra. A particularly
preferred oil component is the oil-in-water emulsion sold under the
trade name of EMULSIGEN PLUS.TM. (comprising a light mineral oil as
well as 0.05% formalin, and 30 mcg/mL gentamicin as preservatives),
available from MVP Laboratories, Ralston, Nebraska, or the VSA-3
adjuvant which is a modified form of the EMULSIGEN PLUS.TM.
adjuvant. Suitable animal oils include, for example, cod liver oil,
halibut oil, menhaden oil, orange roughy oil and shark liver oil,
all of which are available commercially. Suitable vegetable oils,
include, without limitation, canola oil, almond oil, cottonseed
oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil,
soybean oil, and the like.
[0165] Alternatively, a number of aliphatic nitrogenous bases can
be used as adjuvants with the vaccine formulations. For example,
known immunologic adjuvants include amines, quaternary ammonium
compounds, guanidines, benzamidines and thiouroniums (Gall, D.
(1966) Immunology 11:369-386). Specific compounds include
dimethyldioctadecylammonium bromide (DDA) (available from Kodak)
and N,N-dioctadecyl-N,N-bis(2-hydrox- yethyl)propanediamine
("avridine"). The use of DDA as an immunologic adjuvant has been
described; see, e.g., the Kodak Laboratory Chemicals Bulletin
56(1):1-5 (1986); Adv. Drug Deliv. Rev. 5(3):163-187 (1990); J.
Controlled Release 7:123-132 (1988); Clin. Exp. Immunol.
78(2):256-262 (1989); J. Immunol. Methods 97(2):159-164 (1987);
Immunology 58(2):245-250 (1986); and Int. Arch. Allergy Appl.
Immunol. 68(3):201-208 (1982). Avridine is also a well-known
adjuvant. See, e.g., U.S. Pat. No. 4,310,550 to Wolff, III et al.,
which describes the use of N,N-higher
alkyl-N',N'-bis(2-hydroxyethyl)propane diamines in general, and
avridine in particular, as vaccine adjuvants. U.S. Pat. No.
5,151,267 to Babiuk, and Babiuk et al. (1986) Virology 159:57-66,
also relate to the use of avridine as a vaccine adjuvant.
[0166] The vaccine compositions of the present invention can also
include ancillary substances, such as pharmacological agents,
cytokines, or other biological response modifiers. Other ancillary
substances include, but are not limited to, substances to increase
weight gain, muscle mass or muscle strength, such as growth
hormones, growth promoting agents, beta antagonists, partitioning
agents and antibiotics.
[0167] The vaccines of the present invention are normally prepared
as injectables, either as liquid solutions or suspensions, or as
solid forms which are suitable for solution or suspension in liquid
vehicles prior to injection. The preparation may also be emulsified
or the active ingredient encapsulated in liposome vehicles or other
particulate carriers used.
[0168] The vaccine compositions may also be prepared in solid form.
For example, solid particulate formulations can be prepared for
delivery from commercially available needleless injector devices.
Alternatively, solid dose implants can be provided for implantation
into a subject. Controlled or sustained release formulations may
also be used and are made by incorporating the myostatin immunogens
into carriers or vehicles such as liposomes, nonresorbable
impermeable polymers such as ethylenevinyl acetate copolymers and
Hytrel.RTM. copolymers, swellable polymers such as hydrogels, or
resorbable polymers such as collagen and certain polyacids or
polyesters such as those used to make resorbable sutures.
[0169] Furthermore, the immunogens may be formulated into vaccine
compositions in either neutral or salt forms. Pharmaceutically
acceptable salts include the acid addition salts (formed with the
free amino groups of the active polypeptides) and which are formed
with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or organic acids such as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed from free carboxyl
groups may also be derived from inorganic bases such as, for
example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the
like.
