U.S. patent application number 10/449140 was filed with the patent office on 2003-11-06 for beta-like glycoprotein hormone polypeptide and heterodimer.
This patent application is currently assigned to Amgen Inc.. Invention is credited to Cao, Jin, Danilenko, Dimitry M., Gong, Jianhua, Hill, David C., Paszty, Christopher J. R..
Application Number | 20030207403 10/449140 |
Document ID | / |
Family ID | 27393079 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207403 |
Kind Code |
A1 |
Paszty, Christopher J. R. ;
et al. |
November 6, 2003 |
Beta-like glycoprotein hormone polypeptide and heterodimer
Abstract
Novel .beta.10 polypeptides and heterodimers thereof, and
nucleic acid molecules encoding the same are disclosed. The
invention also provides vectors, host cells, selective binding
agents, and methods for producing .beta.10 polypeptides and
heterodimeric forms thereof, specifically .alpha.2/.beta.10. Also
provided for are methods for the treatment, diagnosis,
amelioration, or prevention of diseases with .beta.10 polypeptides
and .alpha.2/.beta.10 heterodimers or their respective binding
agents.
Inventors: |
Paszty, Christopher J. R.;
(Ventura, CA) ; Cao, Jin; (Tarzana, CA) ;
Danilenko, Dimitry M.; (Thousand Oaks, CA) ; Gong,
Jianhua; (Thousand Oaks, CA) ; Hill, David C.;
(Thousand Oaks, CA) |
Correspondence
Address: |
AMGEN INCORPORATED
MAIL STOP 27-4-A
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Amgen Inc.
|
Family ID: |
27393079 |
Appl. No.: |
10/449140 |
Filed: |
May 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10449140 |
May 28, 2003 |
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09818954 |
Mar 27, 2001 |
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09818954 |
Mar 27, 2001 |
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09723970 |
Nov 27, 2000 |
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60199211 |
Apr 24, 2000 |
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60192654 |
Mar 28, 2000 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 514/20.9; 514/9.7; 530/397; 536/23.5 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 29/00 20180101; A61P 37/02 20180101; A61P 37/08 20180101; A61P
3/06 20180101; A61P 17/06 20180101; A61P 9/10 20180101; A61P 31/18
20180101; A61P 25/22 20180101; A61P 21/00 20180101; C07K 2317/34
20130101; A61P 35/00 20180101; A61P 11/06 20180101; C07K 14/59
20130101; A61K 38/00 20130101; C07K 16/26 20130101; C07K 14/575
20130101; G01N 2333/59 20130101; A61P 3/10 20180101; A61P 9/12
20180101; A01K 2217/05 20130101; A61P 15/12 20180101; A61P 19/02
20180101; G01N 33/74 20130101; A61P 17/02 20180101; A61K 48/00
20130101; A61P 1/04 20180101; A61P 31/00 20180101; A61K 35/12
20130101; A61P 15/00 20180101; A61P 3/04 20180101; A61P 5/14
20180101; A61P 15/10 20180101 |
Class at
Publication: |
435/69.1 ;
435/320.1; 435/325; 530/397; 536/23.5; 514/8 |
International
Class: |
C12P 021/02; C12N
005/06; C07K 014/575; A61K 038/24 |
Claims
What is claimed:
1. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) the nucleotide
sequence set forth in SEQ ID No: 2; (b) a nucleotide sequence
encoding the polypeptide set forth in SEQ ID No: 1; (c) a
nucleotide sequence which hybridizes under moderately or highly
stringent conditions to the complement of (a) or (b), wherein the
encoded polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer; and (d) a nucleotide sequence complementary to any of
(a)-(c).
2. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence encoding a polypeptide that is at least about 70, 75, 80,
85, 90, 95, 96, 97, 98, or 99 percent identical to the polypeptide
set forth in SEQ ID NO: 1, wherein the polypeptide, when
heterodimerized to human .alpha.2 polypeptide, has an activity of
the human .alpha.2/.beta.10 heterodimer; (b) a nucleotide sequence
encoding an allelic variant or splice variant of the nucleotide
sequence set forth in SEQ ID NO: 2, wherein the encoded
polypeptide, when heterodimerized to human .alpha.2 polypeptide,
has an activity of the human .alpha.2/.beta.10 heterodimer; (c) a
nucleotide sequence of SEQ ID NO: 2, (a), or (b) encoding a
polypeptide fragment of at least about 25 amino acid residues,
wherein the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer; (d) a nucleotide sequence of SEQ ID NO: 2 or (a)-(c)
comprising a fragment of at least about 16 nucleotides; (e) a
nucleotide sequence which hybridizes under moderately or highly
stringent conditions to the complement of any of (a)-(d), wherein
the encoded polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer; and (f) a nucleotide sequence complementary to any of
(a)-(c).
3. An isolated nucleic acid molecule comprising a nucleotide
sequence selected from the group consisting of: (a) a nucleotide
sequence encoding a polypeptide as set forth in SEQ ID NO: 1 with
at least one conservative amino acid substitution, wherein the
polypeptide, when heterodimerized to human .alpha.2 polypeptide,
has an activity of the human .alpha.2/.beta.10 heterodimer; (b) a
nucleotide sequence encoding a polypeptide as set forth in SEQ ID
NO: 1 with at least one amino acid insertion, wherein the
polypeptide, when heterodimerized to human .alpha.2 polypeptide,
has an activity of the human .alpha.2/.beta.10 heterodimer; (c) a
nucleotide sequence encoding a polypeptide as set forth in SEQ ID
NO: 1 with at least one amino acid deletion, wherein the
polypeptide, when heterodimerized to human .alpha.2 polypeptide,
has an activity of the human .alpha.2/.beta.10 heterodimer; (d) a
nucleotide sequence encoding a polypeptide as set forth in SEQ ID
NO: 1 which has a C- and/or N-terminal truncation, wherein the
polypeptide, when heterodimerized to human .alpha.2 polypeptide,
has an activity of the human .alpha.2/.beta.10 heterodimer; (e) a
nucleotide sequence encoding a polypeptide as set forth in SEQ ID
NO: 1 with at least one modification selected from the group
consisting of amino acid substitutions, amino acid insertions,
amino acid deletions, C-terminal truncation, and N-terminal
truncation, wherein the polypeptide, when heterodimerized to human
.alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer; (f) a nucleotide sequence of (a)-(e)
comprising a fragment of at least about 16 nucleotides; (g) a
nucleotide sequence which hybridizes under moderately or highly
stringent conditions to the complement of any of (a)-(f), wherein
the encoded polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer; and (h) a nucleotide sequence complementary to any of
(a)-(e).
4. A vector comprising the nucleic acid molecule of claims 1, 2, or
3.
5. A host cell comprising the vector of claim 4.
6. The host cell of claim 5 that is a eukaryotic cell.
7. The host cell of claim 5 that is a prokaryotic cell.
8. A process of producing a .beta.10 polypeptide comprising
culturing the host cell of claim 5 under suitable conditions to
express the polypeptide, and optionally isolating the polypeptide
from the culture.
9. A polypeptide produced by the process of claim 8.
10. The process of claim 8, wherein the nucleic acid molecule
comprises promoter DNA other than the promoter DNA for the native
.beta.10 polypeptide operatively linked to the DNA encoding the
.beta.10 polypeptide.
11. The isolated nucleic acid molecule according to claim 2 wherein
the percent identity is determined using a computer program
selected from the group consisting of GAP, BLASTP, BLASTN, FASTA,
BLASTA, BLASTX, BestFit, and the Smith-Waterman algorithm.
12. A process for determining whether a compound modulates .beta.10
polypeptide activity or production comprising exposing a cell
comprising the vector of claim 4 to the compound, and measuring
.beta.10 polypeptide activity or production in said cell.
13. An isolated polypeptide comprising the amino acid sequence set
forth in SEQ ID NO: 3.
14. An isolated polypeptide comprising the amino acid sequence
selected from the group consisting of: (a) the mature amino acid
sequence as set forth in SEQ ID NO: 3, comprising a mature amino
terminus at residue 1, optionally further comprising an
amino-terminal methionine; (b) an amino acid sequence for an
ortholog of SEQ ID NO: 3, wherein the encoded polypeptide, when
heterodimerized to human .alpha.2 polypeptide, has an activity of
the human .alpha.2/.beta.10 heterodimer; (c) an amino acid sequence
that is at least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99
percent identical to the amino acid sequence of SEQ ID NO: 3,
wherein the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer; (d) a fragment of the amino acid sequence set forth in
SEQ ID NO: 3 comprising at least about 25 amino acid residues,
wherein the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer; (e) an amino acid sequence for an allelic variant or
splice variant of either the amino acid sequence set forth in SEQ
ID NO: 3 or at least one of (a)-(c) wherein the polypeptide, when
heterodimerized to human .alpha.2 polypeptide, has an activity of
the human .alpha.2/.beta.10 heterodimer.
15. An isolated polypeptide comprising the amino acid sequence
selected from the group consisting of: (a) the amino acid sequence
set forth in SEQ ID NO: 3 with at least one conservative amino acid
substitution, wherein the polypeptide, when heterodimerized to
human .alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer; (b) the amino acid sequence set
forth in SEQ ID NO: 3 with at least one amino acid insertion,
wherein the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer; (c) the amino acid sequence set forth in SEQ ID NO: 3
with at least one amino acid deletion, wherein the polypeptide,
when heterodimerized to human .alpha.2 polypeptide, has an activity
of the human .alpha.2/.beta.10 heterodimer; (d) the amino acid
sequence set forth in SEQ ID NO: 3 which has a C- and/or N-terminal
truncation, wherein the polypeptide, when heterodimerized to human
.alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer; and (e) the amino acid sequence set
forth in SEQ ID NO: 3, with at least one modification selected from
the group consisting of amino acid substitutions, amino acid
insertions, amino acid deletions, C-terminal truncation, and
N-terminal truncation, wherein the polypeptide, when
heterodimerized to human .alpha.2 polypeptide, has an activity of
the human .alpha.2/.beta.10 heterodimer.
16. An isolated polypeptide encoded by the nucleic acid molecule of
claims 1, 2, or 3.
17. The isolated polypeptide according to claim 14 wherein the
percent identity is determined using a computer program selected
from the group consisting of GAP, BLASTP, BLASTN, FASTA, BLASTA,
BLASTX, BestFit, and the Smith-Waterman algorithm.
18. An antibody produced by immunizing an animal with a peptide
comprising the amino acid sequence of SEQ ID NO: 3.
19. An antibody or fragment thereof that specifically binds the
polypeptide of claims 13, 14, or 15.
20. The antibody of claim 19 that is a monoclonal antibody.
21. A hybridoma that produces a monoclonal antibody that binds to a
peptide comprising the amino acid sequence of SEQ ID NO: 3.
22. A method of detecting or quantitating the amount of .beta.10
polypeptide using an anti-.beta.10 antibody or fragment thereof
which specifically binds the polypeptide of claims 13, 14 or
15.
23. A selective binding agent or fragment thereof that specifically
binds at least one polypeptide, wherein said polypeptide comprises
the amino acid sequence selected from the group consisting of: (a)
the amino acid sequence set forth in SEQ ID NO: 3; and (b) a
fragment of the amino acid sequence set forth in SEQ ID NO: 3; and
(c) a naturally occurring variant of (a) or (b).
24. The selective binding agent of claim 23 that is an antibody or
fragment thereof.
25. The selective binding agent of claim 23 that is a humanized
antibody.
26. The selective binding agent of claim 23 that is a human
antibody or fragment thereof.
27. The selective binding agent of claim 23 that is a polyclonal
antibody or fragment thereof.
28. The selective binding agent of claim 23 that is a monoclonal
antibody or fragment thereof.
29. The selective binding agent of claim 23 that is a chimeric
antibody or fragment thereof.