[0170] The vaccine composition is formulated to contain an
effective amount of the myostatin immunogen, the exact amount being
readily determined by one skilled in the art, wherein the amount
depends on the animal to be treated, the capacity of the animal's
immune system to synthesize antibodies, and the degree of
immunoneutralization of myostatin desired. For purposes of the
present invention, vaccine formulations including approximately 1
.mu.g to about 1 mg, more generally about 5 .mu.g to about 200
.mu.g of immunogen per dose of injected solution should be adequate
to raise an immunological response when administered. If a
peptide-carrier chimera is used, the ratio of immunogen to carrier
in the vaccine formulation will vary based on the particular
carrier and immunogen selected to construct such molecules.
Effective dosages can be readily established by one of ordinary
skill in the art through routine trials establishing dose response
curves.
[0171] The subject is immunized by administration of one of the
above-described vaccine compositions in at least one dose, and
preferably two or more doses. Moreover, the animal may be
administered as many doses as is required to maintain a state of
immunity.
[0172] Any suitable pharmaceutical delivery means may be employed
to deliver the vaccine composition to the vertebrate subject. For
example, conventional needle syringes, spring or compressed gas
(air) injectors (U.S. Pat. Nos. 1,605,763 to Smoot; 3,788,315 to
Laurens; 3,853,125 to Clark et al.; 4,596,556 to Morrow et al.; and
5,062,830 to Dunlap), liquid jet injectors (U.S. Pat. Nos.
2,754,818 to Scherer; 3,330,276 to Gordon; and 4,518,385 to
Lindmayer et al.), and particle injectors (U.S. Pat. Nos. 5,149,655
to McCabe et al. and 5,204,253 to Sanford et al.) are all
appropriate for delivery of the vaccine compositions.
[0173] Preferably, the vaccine composition is administered
intramuscularly, subcutaneously, intravenously, subdermally, or
intradermally, to the subject. If a jet injector is used, a single
jet of the liquid vaccine composition is ejected under high
pressure and velocity, e.g., 1200-1400 PSI, thereby creating an
opening in the skin and penetrating to depths suitable for
immunization.
[0174] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way.
[0175] C. Experimental
EXAMPLE 1
Identification of Immunogenic Myostatin Peptides
[0176] A number of regions of the bovine myostatin molecule were
identified as potentially immunogenic based on computer analysis of
the full-length molecule using various computer programs. One
program used formulates hydropathy scales from the amino acid
sequence of the protein based on the hydrophobic and hydrophilic
properties of each of the 20 amino acids. Hopp and Woods, Proc.
Natl. Acad. Sci. USA (1981) 78:3824-3828. FIG. 17 depicts a
hydrophilicity profile computed using an average group length of
six amino acids. The three highest points of hydrophilicity of the
myostatin molecule were found at amino acid positions 263-268,
which spans the proteolytic cleavage site and has the amino acid
sequence Lys-Arg-Ser-Arg-Arg-Asp (SEQ ID NO:37); positions 31-37
which has the amino acid sequence Lys-Glu-Asn-Val-Glu-Lys-Glu (SEQ
ID NO:38); and positions 106-111 which has the amino acid sequence
Ser-Leu-Lys-Asp-Asp-Asp (SEQ ID NO:39).
[0177] Analysis of the protein was also done using the program
PC/Gene, Release 6.60 (Intelligenetics Inc., Geneva, Switzerland).
Three-dimensional analysis of the myostatin protein was conducted
using the Swiss-Pdb Viewer v2.6
(http://expasy.hcuge.ch/spdbv/mainpage.html).
[0178] From this information, a series of representative DNA
oligomers were designed and constructed with a Beckman Oligo 1000M
DNA Synthesizer using phosphoramidite chemistry. The oligomers were
termed MYOS 1-20. Myos 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 (shown
in FIGS. 2 through 11, respectively) include portions of the coding
stand of DNA while MYOS 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20
include portions of the complimentary strand. The position of these
peptides with reference to the full-length myostatin molecule is
shown in FIG. 12.
[0179] The DNA oligomers coded for peptides with 12 to 23 amino
acids, flanked by 2 amino acid linkers for linkage to a carrier
molecule (see further below). These peptides collectively
represented the entire active portion of the protein, as well as
three individual sections upstream of the proteolytic cleavage site
which releases the active protein.