30. The selective binding agent of claim 23 that is a CDR-grafted
antibody or fragment thereof.
31. The selective binding agent of claim 23 that is an
anti-idiotypic antibody or fragment thereof.
32. The selective binding agent of claim 23 which is a variable
region fragment.
33. The variable region fragment of claim 32 which is a Fab or a
Fab' fragment.
34. A selective binding agent or fragment thereof comprising at
least one complementarity determining region with specificity for a
polypeptide having the amino acid sequence of SEQ ID NO: 3.
35. The selective binding agent of claim 23 which is bound to a
detectable label.
36. The selective binding agent of claim 23 which antagonizes
.beta.10 polypeptide biological activity.
37. A method for treating, preventing, or ameliorating a disease,
condition, or disorder comprising administering to a patient an
effective amount of a selective binding agent according to claim
23.
38. A method for treating, preventing, or ameliorating a thyroid
gland related disease, condition, or disorder comprising
administering to a patient an effective amount of a selective
binding agent according to claim 23.
39. A selective binding agent produced by immunizing an animal with
a polypeptide comprising the amino acid sequence of SEQ ID NO:
3.
40. A hybridoma that produces a selective binding agent capable of
binding a polypeptide according to claims 1, 2, or 3.
41. A composition comprising the polypeptide of claims 13, 14, or
15 and a pharmaceutically acceptable formulation agent.
42. The composition of claim 41 wherein the pharmaceutically
acceptable formulation agent is a carrier, adjuvant, solubilizer,
stabilizer, or anti-oxidant.
43. The composition of claim 41 wherein the polypeptide comprises
the mature amino acid sequence as set forth in SEQ ID NO: 3.
44. A polypeptide comprising a derivative of the polypeptide of
claims 13, 14, or 15.
45. The polypeptide of claim 44 which is covalently modified with a
water-soluble polymer.
46. The polypeptide of claim 45 wherein the water-soluble polymer
is selected from the group consisting of polyethylene glycol,
monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinyl
pyrrolidone) polyethylene glycol, propylene glycol homopolymers,
polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols, and polyvinyl alcohol.
47. A composition comprising a nucleic acid molecule of claims 1,
2, or 3 and a pharmaceutically acceptable formulation agent.
48. A composition of claim 47 wherein said nucleic acid molecule is
contained in a viral vector.
49. A viral vector comprising a nucleic acid molecule of claims 1,
2, or 3.
50. A fusion polypeptide comprising the polypeptide of claims 13,
14, or 15 fused to a heterologous amino acid sequence.
51. The fusion polypeptide of claim 50 wherein the heterologous
amino acid sequence is an IgG constant domain or fragment
thereof.
52. A method for treating, preventing or ameliorating a medical
condition comprising administering to a patient the polypeptide of
claims 13, 14, or 15 or the polypeptide encoded by the nucleic acid
of claims 1, 2, or 3.
53. A method of diagnosing a pathological condition or a
susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or amount of expression of the
polypeptide of claims 13, 14, or 15 or the polypeptide encoded by
the nucleic acid molecule of claims 1, 2, or 3 in a sample; and (b)
diagnosing a pathological condition or a susceptibility to a
pathological condition based on the presence or amount of
expression of the polypeptide.
54. A method of diagnosing a thyroid gland related pathological
condition or a susceptibility to a thyroid gland related
pathological condition in a subject comprising: (a) determining the
presence or amount of expression of the polypeptide of claims 13,
14, or 15 or the polypeptide encoded by the nucleic acid molecule
of claims 1, 2, or 3 in a sample; and (b) diagnosing a thyroid
gland related pathological condition or a susceptibility to a
thyroid gland related pathological condition based on the presence
or amount of expression of the polypeptide.
55. A device, comprising: (a) a membrane suitable for implantation;
and (b) cells encapsulated within said membrane, wherein said cells
secrete a polypeptide of claims 13, 14, or 15, and wherein said
membrane is permeable to said protein and impermeable to materials
detrimental to said cells.
56. A method of identifying a compound which binds to a polypeptide
comprising: (a) contacting the polypeptide of claims 13, 14, or 15
with a compound; and (b) determining the extent of binding of the
polypeptide to the compound.
57. A method of modulating levels of a polypeptide in an animal
comprising administering to the animal the nucleic acid molecule of
claims 1, 2, or 3.
58. A transgenic non-human mammal comprising the nucleic acid
molecule of claims 1, 2, or 3.
59. A heterodimer of human .beta.10 polypeptide and human .alpha.2
polypeptide.
60. A naturally occurring variant of the .alpha.2/.beta.10
heterodimer of claim 59.
61. A vector comprising nucleic acid molecules encoding human
.beta.10 polypeptide and human .alpha.2 polypeptide.
62. A host cell comprising the vector of claim 61.
63. The host cell of claim 62 that is a prokaryotic cell.
64. The host cell of claim 62 that is a eukaryotic cell.
65. A process of producing an .alpha.2/.beta.10 heterodimer
comprising culturing the host cell of claim 62 under suitable
conditions to express the .alpha.2/.beta.10 heterodimer, and
optionally isolating the .alpha.2/.beta.10 heterodimer from the
culture.
66. A heterodimer produced by the process of claim 65.
67. A process for determining whether a compound modulates
.alpha.2/.beta.10 heterodimer activity or production comprising
exposing a cell according to claim 62 to the compound, and
measuring .alpha.2/.beta.10 heterodimer activity or production in
said cell.
68. An antibody produced by immunizing an animal with a human
.alpha.2/.beta.10 heterodimer.
69. An antibody or fragment thereof that specifically binds the
.alpha.2/.beta.10 heterodimer of claim 59 or naturally occurring
variant thereof.
70. The antibody of claim 69 that is a monoclonal antibody.
71. A hybridoma that produces the monoclonal antibody of claim 70
which is specific to an .alpha.2/.beta.10 heterodimer.
72. A method of detecting or quantitating the amount of an
.alpha.2/.beta.10 heterodimer using the antibody of claim 69.
73. A selective binding agent or fragment thereof that specifically
binds at least one of the following: (a) the .alpha.2/.beta.10
heterodimer of claim 59; (b) a fragment of the .alpha.2/.beta.10
heterodimer of claim 59; and (c) a naturally occurring variant of
(a) or (b).
74. The selective binding agent of claim 73 that is an antibody or
fragment thereof.
75. The selective binding agent of claim 73 that is a humanized
antibody.
76. The selective binding agent of claim 73 that is a human
antibody or fragment thereof.
77. The selective binding agent of claim 73 that is a polyclonal
antibody or fragment thereof.
78. The selective binding agent of claim 73 that is a monoclonal
antibody or fragment thereof.
79. The selective binding agent of claim 73 that is a chimeric
antibody or fragment thereof.
80. The selective binding agent of claim 73 that is a CDR-grafted
antibody or fragment thereof.
81. The selective binding agent of claim 73 that is an
anti-idiotypic antibody or fragment thereof.
82. The selective binding agent of claim 73 which is a variable
region fragment.
83. The variable region fragment of claim 82 which is a Fab or a
Fab' fragment.
84. A selective binding agent or fragment thereof comprising at
least one complementarity determining region with specificity for a
human .alpha.2/.beta.10 heterodimer.
85. The selective binding agent of claim 73 which is bound to a
detectable label.
86. The selective binding agent of claim 73 which antagonizes an
.alpha.2/.beta.10 heterodimer biological activity.
87. A method for treating, preventing, or ameliorating a disease,
condition, or disorder comprising administering to a patient an
effective amount of an .alpha.2/.beta.10 heterodimer according to
claim 59, or a selective binding agent that specifically binds said
heterodimer or fragment or naturally occurring variant thereof.
88. A method for treating, preventing, or ameliorating a thyroid
gland related disease, condition, or disorder comprising
administering to a patient an effective amount of an
.alpha.2/.beta.10 heterodimer according to claim 59, or a selective
binding agent that specifically binds said heterodimer or fragment
or naturally occurring variant thereof.
89. A composition comprising the heterodimer of claim 59, or a
naturally occurring variant of said heterodimer, or a fragment of
said heterodimer, or a selective binding agent of any of the
foregoing, and a pharmaceutically acceptable formulation agent.
90. The composition of claim 89 wherein the pharmaceutically
acceptable formulation agent is a carrier, adjuvant, solubilizer,
stabilizer, or anti-oxidant.
91. A method of modulating levels of an .alpha.2/.beta.10
heterodimer in an animal comprising administering to the animal the
nucleic acid molecule of claims 1, 2, or 3.
92. The heterodimer of claim 59 or naturally occurring variant
thereof which is covalently modified with a water-soluble
polymer.
93. The heterodimer of claim 92 wherein the water-soluble polymer
is selected from the group consisting of polyethylene glycol,
monomethoxy-polyethylene glycol, dextran, cellulose, poly-(N-vinyl
pyrrolidone) polyethylene glycol, propylene glycol homopolymers,
polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols, and polyvinyl alcohol.
94. A fusion polypeptide comprising the heterodimer of claim 59 or
naturally occurring variant thereof fused to a heterologous amino
acid sequence.
95. The fusion polypeptide of claim 94 wherein the heterologous
amino acid sequence is an IgG constant domain or fragment
thereof.
96. A method of diagnosing a pathological condition or a
susceptibility to a pathological condition in a subject comprising:
(a) determining the presence or amount of expression of the
heterodimer of claim 59 or naturally occurring variant thereof; and
(b) diagnosing a pathological condition or a susceptibility to a
pathological condition based on the presence or amount of
expression of the heterodimer.
97. A method of diagnosing a thyroid gland related pathological
condition or a susceptibility to a thyroid gland related
pathological condition in a subject comprising: (a) determining the
presence or amount of expression of the heterodimer of claim 59 or
naturally occurring variant thereof; and (b) diagnosing a thyroid
gland related pathological condition or a susceptibility to a
thyroiel gland related pathological condition based on the presence
or amount of expression of the heterodimer.
98. A device, comprising: (a) a membrane suitable for implantation;
and (b) cells encapsulated within said membrane, wherein said cells
secrete an .alpha.2/.beta.10 heterodimer of claim 59 or naturally
occurring variant thereof, and wherein said membrane is permeable
to said heterodimer and impermeable to materials detrimental to
said cells.
99. A method of identifying a compound which binds to a heterodimer
comprising: (a) contacting the heterodimer of claim 59 or naturally
occurring variant thereof with a compound; and (b) determining the
extent of binding of the heterodimer to the compound.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/818,954, filed Mar. 27, 2002, which is a
continuation-in-part of U.S. application Ser. No. 09/723,970, filed
Nov. 27, 2000, which claims the benefit of U.S. Provisional
Application Serial No. 60/199,211, filed Apr. 24, 2000, and U.S.
Provisional Application Serial No. 60/192,654, filed Mar. 28, 2000,
which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a novel beta-like member
(referred to herein as "beta-10" or ".beta.10") of the glycoprotein
hormone family and nucleic acid molecules encoding same. The
invention also relates to a novel heterodimeric glycoprotein
hormone comprising beta-10 and alpha-2 as the subunits. The
invention also relates to vectors, host cells, selective binding
agents, such as antibodies, and methods for producing beta-10
polypeptides and the disclosed beta-10 heterodimer. Also provided
for are methods for the use of beta-10 and the beta-10 heterodimer
and selective beta-10 and beta-10 heterodimer binding agents,
including methods for the diagnosis and treatment of disorders
associated with beta-10 or the beta-10 heterodimer.
BACKGROUND OF THE INVENTION
[0003] As generally accepted in the art, there are currently five
known glycoprotein hormone polypeptides produced in humans:
alpha-subunit, TSH-(thyroid stimulating hormone)-.beta.-subunit,
FSH-(follicle stimulating hormone)-.beta.-subunit, LH-(luteinizing
hormone)-.beta.-subunit, and CG-(chorionic
gonadotropin)-.beta.-subunit; Thotakura and Blithe, Glycobiology,
Volume 5, pages 3-10 (1995); Wondisford et al. in Volume 1,
Endocrinology (edited by L. DeGroot), pages 208-217, W. B. Saunders
Company, Philadelphia, Pa. (1995); Moyle and Campbell, in Volume 1,
Endocrinology (edited by L. DeGroot), pages 230-241, W. B. Saunders
Company, Philadelphia, Pa. (1995). These polypeptides are produced
by single genes, with the exception of the CG-.beta.-subunit which
is encoded by a multigene cluster composed of six homologous
sequences linked to the single LH-.beta.-subunit gene on chromosome
19; Bo and Boime, Journal of Biological Chemistry, vol. 267, pp.
3179-3184 (1992).
[0004] Monomeric alpha-subunit (FAS, or free alpha-subunit) has
hormonal activity and is secreted by the pituitary gland and the
placenta. FAS has been found to play a role in the differentiation
of prolactin producing cells in the pituitary and placenta; see
Begeot et al., Science, vol. 226, pp. 566-568 (1984), Van-Bael and
Denef, Journal of Neuroendocrinology, vol. 8, pp. 99-102 (1996),
and Moy et al., Endocrinology, vol. 137, pp. 1332-1339 (1996); and
also to stimulate placental prolactin secretion; see Blithe et al.,
Endocrinology, vol. 129, pp. 2257-2259 (1991).
[0005] Alpha-subunit also heterodimerizes with each of the four
beta-subunits to form four heterodimeric hormones (TSH, FSH, LH and
CG). TSH, FSH and LH are produced in the pituitary, stored in
secretion granules, and secreted when the appropriate releasing
hormone is produced by the hypothalamus. CG is produced in the
placenta and appears to be secreted constitutively (it is not
stored in secretion granules); see Wondisford et al. in Volume 1,
Endocrinology (ed. L. DeGroot), pp. 208-217, above, and Hall and
Crowley, Jr. in Volume 1, Endocrinology (ed. L. DeGroot), pp.
242-258, W. B. Saunders Company, Philadelphia, Pa. (1995).
[0006] TSH influences basal metabolism by regulating the production
of thyroid hormones and is used clinically for enhancing the
detection and treatment of thyroid carcinoma; see McEvoy, G.(ed.),
AHFS Drug Information, pp. 2041-2042, American Society of
Health-System Pharmacists, Inc., Bethesda, Md. (1998). In addition,
diagnostic tests for measuring TSH levels in the blood are commonly
used for determining the functional status of the thyroid gland
when thyroid gland disorder is suspected.
[0007] FSH and LH play important roles in the maintenance of
reproductive function in males and females (i.e., gonadal
maturation and gonadal steroid production). CG is involved in the
maintenance of pregnancy by stimulating the corpus luteum to
produce steroid hormones during the first trimester. FSH, LH and CG
are used clinically to treat infertility and also as reagents in
assisted reproduction procedures such as in vitro fertilization
(IVF); see McEvoy, G.(ed.), AHFS Drug Information, pp. 2564-2567,
American Society of Health-System Pharmacists, Inc., Bethesda, Md.
(1998). Diagnostic tests for measuring FSH, LH and CG levels are
used for the diagnosis of fertility disorders, as well as to test
for pregnancy.
[0008] Naturally occurring metabolites of the above mentioned
glycoprotein hormone polypeptides have been described, such as the
.beta.-core fragment which is derived from the beta subunit of CG,
but no function has yet been assigned to these metabolites; Moyle
and Campbell in Volume 1 Endocrinology (ed. L. DeGroot) pp.
230-241, above.
[0009] In 1994, the five known glycoprotein hormone polypeptides
were placed into the cystine-knot growth factor structural
superfamily, based on the crystal structure of human CG; Lapthorn
et al., Nature, vol. 369, pp. 455-61 (1994). This superfamily
includes the TGF-.beta. (transforming growth factor beta), NGF
(nerve growth factor) and PDGF (platelet-derived growth factor)
gene families. The cystine-knot is formed by three intramolecular
disulfide bonds, has a very characteristic structure, and is
responsible for the overall three-dimensional structure of all of
the members of the superfamily; Isaacs, Current Opinion in
Structural Biology, vol. 5, pp. 391-395 (1995). A recently
published patent application describes a novel member of the
cystine-knot family (zsig51); Sheppard and Lok,(1999) WIPO patent
application WO99/41377. zsig5 has in fact been determined to be a
new, alpha-like, member of the glycoprotein hormone family and will
thus be referred to here as ".alpha.2" or "alpha-2" [Paszty et al.
(2000) WIPO patent application WO 00/78964].
SUMMARY OF THE INVENTION
[0010] The present invention provides, in part, an isolated
secretable human polypeptide (SEQ ID NO: 1) which is a novel
beta-like member of the glycoprotein hormone family and is herein
designated as "beta-10" or ".beta.10".
[0011] The full length amino acid sequence of human .beta.10 in
accordance with this invention is shown in FIG. 1. The N-terminal
signal peptide predicted for the .beta.10 polypeptide is shown
underlined. The asparagine (N) at position 87 of SEQ ID NO: 1 is
located within a classic NxT glycosylation motif (where x denotes
any amino acid except for proline and T denotes threonine) and is
likely to be glycosylated. The signal peptide cleavage site in the
.beta.10 amino acid sequence is expected to be within the region of
eight amino acids shown boxed in FIG. 1. Signal peptide cleavage at
the site which is most likely to be the authentic in vivo cleavage
site is reflected in the sequence of the "mature" .beta.10
polypeptide (SEQ ID NO: 3).
[0012] The most likely "mature" form (i.e., processed in situ to
remove the signal peptide) of .beta.10 polypeptide was run against
the NonRedundant Protein database using the computer analysis
program known as BLAST to examine homologies (specifically,
commonly occurring or "conserved" amino acid residues) to known
proteins. The top 112 "hits" were found to be various glycoprotein
hormone .beta.-subunits from various mammalian, bird and fish
species. These homologies clearly indicated that .beta.10 is a new
.beta.-like member of the glycoprotein hormone family.
[0013] Further, GAP analysis indicated that the homology of
.beta.10 to the four known human glycoprotein hormone
.beta.-subunits (mentioned above) was 31-37% identity and 42-48%
similarity (see FIG. 2A-D, referred to hereinbelow). The mature
forms of the four known human .beta. glycoprotein hormone
polypeptides contain twelve cysteine residues, which form six
intramolecular disulfide bonds. The mature form of the human
.beta.10 polypeptide of the present invention contains ten cysteine
residues, which are likely to form five intramolecular disulfide
bonds. Using the disulfide bond cysteine pairing of CG-.beta. as a
model, the most likely disulfide bond cysteine pairing for the five
putative disulfide bonds in the .beta.10 polypeptide of this
invention is as follows: C12-C60, C26-C75, C36-C91, C40-C93 and
C96-C103 of SEQ ID NO: 3 (see also FIG. 3).
[0014] The full length amino acid sequence of mouse .beta.10
polypeptide is set forth in SEQ ID NO: 11 and the nucleotide
sequence of the full coding region of the mouse .beta.10 cDNA is
set forth in SEQ ID NO: 12. Signal peptide cleavage at the site
which is most likely to be the authentic in vivo cleavage site is
reflected in the sequence of the "mature" form mouse .beta.10
polypeptide (SEQ ID NO: 13). BestFit analysis indicated that the
amino acid homology of mature form human .beta.10 polypeptide as
compared to mature form mouse .beta.10 polypeptide was 93.4%
identity and 97.2% similarity (see FIG. 4, referred to herein
below).
[0015] Based on the logical inclusion of the .beta.10 polypeptide
of this invention in the glycoprotein hormone family, this
polypeptide could be a monomer (analogous to FAS and .beta.-core
fragment) and/or could form a heterodimer with one or more
glycoprotein hormone family polypeptides (for example heterodimers
.alpha./.beta.10, .beta.10/TSH-.beta.10/LH-.bet- a.). The .beta.10
polypeptide could also form heterodimers with polypeptides which
are distinct from the known glycoprotein hormone polypeptides.
Based on these various possibilities, the .beta.10 polypeptide may
form more than one hormone (i.e., the .beta.10 hormones).
[0016] A heterodimerization assay was used to determine that human
.beta.10 forms a heterodimer with human .alpha.2 polypeptide,
described in the above mentioned WO99/41377 and WO 00/78964 patent
applications, thus discovering and defining a novel heterodimeric
glycoprotein hormone, .alpha.2/.beta.10.
[0017] The general principle of the heterodimerization assay for
secreted proteins, such as the glycoprotein hormones, is
co-transfection of the two distinct genes into mammalian cells,
collection of conditioned media, immunoprecipitation with an
antibody that specifically binds to one of the gene products and
Western blotting of the immunoprecipitate with an antibody that
specifically binds to the other gene product. With the proper
control experiments in place, the presence of a band of the correct
size on the Western would indicate heterodimerization of the two
gene products under the experimental conditions of the assay,
whereas the absence of a band of the correct size on the Western
would indicate that the two gene products did not heterodimerize
under the experimental conditions of the assay. Because the known
heterodimeric glycoprotein hormones (LH, FSH, TSH and CG) can
readily be produced by co-transfection of mammalian cells with the
appropriate genes, this type of mammalian cell based
co-transfection heterodimerization assay is relevant for members of
the glycoprotein hormone family.
[0018] A human .alpha.2-polyHis-tag mammalian expression vector and
a human .beta.10-FLAG-tag mammalian expression vector were
co-transfected into 293 cells and serum free conditioned media was
harvested after 72 hours. Immunoprecipitation was done using
anti-FLAG M2-Agarose affinity beads (Cat# A1205, Sigma, St. Louis,
Mo.). A Western blot of this immunoprecipitate was probed with
affinity purified anti-.alpha.2 rabbit polyclonal antibodies
(example 4) that had been conjugated to Horse Radish Peroxidase
(Linx HRP Rapid Protein Conjugation Kit, cat# K8050-01, Invitrogen
Corp., Carlsbad, Calif.). A strong .alpha.2-polyHis-tag band was
observed using the ECL Western Blot detection kit (cat#RPN 2106,
Amersham Pharmacia Biotech, Piscataway, N.J.). Control experiments
showed that the presence of the strong .alpha.2-polyHis-tag band on
the Western blot was entirely dependent on co-transfection with the
human .beta.10-FLAG-tag mammalian expression vector and the use of
anti-FLAG M2-Agarose affinity beads. No .alpha.2-polyHis-tag band
was observed if either of these 2 components was left out of the
experiment or if plain agarose beads (i.e. without anti-FLAG
antibodies) were used for the immunoprecipitation step.
[0019] Similar to the known heterodimeric glycoprotein hormones
(TSH, FSH, LH and CG) .alpha.2/.beta.10 is a heterodimer of an
alpha-like glycoprotein hormone polypeptide and a beta-like
glycoprotein hormone polypeptide.
[0020] These data also indicate that recombinant, secreted
.alpha.2/.beta.10 heterodimer (without polyHis and FLAG affinity
tags) can be produced in mammalian cells for various therapeutic
and diagnostic utilities as described further below.
[0021] Heterodimeric glycoprotein hormones such as CG can also be
assembled in vitro upon co-incubation of, for example, isolated
alpha-subunit and isolated CG-.beta. subunit under suitable
conditions [see Blithe and Iles, Endocrinology, volume 136, pages
903-910 (1995)]. .alpha.2/.beta.10 heterodimer could similarly be
assembled in vitro upon co-incubation of isolated .alpha.2
polypeptide and isolated .beta.10 polypeptide. Such assembled
.alpha.2/.beta.10 heterodimer could be used for various therapeutic
and diagnostic utilities as described further below.