[0180] In particular, based on computer analysis, three portions of
the active protein were selected as primary immunizing targets. The
first portion was prepared by combining the oligonucleotide pair
designated MYOS 1 and 2 and contained the proteolytic cleavage site
and N-terminus of the active protein. MYOS 1 gave the highest
antigenic determinant rating using the Hopp and Woods computer
program (see FIG. 17). Three-dimensional analysis of the active
portion of myostatin showed that the MYOS 1 peptide is exposed on
the protein surface and is therefore likely to be seen by the
immune system. MYOS 1 also overlaps the proteolytic cleavage site,
which releases the active portion of the protein. Blocking this
site using antibodies thereto prevents cleavage of the protein and
release of the active portion of the protein to prevent its effect
on muscle tissue.
[0181] Two other segments of the active protein (MYOS 5 and 6 and
MYOS 9 and 10) were selected because they appeared to form a loop
and a helix based on a three-dimensional structural analysis. This
loop structure is likely exposed on the protein surface and
therefore able to be seen by the immune system. Antibodies
generated to these portions of the molecule likely bind myostatin
protein and remove it from circulation. The remainder of the active
portion of the protein was reconstructed from the oligonucleotide
pairs (MYOS 3 and 4, MYOS 7 and 8, MYOS 11 and 12, MYOS 13 and 14).
Use of the entire active portion assures the proper
three-dimensional structure to elicit an effective immune response.
One of the regions upstream of the active portion of the protein,
(MYOS 15 and 16) was selected based on computer analysis of likely
antigenic epitopes. The other two upstream portions of the protein
were selected to contain the proteolytic cleavage site (MYOS 19 and
20, which contain the cleavage site and amino acids immediately
upstream of the cleavage site) or to be close to it (MYOS 17 and
18) so an antibody which binds to the site would interfere with the
protease activity.
[0182] Based on comparisons with other known protein sequences,
myostatin has areas of homology with other transforming growth
factor .beta. proteins. Bone morphogenetic protein 6 (BMP-6) has a
great deal of homology to the middle and C-terminus regions of the
active portion of myostatin.
EXAMPLE 2
Construction of pCB150
[0183] The oligomers above were designed to be fused to the
3'-terminus of a polynucleotide encoding a 52 kDa leukotoxin (LKT)
carrier protein, termed "LKT 114" herein. This polynucleotide was
derived from the lktA gene present in plasmid pCB114, described in
U.S. Pat. No. 5,837,268. This plasmid, the nucleotide sequence of
this gene and the corresponding amino acid sequence are shown in
FIGS. 15A-15D herein and also described in U.S. Pat. No. 5,837,268,
incorporated herein by reference in its entirety. The gene encodes
a shortened version of leukotoxin which was developed from the
recombinant leukotoxin gene present in plasmid pAA352 (ATCC
Accession No. 68283 and described in U.S. Pat. No. 5,476,657,
incorporated herein by reference in its entirety) by removal of an
internal DNA fragment of approximately 1300 bp in length. The LKT
114 polypeptide has an estimated molecular weight of 52 kDa and
contains convenient restriction sites for use in producing the
fusion proteins of the present invention.
[0184] Plasmid pCB150, containing the coding sequence for LKT 114,
into which the MYOS oligonucleotides were cloned, was prepared as
follows. The leukotoxin gene was isolated as described in U.S. Pat.
Nos. 5,476,657 and 5,837,268, incorporated herein by reference in
their entireties. In particular, to isolate the leukotoxin gene,
gene libraries of P. haemolytica A1 (strain B122) were constructed
using standard techniques. See, Lo et al., Infect. Immun., supra;
DNA CLONING: Vols. I and II, supra; and Sambrook et al., supra. A
genomic library was constructed in the plasmid vector pUC13 and a
DNA library constructed in the bacteriophage lambda gt11. The
resulting clones were used to transform E. coli and individual
colonies were pooled and screened for reaction with serum from a
calf which had survived a P. haemolytica infection and that had
been boosted with a concentrated culture supernatant of P.
haemolytica to increase anti-leukotoxin antibody levels. Positive
colonies were screened for their ability to produce leukotoxin by
incubating cell lysates with bovine neutrophils and subsequently
measuring release of lactate dehydrogenase from the latter.
[0185] Several positive colonies were identified and these
recombinants were analyzed by restriction endonuclease mapping. One
clone appeared to be identical to a leukotoxin gene cloned
previously. See, Lo et al., Infect. Immun., supra. To confirm this,
smaller fragments were re-cloned and the restriction maps compared.
It was determined that approximately 4 kilobase pairs of DNA had
been cloned. Progressively larger clones were isolated by carrying
out a chromosome walk (5' to 3' direction) in order to isolate
full-length recombinants which were approximately 8 kb in length.
The final construct was termed pAA114. This construct contained the
entire leukotoxin gene sequence.
[0186] lktA, a MaeI restriction endonuclease fragment from pAA114
which contained the entire leukotoxin gene, was treated with the
Klenow fragment of DNA polymerase I plus nucleotide triphosphates
and ligated into the SmaI site of the cloning vector pUC13. This
plasmid was named pAA179. From this, two expression constructs were
made in the ptac-based vector pGH432:lacI digested with SmaI. One,
pAA342, consisted of the 5'-AhaIII fragment of the lktA gene while
the other, pAA345, contained the entire MaeI fragment described
above. The clone pAA342 expressed a truncated leukotoxin peptide at
high levels while pAA345 expressed full length leukotoxin at very
low levels. Therefore, the 3' end of the lktA gene (StyI BamHI
fragment from pAA345) was ligated to StyI BamHI-digested pAA342,
yielding the plasmid pAA352. The P. haemolytica leukotoxin produced
from the pAA352 construct is hereinafter referred to as LKT
352.
[0187] Plasmid pAA352 was then used to prepare a shortened version
of the recombinant leukotoxin polypeptide. The shortened LKT gene
was produced by deleting an internal DNA fragment of approximately
1300 bp in length from the recombinant LKT gene as follows. The
plasmid pCB113, (ATCC Accession No. 69749 and described in U.S.
Pat. No. 5,837,268, incorporated herein by reference in its
entirety) which includes the LKT 352 polypeptide, was digested with
the restriction enzyme BstB1 (New England Biolabs). The resultant
linearized plasmid was then digested with mung-bean nuclease
(Pharmacia) to remove the single stranded protruding termini
produced by the BstB1 digestion. The blunted DNA was then digested
with the restriction enzyme Nael (New England Biolabs), and the
digested DNA was loaded onto a 1% agarose gel where the DNA
fragments were separated by electrophoresis. A large DNA fragment
of approximately 6190 bp was isolated and purified from the agarose
gel using a Gene Clean kit (Bio 101), and the purified fragment was
allowed to ligate to itself using bacteriophage T4 DNA ligase
(Pharmacia). The resulting ligation mix was used to transform
competent E. coli JM105 cells, and positive clones were identified
by their ability to produce an aggregate protein having an
appropriate molecular weight. The recombinant plasmid thus formed
was designated pCB114, (described in U.S. Pat. No. 5,837,268,
incorporated herein by reference in its entirety), and produces a
shortened leukotoxin polypeptide termed "LKT 114".
[0188] Plasmid pCB114 was then used to produce plasmid pSLKT-30.
Plasmid pSLKT-30 was made by cloning the leukotoxin-encoding
fragment from pCB114 by PCR into plasmid pAA352 (ATCC Accession No.
68283 and described in U.S. Pat. No. 5,476,657, incorporated herein
by reference in its entirety). In doing so, mutations were
introduced near the C-terminus, resulting in two amino acid changes
to the native leukotoxin molecule. Thus, a PCR fragment of the
affected area was cloned back into plasmid PSLKT-30. Specifically,
a fragment from pSLKT-30 was created by PCR using LKT6 (SEQ ID
NO:40) as the upstream PCR primer, and LKT13 (SEQ ID NO:41) as the
downstream PCR primer:
1 LKT6: TTA GAG AGT TAT GCC GAA CCC; (SEQ ID NO:40) LKT13: GAT GCC
ATC GCT AGC TAG CTA GGA TCC (SEQ ID NO:41) CCT AGC AAA TTC AAG AGA
AGA TAA ACT TTG ATC CAA CAT TGA.