[0022] Transgenic mice were made that over expressed mouse .alpha.2
alone, mouse .beta.10 alone or the mouse .alpha.2/.beta.10
heterodimer (see example 6). Only those transgenics over expressing
the .alpha.2/.beta.10 heterodimer showed distinct phenotypic
differences as compared to control mice. The .alpha.2/.beta.10
overexpressor transgenic mice exhibited a phenotype characterized
by bilateral thyroid enlargement with multiple follicular papillary
adenomas and resulting hyperthyroidism, as indicated by elevated
serum T4 levels. Other phenotypic changes were felt to be related
to the systemic hyperthyroid state, and included moderate
hepatomegaly, hepatocellular hyperplasia, and slightly decreased
serum cholesterol levels, bilateral renal hypertrophy, and a mild
to moderate leukocytosis with a predominance of lymphocytes (see
example 6). Thus in a normal mouse setting .alpha.2/.beta.10
clearly has a thyroid stimulating hormone (TSH) like activity. Due
to the high level of amino acid conservation between mouse .alpha.2
and human .alpha.2 [88.5% identity and 90.4% similarity for the
predicted mature forms (i.e. without signal peptide)], the high
level of amino acid conservation between mouse .beta.10 and human
.beta.10 [93.4% identity and 97.2% similarity for the predicted
mature forms (i.e. without signal peptide)], and the very high
level of similarity between mouse thyroid gland biology and human
thyroid gland biology, it is anticipated that human
.alpha.2/.beta.10 heterodimer has the same thyroid stimulating
hormone (TSH) like activity as that found for the mouse
.alpha.2/.beta.10 heterodimer. In addition to TSH-like activity,
.alpha.2/.beta.10 may have other, distinct, biological effects in
different physiological settings (i.e., disease conditions), as
described in greater detail further herein.
[0023] TSH influences basal metabolism by regulating the production
of thyroid hormones and is used clinically for enhancing the
detection and treatment of thyroid carcinoma; see McEvoy, G.(ed.),
AHFS Drug Information, pp. 2041-2042, American Society of
Health-System Pharmacists, Inc., Bethesda, Md. (1998). In addition,
diagnostic tests for measuring TSH levels in the blood are commonly
used for determining the functional status of the thyroid gland
when thyroid gland disorder is suspected. It is likely that human
.alpha.2/.beta.10 will have similar clinical utilities as TSH and
will be useful for the treatment and diagnosis of thyroid gland
related diseases and disorders. In addition, human
.alpha.2/.beta.10 may have other therapeutic and diagnostic uses
which are described herein. It is reasonable to surmise that human
.alpha.2/.beta.10 selective binding agents, for example,
antibodies, will have similar clinical utilities to TSH selective
binding agents and will therefore be useful for the treatment and
diagnosis of thyroid gland related diseases and disorders. In
addition, human .alpha.2/.beta.10 selective binding agents may have
other therapeutic and diagnostic uses as described herein.
[0024] This invention also provides for an isolated nucleic acid
molecule comprising a nucleotide sequence selected from the group
consisting of:
[0025] (a) the nucleotide sequence set forth in SEQ ID NO: 2;
[0026] (b) a nucleotide sequence encoding the polypeptide set forth
in SEQ ID NO: 1;
[0027] (c) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of (a) or (b),
wherein the encoded polypeptide, when heterodimerized to human
.alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer; and
[0028] (d) a nucleotide sequence complementary to any of
(a)-(c).
[0029] The invention also provides for an isolated nucleic acid
molecule comprising a nucleotide sequence selected from the group
consisting of:
[0030] (a) a nucleotide sequence encoding a polypeptide that is at
least about 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent
identical to the polypeptide as set forth in SEQ ID NO: 1, wherein
the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer;
[0031] (b) a nucleotide sequence encoding an allelic variant or
splice variant of the nucleotide sequence set forth in SEQ ID NO:
2, wherein the encoded polypeptide, when heterodimerized to human
.alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer;
[0032] (c) a nucleotide sequence of SEQ ID NO: 2, (a), or (b)
encoding a polypeptide fragment of at least about 25 amino acid
residues, wherein the polypeptide, when heterodimerized to human
.alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer;
[0033] (d) a nucleotide sequence of SEQ ID NO: 2 or (a)-(c)
comprising a fragment of at least about 16 nucleotides;
[0034] (e) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of any of (a)-(d),
wherein the encoded polypeptide, when heterodimerized to human
.alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer; and
[0035] (f) a nucleotide sequence complementary to any of
(a)-(d).
[0036] The invention further provides for an isolated nucleic acid
molecule comprising a nucleotide sequence selected from the group
consisting of:
[0037] (a) a nucleotide sequence encoding a polypeptide as set
forth in SEQ ID NO: 1 with at least one conservative amino acid
substitution, wherein the polypeptide, when heterodimerized to
human .alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer;
[0038] (b) a nucleotide sequence encoding a polypeptide as set
forth in SEQ ID NO: 1 with at least one amino acid insertion,
wherein the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer;
[0039] (c) a nucleotide sequence encoding a polypeptide as set
forth in SEQ ID NO: 1 with at least one amino acid deletion,
wherein the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer;
[0040] (d) a nucleotide sequence encoding a polypeptide as set
forth in SEQ ID NO: 1 which has a C- and/or N-terminal truncation,
wherein the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer;
[0041] (e) a nucleotide sequence encoding a polypeptide as set
forth in SEQ ID NO: 1 with at least one modification selected from
the group consisting of amino acid substitutions, amino acid
insertions, amino acid deletions, C-terminal truncation, and
N-terminal truncation, wherein the polypeptide, when
heterodimerized to human .alpha.2 polypeptide, has an activity of
the human .alpha.2/.beta.10 heterodimer;
[0042] (f) a nucleotide sequence of (a)-(e) comprising a fragment
of at least about 16 nucleotides;
[0043] (g) a nucleotide sequence which hybridizes under moderately
or highly stringent conditions to the complement of any of (a)-(f),
wherein the encoded polypeptide, when heterodimerized to human
.alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer; and
[0044] (h) a nucleotide sequence complementary to any of
(a)-(e).
[0045] The invention also provides for an isolated polypeptide
comprising the amino acid sequence selected from the group
consisting of:
[0046] (a) the mature amino acid sequence set forth in SEQ ID NO:
3, and optionally further comprising an amino-terminal
methionine;
[0047] (b) an amino acid sequence for an ortholog of SEQ ID NO: 3,
wherein the encoded polypeptide, when heterodimerized to human
.alpha.2 polypeptide, has an activity of the human
.alpha.2/.beta.10 heterodimer;
[0048] (c) an amino acid sequence that is at least about 70, 75,
80, 85, 90, 95, 96, 97, 98, or 99 percent identical to the amino
acid sequence of SEQ ID NO: 3, wherein the polypeptide, when
heterodimerized to human .alpha.2 polypeptide, has an activity of
the human .alpha.2/.beta.10 heterodimer;
[0049] (d) a fragment of the amino acid sequence set forth in SEQ
ID NO: 3 comprising at least about 25 amino acid residues, wherein
the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer;
[0050] (e) an amino acid sequence for an allelic variant or splice
variant of either the amino acid sequence as set forth in SEQ ID
NO: 3, or at least one of (a)-(c) wherein the polypeptide, when
heterodimerized to human .alpha.2 polypeptide, has an activity of
the human .alpha.2/.beta.10 heterodimer.
[0051] The invention further provides for an isolated polypeptide
comprising the amino acid sequence selected from the group
consisting of:
[0052] (a) the amino acid sequence as set forth in SEQ ID NO: 3
with at least one conservative amino acid substitution, wherein the
polypeptide, when heterodimerized to human .alpha.2 polypeptide,
has an activity of the human .alpha.2/.beta.10 heterodimer;
[0053] (b) the amino acid sequence as set forth in SEQ ID NO: 3
with at least one amino acid insertion, wherein the polypeptide,
when heterodimerized to human .alpha.2 polypeptide, has an activity
of the human .alpha.2/.beta.10 heterodimer;
[0054] (c) the amino acid sequence as set forth in SEQ ID NO: 3
with at least one amino acid deletion, wherein the polypeptide,
when heterodimerized to human .alpha.2 polypeptide, has an activity
of the human .alpha.2/.beta.10 heterodimer;
[0055] (d) the amino acid sequence as set forth in SEQ ID NO: 3
which has a C- and/or N-terminal truncation, wherein the
polypeptide, when heterodimerized to human .alpha.2 polypeptide,
has an activity of the human .alpha.2/.beta.10 heterodimer; and
[0056] (e) the amino acid sequence as set forth in SEQ ID NO: 3,
with at least one modification selected from the group consisting
of amino acid substitutions, amino acid insertions, amino acid
deletions, C-terminal truncation, and N-terminal truncation,
wherein the polypeptide, when heterodimerized to human .alpha.2
polypeptide, has an activity of the human .alpha.2/.beta.10
heterodimer.
[0057] Also provided are fusion polypeptides comprising the amino
acid sequences of (a)-(e) above.
[0058] The present invention also provides for an expression vector
comprising the isolated nucleic acid molecules as set forth herein,
recombinant host cells comprising recombinant nucleic acid
molecules as set forth herein, and a method of producing a .beta.10
polypeptide or an .alpha.2/.beta.10 heterodimer of this invention
comprising culturing the host cells and optionally isolating the
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer so
produced.
[0059] A transgenic non-human animal comprising a nucleic acid
molecule(s) encoding a .beta.10 polypeptide or
.alpha.2/.beta.10heterodimer of this invention is also encompassed
by the invention. The nucleic acid molecules are introduced into
the animal in a manner that allows expression and increased levels
of .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer, which may
include increased circulating levels. The transgenic non-human
animal is preferably a mammal.
[0060] Also provided are derivatives of the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer of the present invention.
[0061] Additionally provided are selective binding agents such as
antibodies and peptides capable of specifically binding the
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer of the
invention. Such antibodies and peptides may be agonistic or
antagonistic to an activity of the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer.
[0062] Pharmaceutical compositions comprising the nucleotides,
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer, or selective
binding agents of the present invention and one or more
pharmaceutically acceptable formulation agents are also encompassed
by the invention. The pharmaceutical compositions are used to
provide therapeutically effective amounts of the nucleotides or
polypeptides of the present invention. The invention is also
directed to methods of using the nucleic acid molecules, .beta.10
polypeptide, .alpha.2/.beta.10 heterodimer and selective binding
agents.
[0063] The nucleic acid molecules, .beta.10 polypeptide,
.alpha.2/.beta.10 heterodimer and selective binding agents of the
present invention may be used to treat, prevent, ameliorate, and/or
detect diseases and disorders, including those recited herein.
[0064] The present invention also provides a method of assaying
test molecules to identify a test molecule which binds to a
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer. The method
comprises contacting the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer with a test molecule and determining the extent of
binding of the test molecule to the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer. The method further comprises
determining whether such test molecules are agonists or antagonists
of the .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer. The
present invention further provides a method of testing the impact
of molecules on the expression of the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer or on the activity of the .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer.
[0065] Methods of regulating expression and modulating (i.e.,
increasing or decreasing) levels of a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer of this invention are also
encompassed by the invention. One method comprises administering to
an animal a nucleic acid molecule(s) encoding such a .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer. In another method, a
nucleic acid molecule comprising elements that regulate or modulate
expression of the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer of this invention may be administered. Examples of
these methods include gene therapy, cell therapy, and anti-sense
therapy as further described herein.
BRIEF DESCRIPTION OF THE FIGURES
[0066] FIG. 1 depicts in linear array the full coding region of
human .beta.10 polypeptide in accordance with this invention (SEQ
ID NO: 1). The predicted signal peptide region is underlined and
the region containing the predicted signal peptide cleavage site is
boxed. The asparagine (N) residue that is located within the
classic NxT glycosylation motif, and which is very likely to be
glycosylated, is shown in larger font. The corresponding nucleic
acid sequence which encodes this polypeptide (SEQ ID NO: 2)
comprises nucleotides 1-390, inclusive, of the nucleic acid
sequence shown in this Figure.