[0189] The fragment contained the desired change and the Nsil and
Ncol restriction sites. The isolated fragment was digested using
the restriction enzymes Nsil and Ncol, as was the plasmid pSLKT-30.
The Nsil/Ncol fragment was removed from the plasmid and replaced
with the PCR fragment, resulting in the mutation back to the
original sequence. The plasmid was termed pCB150. A diagram of
plasmid pCB150 is shown in FIG. 14. The nucleic acid sequence of
LKT 114 from plasmid pCB150 is shown in FIGS. 15A-15D.
EXAMPLE 3
Construction of LKT-Myostatin Peptide Multimer Fusions
[0190] Multiple copies of each oligomer pair described in Example 1
were used to prepare tandem repeats of coding sequences for
myostatin peptide multimers joined to the LKT 114 gene. The entire
active portion of the protein was also reconstructed and fused to
LKT 114 for use as an immunizing agent.
[0191] Representative LKT-myostatin peptide fusions were
constructed as follows. Oligonucleotide pairs from Example 1 were
annealed and ligated into the vector pUCl9 (Pharmacia) which had
been digested with the restriction endonuclease HincII. The ligated
DNA was used to transform E. coli strain TOP10F' (Invitrogen).
Transformants containing the oligonucleotide inserts were
identified by PCR and restriction endonuclease mapping.
[0192] The oligonucleotide pairs were designed to be linked
together by ligating the BamHI site at the front end of one
oligonucleotide pair to the BglII site at the back end of a second
copy of the oligonucleotide pair. The restriction sites at the
point of ligation were disabled leaving a single BamHI site at the
front end of the repeat and a single BglII site at the back end of
the repeat. Tandem repeats of each oligonucleotide pair were
constructed by digesting the oligonucleotide-containing plasmid
with the restriction endonucleases BamHI and BglII to release the
inserted oligonucleotide fragment. This fragment was then ligated
back into the oligonucleotide-containing plasmid, which had been
digested with the restriction endonuclease BglII. The ligated DNA
was used to transform E. coli strain TOP10F'. Transformants
containing repeats of the oligonucleotide inserts were identified
by PCR and restriction endonuclease mapping. This process was
repeated until pUC19 plasmids containing at least four repeating
copies and up to 8 copies of each oligonucleotide pair in the
correct orientation were produced.
[0193] In addition to being linked to themselves, some of the
oligonucleotide pairs were also designed to link to each other to
recreate the active region of the myostatin protein as closely as
possible. This was done by ligating BamHI, BglII cut
oligonucleotide pair MYOS 3/4 in to the BglII site behind
oligonucleotide pair MYOS 1/2 in the pUC19 vector. This was
followed by ligating in oligonucleotide pair MYOS 5/6 cut with
BstBI and BglII into the vector containing the reconstructed
myostatin active region cut with BstBI and BglII. Oligonucleotide
pair MYOS 7/8 was cut with BamHI and BglII and ligated into the
vector containing the reconstructed myostatin active region at the
BglII site. Oligonucleotide pair MYOS {fraction (9/10)} containing
vector was cut with EcoRI and the EcoRI fragment from the pUC19
myostatin reconstruction was ligated in. Oligonucleotide pair MYOS
{fraction (11/12)} was cut with BamHI and BglII and ligated into
the vector containing the reconstructed myostatin active region at
the BglII site. This was followed by ligating in oligonucleotide
pair MYOS {fraction (13/14)} cut with BsmI and BglII into the
vector containing the reconstructed myostatin active region cut
with BsmI and BglII. This completed the reconstruction of the
coding sequence for the myostatin active region, which contained
three sets of two amino acid linkers inserted into the myostatin
active region sequence at positions 55-60, 139-144 and 241-246 and
at the C-terminus (see FIG. 13).