[0067] FIG. 2A-2D illustrates the relatedness of the known human
glycoprotein hormone .beta.-subunit polypeptides (prior art) and
the .beta.10 polypeptide of this invention. The mature form of
.beta.10 used for these comparisons (SEQ ID NO: 3) most likely
represents the authentic in vivo form of .beta.10 polypeptide.
FIGS. 2A-D comprise the GAP output showing the amino acid homology
between the mature form of .beta.10 and respectively, TSH-(thyroid
stimulating hormone)-.beta.-subunit, FSH-(follicle stimulating
hormone)-.beta.-subunit, LH-(luteinizing hormone)-.beta.-subunit,
and CG-(chorionic gonadotropin)-.beta.-subunit.
[0068] FIG. 3 shows the likely disulfide bond cysteine (C) pairs of
the five putative disulfide bonds in the most likely mature form of
human .beta.10 (SEQ ID NO: 3). The ten cysteine residues are shown
in large font and the disulfide bonds are drawn as solid lines. The
three disulfide bonds (C12-C60, C36-C91, C40-C93) that form the
cystine-knot are drawn above the amino acid sequence, and the two
additional disulfide bonds (C26-C75, C96-C103) are drawn below the
amino acid sequence.
[0069] FIG. 4 is the BestFit output showing the amino acid homology
between the mature form of human .beta.10 and the mature form of
mouse .beta.10. The mature form of human .beta.10 used for this
comparison (SEQ ID NO: 3) most likely represents the authentic in
vivo form of human .beta.10 polypeptide. The mature form of mouse
.beta.10 used for this comparison (SEQ ID NO: 13) most likely
represents the authentic in vivo form of mouse .beta.10
polypeptide.
DETAILED DESCRIPTION OF THE INVENTION
[0070] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All references cited in this application are
expressly incorporated by reference herein.
Definitions
[0071] The terms ".beta.10 gene" or ".beta.10 nucleic acid
molecule" or "polynucleotide" refers to a nucleic acid molecule
comprising or consisting of a nucleotide sequence as set forth in
SEQ ID NO: 2, a nucleotide sequence encoding the polypeptide as set
forth in SEQ ID NO: 1, a nucleotide of the DNA insert in ATCC
deposit no. PTA-1210, and nucleic acid molecules as defined
herein.
[0072] The term ".beta.10 polypeptide" refers to a polypeptide
comprising the amino acid sequence of SEQ ID NO: 3, and related
polypeptides. Related polypeptides include: .beta.10 polypeptide
allelic variants, .beta.10 polypeptide orthologs, .beta.10
polypeptide splice variants, .beta.10 polypeptide variants and
.beta.10 polypeptide derivatives. .beta.10 polypeptides may be
mature polypeptides, as defined herein, and may or may not have an
amino terminal methionine residue, depending on the method by which
they are prepared.
[0073] The term ".beta.10 polypeptide allelic variant" refers to
one of several possible naturally occurring alternate forms of a
gene occupying a given locus on a chromosome of an organism or a
population of organisms.
[0074] The term ".beta.10 polypeptide derivatives" refers to the
polypeptide set forth in SEQ ID NO: 3, polypeptide allelic variants
thereof, polypeptide orthologs thereof, polypeptide splice variants
thereof, or polypeptide variants thereof, as defined herein, that
have been chemically modified.
[0075] The term ".beta.10 polypeptide fragment" refers to a
polypeptide that comprises a truncation at the amino terminus (with
or without a leader sequence) and/or a truncation at the carboxy
terminus of the polypeptide set forth in SEQ ID NO: 3, polypeptide
allelic variants thereof, polypeptide orthologs thereof,
polypeptide splice variants thereof and/or a polypeptide variant
thereof having one or more amino acid additions or substitutions or
internal deletions (wherein the resulting polypeptide is at least 6
amino acids or more in length) as compared to the .beta.10
polypeptide amino acid sequence set forth in SEQ ID NO: 3.
Polypeptide fragments may result from alternative RNA splicing or
from in vivo protease activity. In preferred embodiments,
truncations comprise about 10 amino acids, or about 20 amino acids,
or about 50 amino acids, or about 75 amino acids, or about 100
amino acids, or more than about 100 amino acids. The polypeptide
fragments so produced will comprise about 25 contiguous amino
acids, or about 50 amino acids, or about 75 amino acids, or about
100 amino acids, or about 150 amino acids, or about 200 amino
acids. Such polypeptide fragments may optionally comprise an amino
terminal methionine residue. It will be appreciated that such
fragments can be used, for example, to generate antibodies to
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer.
[0076] The term ".beta.10 fusion polypeptide" refers to a fusion of
one or more amino acids (such as a heterologous peptide or
polypeptide) at the amino or carboxy terminus of the polypeptide
set forth in SEQ ID NO: 3, polypeptide allelic variants,
polypeptide orthologs, polypeptide splice variants, or polypeptide
variants having one or more amino acid deletions, substitutions or
internal additions as compared to the .beta.10 polypeptide amino
acid sequence set forth in SEQ ID NO: 3.
[0077] The term ".beta.10 polypeptide ortholog" refers to a
polypeptide from another species that corresponds to the .beta.10
polypeptide amino acid sequence set forth in SEQ ID NO: 3. For
example, mouse and human .beta.10 polypeptides are considered
orthologs of each other.
[0078] The term ".beta.10 polypeptide splice variant" refers to a
nucleic acid molecule, usually RNA, which is generated by
alternative processing of intron sequences in an RNA transcript of
the .beta.10 polypeptide amino acid sequence set forth in SEQ ID
NO: 3.
[0079] The term ".beta.10 polypeptide variants" refers to
.beta.10-like polypeptides comprising amino acid sequences having
one or more amino acid sequence substitutions, deletions (such as
internal deletions and/or polypeptide fragments), and/or additions
(such as internal additions and/or fusion polypeptides) as compared
to the .beta.10 polypeptide amino acid sequence set forth in SEQ ID
NO: 3. Variants may be naturally occurring (e.g., .beta.10-like
polypeptide allelic variants, polypeptide orthologs and polypeptide
splice variants) or artificially constructed. Such polypeptide
variants may be prepared from the corresponding nucleic acid
molecules having a DNA sequence that varies accordingly from the
DNA sequence set forth in SEQ ID NO: 2. In preferred embodiments,
the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or
from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or
from 1 to 75, or from 1 to 100, or more than 100 amino acid
substitutions, insertions, additions and/or deletions, wherein the
substitutions may be conservative, or non-conservative, or any
combination thereof.
[0080] The term ".alpha.2/.beta.10 heterodimer" refers to a
heterodimer of the .beta.10 polypeptide and the .alpha.2
polypeptide.
[0081] The term "antigen" refers to a molecule or a portion of a
molecule capable of being bound by a selective binding agent, such
as an antibody, and additionally capable of being used in an animal
to produce antibodies capable of binding to an epitope of that
antigen. An antigen may have one or more epitopes.
[0082] The term "biologically active .beta.10 polypeptides" refers
to .beta.10-like polypeptides that, when heterodimerized to human
.alpha.2 polypeptide, have an activity of the human
.alpha.2/.beta.10 heterodimer.
[0083] The terms "effective amount" and "therapeutically effective
amount" each refer to the amount of a .beta.10 nucleic acid
molecule or a .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
of this invention used to support an observable level of one or
more biological activities of the .alpha.2/.beta.10 heterodimer
described herein.
[0084] The term "expression vector" refers to a vector which is
suitable for use in a host cell and contains nucleic acid sequences
which direct and/or control the expression of heterologous nucleic
acid sequences. Expression includes, but is not limited to,
processes such as transcription, translation, and RNA splicing, if
introns are present.
[0085] The term "host cell" is used to refer to a cell which has
been transformed, or is capable of being transformed with a nucleic
acid sequence and then of expressing a selected gene of interest.
The term includes the progeny of the parent cell, whether or not
the progeny is identical in morphology or in genetic make-up to the
original parent, so long as the selected gene is present.
[0086] The term "identity" as known in the art, refers to a
relationship between the sequences of two or more polypeptide
molecules or two or more nucleic acid molecules, as determined by
comparing the sequences. In the art, "identity" also means the
degree of sequence relatedness between nucleic acid molecules or
polypeptides, as the case may be, as determined by the match
between strings of two or more nucleotide or two or more amino acid
sequences. "Identity" measures the percent of identical matches
between the smaller of two or more sequences with gap alignments
(if any) addressed by a particular mathematical model or computer
program (i.e., "algorithms").
[0087] The term "similarity" is a related concept, but in contrast
to "identity", refers to a measure of similarity which includes
both identical matches and conservative substitution matches. If
two polypeptide sequences have, for example, 10/20 identical amino
acids, and the remainder are all non-conservative substitutions,
then the percent identity and similarity would both be 50%. If in
the same example, there are 5 more positions where there are
conservative substitutions, then the percent identity remains 50%,
but the per cent similarity would be 75% (15/20). Therefore, in
cases where there are conservative substitutions, the degree of
similarity between two polypeptides will be higher than the percent
identity between those two polypeptides.
[0088] The term "isolated nucleic acid molecule" refers to a
nucleic acid molecule of the invention that is free from at least
one contaminating nucleic acid molecule with which it is naturally
associated Preferably, the isolated nucleic acid molecule of the
present invention is substantially free from any other
contaminating nucleic acid molecule(s) or other contaminants that
are found in its natural environment which would interfere with its
use in polypeptide production or its therapeutic, diagnostic,
prophylactic or research use.
[0089] The term "isolated polypeptide" refers to a polypeptide of
the present invention that is free from at least one contaminating
polypeptide or other contaminants that are found in its natural
environment. Preferably, the isolated polypeptide is substantially
free from any other contaminating polypeptides or other
contaminants that are found in its natural environment which would
interfere with its therapeutic, diagnostic, prophylactic or
research use.
[0090] The term "mature .beta.10 polypeptide" refers to a .beta.10
polypeptide lacking a leader sequence. A mature .beta.10
polypeptide may also include other modifications such as
proteolytic processing of the amino terminus (with or without a
leader sequence) and/or the carboxy terminus, cleavage of a smaller
polypeptide from a larger precursor, N-linked and/or O-linked
glycosylation, and the like.
[0091] The term "nucleic acid sequence" or "nucleic acid molecule"
refers to a DNA or RNA sequence. The term encompasses molecules
formed from any of the known base analogs of DNA and RNA such as,
but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,
aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,
N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyamino-methyl-2-thiou- racil,
beta-D-mannosylqueosine, 5-methoxycarbonyl-methyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
[0092] The term "naturally occurring" or "native" when used in
connection with biological materials such as nucleic acid
molecules, polypeptides, host cells, and the like, refers to
materials which are found in nature and are not manipulated by man.
Similarly, "non-naturally occurring" or "non-native" as used herein
refers to a material that is not found in nature or that has been
structurally modified or synthesized by man.
[0093] The term "operably linked" is used herein to refer to an
arrangement of flanking sequences wherein the flanking sequences so
described are configured or assembled so as to perform their usual
function. Thus, a flanking sequence operably linked to a coding
sequence may be capable of effecting the replication, transcription
and/or translation of the coding sequence. For example, a coding
sequence is operably linked to a promoter when the promoter is
capable of directing transcription of that coding sequence. A
flanking sequence need not be contiguous with the coding sequence,
so long as it functions correctly. Thus, for example, intervening
untranslated yet transcribed sequences can be present between a
promoter sequence and the coding sequence and the promoter sequence
can still be considered "operably linked" to the coding
sequence.