[0194] The multiple copies of each oligonucleotide pair and the
myostatin active region reconstruction were then released from the
pUC19 plasmid by digestion with the restriction endonucleases BamHI
and BglII. These DNA fragments were then ligated into the plasmid
pCB150. Plasmid pCB150 was digested with the restriction
endonuclease BamHI. The ligated DNA was used to transform E. coli
strain TOP10F'. Transformants containing the oligonucleotide
inserts were identified by PCR and restriction endonuclease
mapping. The recombinant plasmids were designated pJS121, pJS122,
pJS123, pJS124, pJS125, pJS126, pJS127, pJS128, pJS129, pJS130, and
pCB317.
[0195] The plasmid pJS121 contains 6 repeating copies of
oligonucleotide pair MYOS 1/2 fused to LKT 114. The plasmid pJS122
contains 8 repeating copies of oligonucleotide pair MYOS 3/4 fused
to LKT 114. The plasmid pJS123 contains 8 repeating copies of
oligonucleotide pair MYOS 5/6 fused to LKT 114. The plasmid pJS124
contains 8 repeating copies of oligonucleotide pair MYOS 7/8 fused
to LKT 114. The plasmid pJS125 contains 6 repeating copies of
oligonucleotide pair MYOS {fraction (9/10)} fused to LKT 114. The
plasmid pJS126 contains 4 repeating copies of oligonucleotide pair
MYOS {fraction (11/12)} fused to LKT 114. The plasmid pJS127
contains 6 repeating copies of oligonucleotide pair MYOS {fraction
(13/14)} fused to LKT 114. The plasmid pJS128 contains 4 repeating
copies of oligonucleotide pair MYOS {fraction (15/16)} fused to LKT
114. The plasmid pJS129 contains 8 repeating copies of
oligonucleotide pair MYOS {fraction (17/18)} fused to LKT 114. The
plasmid pJS130 contains 4 repeating copies of oligonucleotide pair
MYOS {fraction (19/20)} fused to LKT 114. The plasmid pCB317
contains a single copy of the myostatin active region
reconstruction fused to LKT 114.
EXAMPLE 4
Purification of LKT-Myostatin Peptide Fusions
[0196] The recombinant LKT-myostatin peptide fusion proteins from
above were expressed as inclusion bodies and purified using the
following procedure. A loop of cells from each frozen stock was
inoculated into 10 ml of TB broth in a 50 ml Erlenmeyer flask. The
TB broth was supplemented with 100 .mu.g/ml of ampicillin and
incubated at 37.degree. C. for 12-16 hours on an Innova 4000 shaker
at 250 rpm. The culture was used to inoculate one liter of TB broth
in a 4L Erlenmeyer flask. The TB broth was supplemented with 100
.mu.g/ml of ampicillin and incubated at 37.degree. C. for
approximately 3 hours on an Innova 4000 shaker at 250 rpm. 1 ml of
a 1M IPTG (isoprpyl-B,D-thiogalactopyranoside) solution was then
added to the culture to induce recombinant protein production. The
culture was then incubated for a further two hours. The cells were
harvested by centrifugation for 10 min at 6000 rpm in 3.times. 500
ml polypropylene bottles using a JA 10 rotor in an Avanti J25
centrifuge. The cell pellet was resuspended in 40 ml of 25%
sucrose, 50 mM Tris-hydrochloride, pH 8.0 and frozen at -70.degree.
C. for 15 min. The frozen cells were thawed at room temperature and
mixed with 10 ml of Lysozyme (Sigma, 10 mg/ml in 250 mM
Tris-hydrochloride, pH 8.0). After incubation for 15 min on ice,
300 ml of lysis buffer (2% Triton X100, 50 mM EDTA, 100 mM
Tris-hydrochloride, pH 8.0) was added and mixed by shaking. The
lysed cell suspension was then sonicated for 4.times. 30 second
bursts at full power with a large probe on a Misonix sonicator. The
solution was split into 2.times. 250 ml centrifuge bottles and
centrifuged for 25 min at 10000 rpm in a JA 14 rotor. The inclusion
body pellets were washed by resuspending in 100 ml of
double-distilled water and centrifuging to collect the inclusion
bodies. This washing procedure was repeated once more and the final
inclusion body pellet was suspended in 10 ml of double-distilled
water and stored at -20.degree. C. until needed.
[0197] All of the isolated fusion proteins were tested by SDS-PAGE
to determine their identity by molecular weight, concentration and
purity, by comparing the proteins to known standards. 10 .mu.l
aliquots of each fusion protein were solubilized with 10 .mu.l of
8M Urea and 2 .mu.l of the solubilized protein was then mixed with
100 .mu.l of 1.times.SDS-PAGE loading buffer. The loading buffer
samples were heated to 94.degree. C. for 5 min and run on a 10%
polyacrylamide gel. Recombinant LKT 114 from pCB150 was also run as
a control.
EXAMPLE 5
In Vivo Biologic Effect of LKT-Myostatin Peptide Fusion
Proteins
[0198] To test the ability of the fusion proteins comprising
multiple copies of various peptides of myostatin fused to a carrier
protein to manifest a biologic effect in vivo, the following
vaccination trial was preformed. Recombinant LKT-myostatin peptide
fusion proteins were prepared as described above. Vaccines for each
were prepared by solubilizing each of the fusion proteins in a
final concentration of 6M Urea (used for the first injection) or 4M
Guanidine-HCl (used for all subsequent injections). To 2.5 ml (used
for the first two injections) or 1.5 ml (used for the last
injection) aliquots of VSA-3 adjuvant (a modified Emulsigen Plus
adjuvant) 1250 .mu.g of each solubilized protein was added and
mixed by 5.times.5 sec bursts with a Misonix sonicator with a
microtip probe at a power setting of 5. To these mixtures, 50 .mu.l
of a 1% Thimerosal solution and PBS pH 7.4 (Phosphate Buffer
Saline) to a final volume of 5 ml were added and the mixtures
sonicated again. A volume of 200 .mu.l was used for each injection.
Each injection contained 50 .mu.g of fusion protein. This initial
injection (day 0) was given at 3-4 weeks of age with subsequent
injections at days 28 and 56.
[0199] Fourteen treatment groups each contained 15 CDl Swiss mice.
The treatment groups were as follows (see Table 1): Group 1 no
vaccination control, Group 2 adjuvant only control, Group 3 pCB150
carrier protein control, Groups 4 to 13 pJS121 to pJS130 test
proteins, Group 14 pCB317 test protein. The mice were weighed
weekly to determine weight gain over the course of the 98 day
experiment. The results of this trial are summarized in Table 2 and
FIG. 18. Guanidine-HCl was used as the solubilizing agent of choice
as it appeared to provide improved protein stability over Urea in
the vaccine formulation. The concentration of VSA-3 was reduced
from 50% to 30% in the vaccine formulation in an effort to reduce
injection site reactions.
2TABLE 1 Treatment Group Myos Oligo Plasmid 1 -- -- 2 -- -- 3 --
pCB150 4 1 pJS121 5 3 pSJ122 6 5 pSJ123 7 7 pJS124 8 9 pJS125 9 11
pJS126 10 13 pJS127 11 15 pJS128 12 17 pJS129 13 19 pJS130 14
reconstruction pCB317
[0200]
3 TABLE 2 Mean Group Mean Group Mean Group Weight Gain Treatment
Weight Day Weight Day Through Day Group 0 .+-. SEM 98 .+-. SEM 84
.+-. SEM 1 Control 16.67 .+-. 0.32 29.33 .+-. 0.71 12.67 .+-. 0.65
2 Control 16.11 .+-. 0.25 29.09 .+-. 0.79 12.99 .+-. 0.72 3 Control
16.05 .+-. 0.34 29.33 .+-. 0.70 13.27 .+-. 0.61 4 Test 16.39 .+-.