[0094] The term "pharmaceutically acceptable carrier" or
"physiologically acceptable carrier" as used herein refers to one
or more formulation materials suitable for accomplishing or
enhancing the delivery of a .beta.10 nucleic acid molecule,
.beta.10 polypeptide, .alpha.2/.beta.10 heterodimer or selective
binding agents of the present invention as a pharmaceutical
composition.
[0095] The term "selective binding agent" refers to a molecule or
molecules having specificity for the .beta.10 polypeptide and/or
.alpha.2/.beta.10 heterodimer of this invention. As used herein,
the terms, "specific" and "specificity" refer to the ability of the
selective binding agents to bind to human .beta.10 polypeptide
and/or .alpha.2/.beta.10 heterodimer and not to bind to human
non-.beta.10 polypeptide and/or non-.alpha.2/.beta.10 heterodimer.
It will be appreciated, however, that the selective binding agents
may also bind orthologs of the polypeptide set forth in SEQ ID NO:
3, and/or orthologs of human .alpha.2/.beta.10 heterodimer, that
is, interspecies versions thereof, such as mouse and rat
polypeptides.
[0096] The term "transduction" is used to refer to the transfer of
genes from one bacterium to another, usually by a phage.
"Transduction" also refers to the acquisition and transfer of
eukaryotic cellular sequences by retroviruses.
[0097] The term "transfection" is used to refer to the uptake of
foreign or exogenous DNA by a cell, and a cell has been
"transfected" when the exogenous DNA has been introduced inside the
cell membrane. A number of transfection techniques are well known
in the art and are disclosed herein. See, for example, Graham et
al., Virology, 52:456 (1973); Sambrook et al., Molecular Cloning, a
laboratory Manual, Cold Spring Harbor Laboratories (New York,
1989); Davis et al., Basic Methods in Molecular Biology, Elsevier,
1986; and Chu et al., Gene, 13:197 (1981). Such techniques can be
used to introduce one or more exogenous DNA moieties into suitable
host cells.
[0098] The term "transformation" as used herein refers to a change
in a cell's genetic characteristics, and a cell has been
transformed when it has been modified to contain a new DNA. For
example, a cell is transformed where it is genetically modified
from its native state. Following transfection or transduction, the
transforming DNA may recombine with that of the cell by physically
integrating into a chromosome of the cell, may be maintained
transiently as an episomal element without being replicated, or may
replicate independently as a plasmid. A cell is considered to have
been stably transformed when the DNA is replicated with the
division of the cell.
[0099] The term "vector" is used to refer to any molecule (e.g.,
nucleic acid, plasmid, or virus) used to transfer coding
information to a host cell.
Relatedness of Nucleic Acid Molecules and/or Polypeptides
[0100] It is understood that related nucleic acid molecules include
allelic or splice variants of the nucleic acid molecule of SEQ ID
NO: 2, and include sequences which are complementary to any of the
above nucleotide sequences. Related nucleic acid molecules also
include a nucleotide sequence encoding a polypeptide comprising or
consisting essentially of a substitution, modification, addition
and/or a deletion of one or more amino acid residues compared to
the polypeptide in SEQ ID NO: 1.
[0101] Fragments include nucleic acid molecules which encode a
polypeptide of at least about 25 amino acid residues, or about 50,
or about 75, or about 100, or greater than about 100 amino acid
residues of the polypeptide of SEQ ID NO: 1.
[0102] In addition, related .beta.10 nucleic acid molecules include
those molecules which comprise nucleotide sequences which hybridize
under moderately or highly stringent conditions as defined herein
with the fully complementary sequence of the nucleic acid molecule
of SEQ ID NO: 2, or of a molecule encoding a polypeptide, which
polypeptide comprises the amino acid sequence of SEQ ID NO: 1, or
of a nucleic acid fragment as defined herein, or of a nucleic acid
fragment encoding a polypeptide as defined herein. Hybridization
probes may be prepared using the .beta.10 sequences provided herein
to screen cDNA, genomic or synthetic DNA libraries for related
sequences. Regions of the DNA and/or amino acid sequence of the
.beta.10 polypeptide that exhibit significant identity to known
sequences are readily determined using sequence alignment
algorithms as described herein and those regions may be used to
design probes for screening.
[0103] The term "highly stringent conditions" refers to those
conditions that are designed to permit hybridization of DNA strands
whose sequences are highly complementary, and to exclude
hybridization of significantly mismatched DNAs. Hybridization
stringency is principally determined by temperature, ionic
strength, and the concentration of denaturing agents such as
formamide. Examples of "highly stringent conditions" for
hybridization and washing are 0.015M sodium chloride, 0.0015M
sodium citrate at 65-68.degree. C. or 0.015M sodium chloride,
0.0015M sodium citrate, and 50% formamide at 42.degree. C. See
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, 2.sup.nd Ed., Cold Spring Harbor Laboratory, (Cold Spring
Harbor, N.Y. 1989); Anderson et al., Nucleic Acid Hybridisation: a
practical approach, Ch. 4, IRL Press Limited (Oxford, England).
[0104] More stringent conditions (such as higher temperature, lower
ionic strength, higher formamide, or other denaturing agent) may
also be used, however, the rate of hybridization will be affected.
Other agents may be included in the hybridization and washing
buffers for the purpose of reducing non-specific and/or background
hybridization. Examples are 0.1% bovine serum albumin, 0.1%
polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium
dodecylsulfate (NaDodSO.sub.4 or SDS), ficoll, Denhardt's solution,
sonicated salmon sperm DNA (or other non-complementary DNA), and
dextran sulfate, although other suitable agents can also be used.
The concentration and types of these additives can be changed
without substantially affecting the stringency of the hybridization
conditions. Hybridization experiments are usually carried out at pH
6.8-7.4, however, at typical ionic strength conditions, the rate of
hybridization is nearly independent of pH. See Anderson et al.,
Nucleic Acid Hybridisation: a Practical Approach, Ch. 4, IRL Press
Limited (Oxford, England).
[0105] Factors affecting the stability of a DNA duplex include base
composition, length, and degree of base pair mismatch.
Hybridization conditions can be adjusted by one skilled in the art
in order to accommodate these variables and allow DNAs of different
sequence relatedness to form hybrids. The melting temperature of a
perfectly matched DNA duplex can be estimated by the following
equation:
T.sub.m(.degree.
C.)=81.5+16.6(log[Na+])+0.41(%G+C)-600/N-0.72(%formamide)
[0106] where N is the length of the duplex formed, [Na+] is the
molar concentration of the sodium ion in the hybridization or
washing solution, %G+C is the percentage of (guanine+cytosine)
bases in the hybrid. For imperfectly matched hybrids, the melting
temperature is reduced by approximately 1.degree. C. for each 1%
mismatch.
[0107] The term "moderately stringent conditions" refers to
conditions under which a DNA duplex with a greater degree of base
pair mismatching than could occur under "highly stringent
conditions" is able to form. Examples of typical "moderately
stringent conditions" are 0.015M sodium chloride, 0.0015M sodium
citrate at 50-65.degree. C. or 0.015M sodium chloride, 0.0015M
sodium citrate, and 20% formamide at 37-50.degree. C. By way of
example, a "moderately stringent" condition of 50.degree. C. in
0.015 M sodium ion will allow about a 21% mismatch.
[0108] It will be appreciated by those skilled in the art that
there is no absolute distinction between "highly" and "moderately"
stringent conditions. For example, at 0.015M sodium ion (no
formamide), the melting temperature of perfectly matched long DNA
is about 71.degree. C. With a wash at 65.degree. C. (at the same
ionic strength), this would allow for approximately a 6% mismatch.
To capture more distantly related sequences, one skilled in the art
can simply lower the temperature or raise the ionic strength.
[0109] A good estimate of the melting temperature in 1M NaCl* for
oligonucleotide probes up to about 20 nucleotides is given by:
Tm=2.degree. C. per A-T base pair+4.degree. C. per G-C base
pair
[0110] *The sodium ion concentration in 6.times. salt sodium
citrate (SSC) is 1M. See Suggs et al., Developmental Biology Using
Purified Genes, p. 683, Brown and Fox (eds.) (1981).
[0111] High stringency washing conditions for oligonucleotides are
usually at a temperature of 0-5.degree. C. below the Tm of the
oligonucleotide in 6.times.SSC, 0.1% SDS.
[0112] In another embodiment, related nucleic acid molecules
comprise or consist of a nucleotide sequence that is about 70
percent identical to the nucleotide sequence of SEQ ID NO: 2, or
comprise or consist essentially of a nucleotide sequence encoding a
polypeptide that is about 70 percent identical to the polypeptide
of SEQ ID NO: 1. In preferred embodiments, the nucleotide sequences
are about 70 percent, 75 percent, 80 percent, or about 85 percent,
or about 90 percent, or about 95, 96, 97, 98, or 99 percent
identical to the nucleotide sequence as shown in SEQ ID NO: 2, or
the nucleotide sequences encode a polypeptide that is about 70
percent, 75 percent, 80 percent, or about 85 percent, or about 90
percent, or about 95, 96, 97, 98, or 99 percent identical to the
polypeptide sequence as set forth in SEQ ID NO: 1.
[0113] Differences in the nucleic acid sequence may result in
conservative and/or non-conservative modifications of the amino
acid sequence relative to the amino acid sequence of SEQ ID NO:
1.
[0114] Conservative modifications to the amino acid sequence of SEQ
ID NO: 1 (and the corresponding modifications to the encoding
nucleotides) will produce .beta.10-like polypeptides in accordance
with this invention having functional and chemical characteristics
similar to those of the naturally occurring .beta.10 polypeptide
hereof. In contrast, substantial modifications in the functional
and/or chemical characteristics of the .beta.10 polypeptide may be
accomplished by selecting substitutions in the amino acid sequence
of SEQ ID NO: 1 that differ significantly in their effect on
maintaining (a) the structure of the molecular backbone in the area
of the substitution, for example, as a sheet or helical
conformation, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain.
[0115] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
nonnative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis."
[0116] Conservative amino acid substitutions also encompass
non-naturally occurring amino acid residues which are typically
incorporated by chemical peptide synthesis rather than by synthesis
in biological systems. These include peptidomimetics, and other
reversed or inverted forms of amino acid moieties.
[0117] Naturally occurring residues may be divided into classes
based on common side chain properties:
[0118] 1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;
[0119] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0120] 3) acidic: Asp, Glu;
[0121] 4) basic: His, Lys, Arg;
[0122] 5) residues that influence chain orientation: Gly, Pro;
and
[0123] 6) aromatic: Trp, Tyr, Phe.
[0124] For example, non-conservative substitutions may involve the
exchange of a member of one of these classes for a member from
another class. Such substituted residues may be introduced into
regions of the human .beta.10 polypeptide that are homologous with
non-human .beta.10 polypeptide orthologs, or into the
non-homologous regions of the molecule.
[0125] In making such changes, the hydropathic index of amino acids
may be considered. Each amino acid has been assigned a hydropathic
index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0126] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art. Kyte et al., J. Mol. Biol., 157:105-131
(1982). It is known that certain amino acids may be substituted for
other amino acids having a similar hydropathic index or score and
still retain a similar biological activity. In making changes based
upon the hydropathic index, the substitution of amino acids whose
hydropathic indices are within .+-.2 is preferred, those which are
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
[0127] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functionally
equivalent protein or peptide thereby created is intended for use
in immunological embodiments, as in the present case. The greatest
local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, i.e., with a biological property
of the protein.
[0128] The following hydrophilicity values have been assigned to
amino acid residues: arginine (+3.0); lysine (+3.0); aspartate
(+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline
(-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
In making changes based upon similar hydrophilicity values, the
substitution of amino acids whose hydrophilicity values are within
.+-.2 is preferred, those which are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred. One may also identify epitopes from primary amino acid
sequences on the basis of hydrophilicity. These regions are also
referred to as "epitopic core regions."