0.37 30.02 .+-. 0.60 13.63 .+-. 0.60 5 Test 15.52 .+-. 0.35 30.48
.+-. 0.84 14.96 .+-. 0.72 6 Test 15.78 .+-. 0.33 30.84 .+-. 0.99
15.06 .+-. 0.90 7 Test 15.72 .+-. 0.27 30.36 .+-. 0.76 14.64 .+-.
0.65 8 Test 15.46 .+-. 0.25 29.42 .+-. 0.84 13.96 .+-. 0.79 9 Test
15.32 .+-. 0.32 29.48 .+-. 0.54 14.16 .+-. 0.50 10 Test 16.44 .+-.
0.31 31.27 .+-. 0.92 14.85 .+-. 0.92 11 Test 16.30 .+-. 0.41 31.02
.+-. 0.70 14.72 .+-. 0.75 12 Test 15.54 .+-. 0.28 30.73 .+-. 0.71
15.19 .+-. 0.69 13 Test 15.57 .+-. 0.30 31.04 .+-. 0.96 15.47 .+-.
0.99 14 Test 15.51 .+-. 0.20 29.25 .+-. 0.62 13.73 .+-. 0.56
EXAMPLE 6
Statistical Analysis of Trial Results
[0201] Statistical analysis of the trial results was performed
using a statistical software package (Statistix Version 1.0). In
this trial, all control groups had very similar mean total weights,
while several test groups had elevated mean total weights. A
one-way ANOVA on the weight gain over the 98 days of the experiment
was performed. An LSD comparison of means test indicated that
treatment group 13 was significantly different from any of the
control groups. Test groups 12 and 6 were significantly different
from two of the three control groups. The treatment groups were
also analyzed by grouping them into controls (groups 1-3) and test
group (groups 4-14). A one-way ANOVA on the weight gains from these
two groups was performed. An LSD comparison of means test indicated
that the group that received a test treatment was significantly
different from the control group.
[0202] Deposits of Strains Useful in Practicing the Invention
[0203] A deposit of biologically pure cultures of the following
strains was made with the American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va. The accession number
indicated was assigned after successful viability testing, and the
requisite fees were paid. The deposits were made under the
provisions of the Budapest Treaty on the International Recognition
of the Deposit of Microorganisms for the Purpose of Patent
Procedure and the Regulations thereunder (Budapest Treaty). This
assures maintenance of viable cultures for a period of thirty (30)
years from the date of deposit and at least five (5) years after
the most recent request for the furnishing of a sample of the
deposit by the depository. The organisms will be made available by
the ATCC under the terms of the Budapest Treaty, which assures
permanent and unrestricted availability of the cultures to one
determined by the U.S. Commissioner of Patents and Trademarks to be
entitled thereto according to 35 U.S.C. .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 C.F.R.
.sctn.1.12). Upon the granting of a patent, all restrictions on the
availability to the public of the deposited cultures will be
irrevocably removed.
[0204] These deposits are provided merely as convenience to those
of skill in the art, and are not an admission that a deposit is
required under 35 U.S.C. .sctn.112. The nucleic acid sequences of
these plasmids, as well as the amino acid sequences of the
polypeptides encoded thereby, are incorporated herein by reference
and are controlling in the event of any conflict with the
description herein. A license may be required to make, use, or sell
the deposited materials, and no such license is hereby granted.
4 Strain Deposit Date ATCC No. pAA352 in E. coil W1485 March 30,
1990 68283 pCB113 in E. coil JM105 February 1, 1995 69749 pCB150 in
E. coil TOP10F' pJS123 in E. coil TOP10F' pJS127 in E. coil TOP10F'
pJS130 in E. coil TOP10F'
[0205] Thus, immunogenic myostatin peptides, multimers and
immunoconjugates are disclosed, as are methods of making and using
the same. Although preferred embodiments of the subject invention
have been described in some detail, it is understood that obvious
variations can be made without departing from the spirit and the
scope of the invention as defined by the appended claims.
* * * * *
References