[0129] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of the
.beta.10 polypeptide or to increase or decrease the affinity of the
.beta.10 polypeptides or the .alpha.2/.beta.10 heterodimers
described herein.
[0130] Exemplary amino acid substitutions are set forth in Table
I.
1TABLE I Amino Acid Substitutions Original Exemplary Preferred
Residues Substitutions Substitutions Ala Val, Leu, Ile Val Arg Lys,
Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn
Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu,
Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile Ile Val,
Met, Ala, Phe Lys Arg, Arg 1,4 Diaminobutyric Acid, Gln, Asn Met
Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Leu Tyr Pro Ala Gly Ser
Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr,
Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine
[0131] A skilled artisan will be able to determine suitable
variants of the polypeptides of SEQ ID NOs: 1 or 3 using well known
techniques. For identifying suitable areas of the molecule that may
be changed without destroying activity, one skilled in the art may
target areas not believed to be important for activity. For
example, when similar polypeptides with similar activities from the
same species or from other species are known, one skilled in the
art may compare the amino acid sequence of a .beta.10 polypeptide
to such similar polypeptides With such a comparison, one can
identify residues and portions of the molecules that are conserved
among similar polypeptides. It will be appreciated that changes in
areas of a .beta.10 polypeptide that are not conserved relative to
such similar polypeptides would be less likely to adversely affect
the biological activity and/or structure of the .beta.10
polypeptide or the .alpha.2/.beta.10 heterodimer. One skilled in
the art would also know that, even in relatively conserved regions,
one may substitute chemically similar amino acids for the naturally
occurring residues while retaining activity (conservative amino
acid residue substitutions). Therefore, even areas that may be
important for biological activity or for structure may be subject
to conservative amino acid substitutions without destroying the
biological activity or without adversely affecting the polypeptide
structure.
[0132] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar
polypeptides that are important for activity or structure. In view
of such a comparison, one can predict the importance of amino acid
residues in a .beta.10 polypeptide that correspond to amino acid
residues that are important for activity or structure in similar
polypeptides. One skilled in the art may opt for chemically similar
amino acid substitutions for such predicted important amino acid
residues of the .beta.10 polypeptide.
[0133] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of that
information, one skilled in the art may predict the alignment of
amino acid residues of a .beta.10 polypeptide with respect to its
three dimensional structure. One skilled in the art may choose not
to make radical changes to amino acid residues predicted to be on
the surface of the protein, since such residues may be involved in
important interactions with other molecules. Moreover, one skilled
in the art may generate test variants containing a single amino
acid substitution at each desired amino acid residue. The variants
can then be screened using activity assays know to those skilled in
the art. Such variants could be used to gather information about
suitable variants. For example, if one discovered that a change to
a particular amino acid residue resulted in destroyed, undesirably
reduced, or unsuitable activity, variants with such a change would
be avoided. In other words, based on information gathered from such
routine experiments, one skilled in the art can readily determine
the amino acids where further substitutions should be avoided
either alone or in combination with other mutations.
[0134] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4):422-427 (1996), Chou et al., Biochemistry,
13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222
(1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol.,
47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and
Chou et al., Biophys. J., 26:367-384 (1979). Moreover, computer
programs are currently available to assist with predicting
secondary structure. One method of predicting secondary structure
is based upon homology modeling. For example, two polypeptides or
proteins which have a sequence identity of greater than 30%, or
similarity greater than 40% often have similar structural
topologies. The recent growth of the protein structural data base
(PDB) has provided enhanced predictability of secondary structure,
including the potential number of folds within a polypeptide's or
protein's structure. See Holm et al., Nucl. Acid. Res.,
27(1):244-247 (1999). It has been suggested (Brenner et al., Curr.
Op. Struct. Biol., 7(3):369-376 (1997)) that there are a limited
number of folds in a given polypeptide or protein and that once a
critical number of structures have been resolved, structural
prediction will gain dramatically in accuracy.
[0135] Additional methods of predicting secondary structure include
"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87
(1997); Sippl et al., Structure, 4(1):15-9 (1996)), "profile
analysis" (Bowie et al., Science, 253:164-170 (1991); Gribskov et
al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat.
Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage"
(See Home, supra, and Brenner, supra).
[0136] Preferred .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer variants in accordance with this invention include
glycosylation variants wherein the number and/or type of
glycosylation sites has been altered compared to the amino acid
sequence set forth in SEQ ID NO: 1. In one embodiment, 10
polypeptide or .alpha.2/.beta.10 heterodimer variants according to
this invention comprise a greater or a lesser number of N-linked
glycosylation sites than the amino acid sequence set forth in SEQ
ID NO: 1. An N-linked glycosylation site is characterized by the
sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue
designated as X may be any amino acid residue except proline. The
substitution(s) of amino acid residues to create this sequence
provides a potential new site for the addition of an N-linked
carbohydrate chain. Alternatively, substitutions which eliminate
this sequence will remove an existing N-linked carbohydrate chain.
Also provided is a rearrangement of N-linked carbohydrate chains
wherein one or more N-linked glycosylation sites (typically those
that are naturally occurring) are eliminated and one or more new
N-linked sites are created. Additional preferred .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer variants include
cysteine variants, wherein one or more cysteine residues are
deleted from or substituted for another amino acid (e.g., serine)
as compared to the amino acid sequence set forth in SEQ ID NO: 1.
Cysteine variants are useful when the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer must be refolded into a biologically
active conformation such as after the isolation of insoluble
inclusion bodies. Cysteine variants generally have fewer cysteine
residues than the native protein, and typically have an even number
to minimize interactions resulting from unpaired cysteines.
[0137] In addition, the polypeptide comprising the amino acid
sequence of SEQ ID NO: 3 or a polypeptide variant thereof may be
fused to a heterologous polypeptide, such as but not limited to
.alpha.2, to form a heterodimer. Heterologous peptides and
polypeptides include, but are not limited to: an epitope to allow
for the detection and/or isolation of a .beta.10 fusion polypeptide
or an .alpha.2/.beta.10 heterodimer; a transmembrane receptor
protein or a portion thereof, such as an extracellular domain, or a
transmembrane and intracellular domain; a ligand or a portion
thereof which binds to a transmembtane receptor protein; an enzyme
or portion thereof which is catalytically active; a polypeptide or
peptide which promotes oligomerization, such as a leucine zipper
domain; a polypeptide with which .beta.10 normally dimerizes, such
as .alpha.2; a polypeptide or peptide which increases stability,
such as an immunoglobulin constant region; and a polypeptide which
has a therapeutic activity different from the polypeptide
comprising the amino acid sequence set forth in SEQ ID NO: 3 or a
polypeptide variant thereof.
[0138] Fusions can be made either at the amino terminus or at the
carboxy terminus of the polypeptide comprising the amino acid
sequence set forth in SEQ ID NO: 3 or a polypeptide variant.
Fusions may be direct with no linker or adapter molecule or
indirect using a linker or adapter molecule. A linker or adapter
molecule may be one or more amino acid residues, typically up to
about 20 to about 50 amino acid residues. A linker or adapter
molecule may also be designed with a cleavage site for a DNA
restriction endonuclease or for a protease to allow for the
separation of the fused moieties. It will be appreciated that once
constructed, the fusion polypeptides can be derivatized according
to the methods described herein.
[0139] In a further embodiment of the invention, the polypeptide
comprising the amino acid sequence of SEQ ID NO: 3 or a polypeptide
variant is fused to one or more domains of an Fc region of human
IgG. Antibodies comprise two functionally independent parts, a
variable domain known as "Fab", which binds antigen, and a constant
domain known as "Fc", which is involved in effector functions such
as complement activation and attack by phagocytic cells. An Fc has
a long serum half-life, whereas an Fab is short-lived. Capon et
al., Nature, 337:525-31 (1989). When constructed together with a
therapeutic protein, an Fc domain can provide longer half-life or
incorporate such functions as Fc receptor binding, protein A
binding, complement fixation and perhaps even placental transfer.
Id. Table II summarizes the use of certain Fc fusions known in the
art.
2TABLE II Fc Fusion with Therapeutic Proteins Form of Fusion
Therapeutic Fc partner implications Reference IgG1 N-terminus
Hodgkin's U.S. Pat. No. of CD30-L disease; 5,480,981 anaplastic
lymphoma; T-cell leukemia Murine IL-10 anti- Zheng et al.
Fc.gamma.2a inflammatory; (1995), J. transplant Immunol., 154:
rejection 5590-5600 IgG1 TNF septic shock Fisher et al. receptor
(1996), N. Engl. J. Med., 334: 1697-1702; Van Zee et al., (1996),
J. Immunol., 156: 2221-2230 IgG, IgA, TNF inflammation, U.S. Pat.
No. IgM, or receptor autoimmune 5,808,029, IgE disorders issued
(excluding Sep. 15, 1998 the first domain) IgG1 CD4 AIDS Capon et
al. receptor (1989), Nature 337: 525-531 IgG1, N-terminus
anti-cancer, Harvill et al. IgG3 of IL-2 antiviral (1995),
Immunotech., 1: 95-105 IgG1 C-terminus osteoarthritis; WO 97/23614,
of OPG bone density published Jul. 3, 1997 IgG1 N-terminus
anti-obesity PCT/US of leptin 97/23183, filed Dec. 11, 1997 Human
Ig CTLA-4 autoimmune Linsley (1991), c.gamma.1 disorders J. Exp.
Med., 174: 561-569
[0140] In one example, all or a portion of the human IgG hinge, CH2
and CH3 regions may be fused at either the N-terminus or C-terminus
of a .beta.10 polypeptide of this invention using methods known to
the skilled artisan. The resulting .beta.10-fusion polypeptide or
.alpha.2/.beta.10-fusion polypeptide may be purified by use of a
Protein A affinity column. Peptides and proteins fused to an Fc 10
region have been found to exhibit a substantially greater half-life
in vivo than the unfused counterpart. Also, a fusion to an Fc
region allows for dimerization/multimerization of the fusion
polypeptide. The Fc region may be a naturally occurring Fc region,
or may be altered to improve certain qualities, such as therapeutic
qualities, circulation time, reduce aggregation, etc.
[0141] Identity and similarity of related nucleic acid molecules
and polypeptides can be readily calculated by known methods. Such
methods include, but are not limited to, those described in
Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and
Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press,
1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J.,
eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM
J. Applied Math., 48:1073 (1988).
[0142] Preferred methods to determine identity and/or similarity
are designed to give the largest match between the sequences
tested. Methods to determine identity and similarity are described
in publicly available computer programs. Preferred computer program
methods to determine identity and similarity between two sequences
include, but are not limited to, the GCG program package, including
GAP (Devereux et al., Nucl. Acid. Res., 12:387 (1984); Genetics
Computer Group, University of Wisconsin, Madison, Wis.), BLASTP,
BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410
(1990)). The BLASTX program is publicly available from the National
Center for Biotechnology Information (NCBT) and other sources
(BLAST Manual, Altschul et al. NCB/NLM/NIH Bethesda, Md. 20894;
Altschul et al., supra). The well known Smith Waterman algorithm
may also be used to determine identity.
[0143] Certain alignment schemes for aligning two amino acid
sequences may result in the matching of only a short region of the
two sequences, and this small aligned region may have very high
sequence identity even though there is no significant relationship
between the two full length sequences. Accordingly, in a preferred
embodiment, the selected alignment method (GAP program) will result
in an alignment that spans at least fifty (50) contiguous amino
acids of the target polypeptide.
[0144] For example, using the computer algorithm GAP (Genetics
Computer Group, University of Wisconsin, Madison, Wis.), two
polypeptides for which the percent sequence identity is to be
determined are aligned for optimal matching of their respective
amino acids (the "matched span", as determined by the algorithm). A
gap opening penalty (which is calculated as 3.times. the average
diagonal; the "average diagonal" is the average of the diagonal of
the comparison matrix being used; the "diagonal" is the score or
number assigned to each perfect amino acid match by the particular
comparison matrix) and a gap extension penalty (which is usually
{fraction (1/10)} times the gap opening penalty), as well as a
comparison matrix such as PAM 250 or BLOSUM 62 are used in
conjunction with the algorithm. A standard comparison matrix (see
Dayhoff et al., Atlas of Protein Sequence and Structure, vol. 5,
supp.3 (1978) for the PAM 250 comparison matrix; Henikoff et al.,
Proc. Natl. Acad. Sci USA, 89:10915-10919 (1992) for the BLOSUM 62
comparison matrix) is also used by the algorithm.
[0145] Preferred parameters for a polypeptide sequence comparison
include the following:
[0146] Algorithm: Needleman et al., J. Mol. Biol., 48:443-453
(1970);
[0147] Comparison matrix: BLOSUM 62 from Henikoff et al., Proc.
Natl. Acad. Sci. USA, 89:10915-10919 (1992);
[0148] Gap Penalty: 12
[0149] Gap Length Penalty: 4
[0150] Threshold of Similarity: 0
[0151] The GAP program is useful with the above parameters. The
aforementioned parameters are the default parameters for
polypeptide comparisons (along with no penalty for end gaps) using
the GAP algorithm.
[0152] Preferred parameters for nucleic acid molecule sequence
comparisons include the following:
[0153] Algorithm: Needleman et al., J. Mol Biol., 48:443-453
(1970);
[0154] Comparison matrix: matches=+10, mismatch=0
[0155] Gap Penalty: 50
[0156] Gap Length Penalty: 3
[0157] The GAP program is also useful with the above parameters.
The aforementioned parameters are the default parameters for
nucleic acid molecule comparisons.
[0158] Other exemplary algorithms, gap opening penalties, gap
extension penalties, comparison matrices, thresholds of similarity,
etc. may be used, including those set forth in the Program Manual,
Wisconsin Package, Version 9, September, 1997. The particular
choices to be made will be apparent to those of skill in the art
and will depend on the specific comparison to be made, such as DNA
to DNA, protein to protein, protein to DNA; and additionally,
whether the comparison is between given pairs of sequences (in
which case GAP or BestFit are generally preferred) or between one
sequence and a large database of sequences (in which case FASTA or
BLASTA are preferred).
Synthesis
[0159] It will be appreciated by those skilled in the art the
nucleic acid and polypeptide molecules described herein may be
produced by recombinant and other means.
Nucleic Acid Molecules
[0160] The nucleic acid molecules encode a polypeptide comprising
the amino acid sequence of a .beta.10 polypeptide of this invention
can readily be obtained in a variety of ways including, without
limitation, chemical synthesis, cDNA or genomic library screening,
expression library screening and/or PCR amplification of cDNA.
[0161] Recombinant DNA methods used herein are generally those set
forth in Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989), and/or Ausubel et al., eds., Current Protocols in Molecular
Biology, Green Publishers Inc. and Wiley and Sons, N.Y. (1994). The
present invention provides for nucleic acid molecules as described
herein and methods for obtaining the molecules.
[0162] Where a gene encoding the amino acid sequence of a .beta.10
polypeptide has been identified from one species, all or a portion
of that gene may be used as a probe to identify orthologs or
related genes from the same species. The probes or primers may be
used to screen cDNA libraries from various tissue sources believed
to express the .beta.10 polypeptide. In addition, part or all of a
nucleic acid molecule having the sequence set forth in SEQ ID NO: 2
may be used to screen a genomic library to identify and isolate a
gene encoding the amino acid sequence of a .beta.10 polypeptide.
Typically, conditions of moderate or high stringency will be
employed for screening to minimize the number of false positives
obtained from the screen.
[0163] Nucleic acid molecules encoding the amino acid sequence of a
.beta.10 polypeptide may also be identified by expression cloning
which employs the detection of positive clones based upon a
property of the expressed protein. Typically, nucleic acid
libraries are screened by the binding of an antibody or other
binding partner (e.g., receptor or ligand) to cloned proteins which
are expressed and displayed on a host cell surface. The antibody or
binding partner is modified with a detectable label to identify
those cells expressing the desired clone.
[0164] Recombinant expression techniques conducted in accordance
with the descriptions set forth below may be followed to produce
these polynucleotides and to express the encoded polypeptides. For
example, by inserting a nucleic acid sequence which encodes the
amino acid sequence of a .beta.10 polypeptide into an appropriate
vector, one skilled in the art can readily produce large quantities
of the desired nucleotide sequence. The sequences can then be used
to generate detection probes or amplification primers.
Alternatively, a polynucleotide encoding the amino acid sequence of
a .beta.10 polypeptide can be inserted into an expression vector.
By introducing the expression vector into an appropriate host, the
encoded .beta.10 polypeptide may be produced in large amounts.
[0165] Another method for obtaining a suitable nucleic acid
sequence is the polymerase chain reaction (PCR). In this method,
cDNA is prepared from poly(A)+RNA or total RNA using the enzyme
reverse transcriptase. Two primers, typically complementary to two
separate regions of cDNA (oligonucleotides) encoding the amino acid
sequence of a .beta.10 polypeptide, are then added to the cDNA
along with a polymerase such as Taq polymerase, and the polymerase
amplifies the cDNA region between the two primers.
[0166] Another means of preparing a nucleic acid molecule encoding
the amino acid sequence of a .beta.10 polypeptide is chemical
synthesis using methods well known to the skilled artisan such as
those described by Engels et al., Angew. Chem. Intl. Ed.,
28:716-734 (1989). These methods include, inter alia, the
phosphotriester, phosphoramidite, and H-phosphonate methods for
nucleic acid synthesis. A preferred method for such chemical
synthesis is polymer-supported synthesis using standard
phosphoramidite chemistry. Typically, the DNA encoding the amino
acid sequence of a .beta.10 polypeptide will be several hundred
nucleotides in length. Nucleic acids larger than about 100
nucleotides can be synthesized as several fragments using these
methods. The fragments can then be ligated together to form the
full length nucleotide sequence of a .beta.10 polypeptide. Usually,
the DNA fragment encoding the amino terminus of the polypeptide
will have an ATG, which encodes a methionine residue. This
methionine may or may not be present on the mature form of the
.beta.10 polypeptide, depending on whether the polypeptide produced
in the host cell is designed to be secreted from that cell. Other
methods known to the skilled artisan may be used as well.
[0167] In certain embodiments, nucleic acid variants contain codons
which have been altered for the optimal expression of .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer in a given host cell.
Particular codon alterations will depend upon the .beta.10
polypeptide(s) and host cell(s) selected for expression. Such
"codon optimization" can be carried out by a variety of methods,
for example, by selecting codons which are preferred for use in
highly expressed genes in a given host cell. Computer algorithms
which incorporate codon frequency tables such as "Ecohigh.cod" for
codon preference of highly expressed bacterial genes may be used
and are provided by the University of Wisconsin Package Version
9.0, Genetics Computer Group, Madison, Wis. Other useful codon
frequency tables include "Celegans_high.cod", "Celegans_low.cod",
"Drosophila_high.cod", "Human_high cod", "Maize_high.cod", and
"Yeast_high.cod".
Vectors and Host Cells
[0168] When contemplating expression of an .alpha.2/.beta.10
heterodimer, it should be understood that the .beta.10 polypeptide
expression vector as well as an expression vector encoding .alpha.2
polypeptide can both be introduced (for example, transformed,
co-transformed, transfected, co-transfected, transduced,
co-transduced) into a host cell, cell line, tissue, organ, animal
or plant. It is also understood that introduction of a .beta.10
polypeptide expression vector alone into a host cell, cell line,
tissue, organ, animal or plant that already produces .alpha.2
polypeptide can result in de novo or enhanced production of an
.alpha.2/.beta.10 heterodimer. As described above in the
heterodimerization assay recombinant polyHis and FLAG tagged
.alpha.2/.beta.10 heterodimer was produced by co-transfection of
mammalian cells. Heterodimeric glycoprotein hormones such as CG can
also be assembled in vitro upon co-incubation of, for example,
isolated alpha-subunit and isolated CG-.beta. subunit under
suitable conditions [see Blithe and Iles, Endocrinology, volume
136, pages 903-910 (1995)]. .alpha.2/.beta.10 heterodimer could
similarly be assembled in vitro upon incubation of isolated
.alpha.2 polypeptide and isolated .beta.10 polypeptide. The result
might be a mixture of .alpha.2 polypeptide, .beta.10 polypeptide
and .alpha.2/.beta.10 heterodimer. Each of these products could be
isolated in purified form using conventional methods such as size
exclusion chromatography and/or immunoaffinity chromatography. In
this regard .alpha.2 polypeptide alone and .beta.10 polypeptide
alone can be separately produced and secreted from mammalian cells
as described below. A human .alpha.2-polyHis-tag mammalian
expression vector was transfected into 293 cells and serum free
conditioned media was harvested after 72 hours. Western blot of
this conditioned media was probed with affinity purified
anti-.alpha.2 rabbit polyclonal antibodies (example 4) that had
been conjugated to Horse Radish Peroxidase (Linx HRP Rapid Protein
Conjugation Kit, cat# K8050-01, Invitrogen Corp., Carlsbad,
Calif.). A strong band was observed using the ECL Western Blot
detection kit (cat#RPN 2106, Amersham Pharmacia Biotech,
Piscataway, N.J.) demonstrating that a human .alpha.2 polypeptide
could be secreted in the absence of .beta.10 polypeptide. A human
.beta.10-FLAG-tag mammalian expression vector was transfected into
293 cells and serum free conditioned media was harvested after 72
hours. Western blot of this conditioned media was probed with a
biotinylated anti-FLAG M2 monoclonal antibody (cat# F9291, Sigma,
St. Louis, Mo.) and then probed with Streptavidin linked Horse
Radish Peroxidase (RPN 1231, Amersham Life Sciences). A strong band
was observed using the ECL Western Blot detection kit (cat#RPN
2106, Amersham Pharmacia Biotech, Piscataway, N.J.) demonstrating
that a human .beta.10 polypeptide could be secreted in the absence
of .alpha.2 polypeptide.
[0169] A nucleic acid molecule encoding the amino acid sequence of
a .beta.10 polypeptide may be inserted into an appropriate
expression vector using standard ligation techniques. The vector is
typically selected to be functional in the particular host cell
employed (i.e., the vector is compatible with the host cell
machinery such that amplification of the gene and/or expression of
the gene can occur). A nucleic acid molecule encoding the amino
acid sequence of a .beta.10 polypeptide according to this invention
may be amplified/expressed in prokaryotic, yeast, insect
(baculovirus systems), and/or eukaryotic host cells. Selection of
the host cell will depend in part on whether the .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer is to be
post-translationally modified (e.g., glycosylated and/or
phosphorylated). If so, yeast, insect, or mammalian host cells are
preferable. For a review of expression vectors, see Meth. Enz.,
v.185, D. V. Goeddel, ed. Academic Press Inc., San Diego, Calif.
(1990).
[0170] Typically, expression vectors used in any of the host cells
will contain sequences for plasmid maintenance and for cloning and
expression of exogenous nucleotide sequences. Such sequences,
collectively referred to as "flanking sequences" in certain
embodiments will typically include one or more of the following
nucleotide sequences: a promoter, one or more enhancer sequences,
an origin of replication, a transcriptional termination sequence, a
complete intron sequence containing a donor and acceptor splice
site, a sequence encoding a leader sequence for polypeptide
secretion, a ribosome binding site, a polyadenylation sequence, a
polylinker region for inserting the nucleic acid encoding the
polypeptide to be expressed, and a selectable marker element. Each
of these sequences is discussed below.
[0171] Optionally, the vector may contain a "tag"-encoding
sequence, i.e., an oligonucleotide molecule located at the 5' or 3'
end of the .beta.10 polypeptide coding sequence; the
oligonucleotide sequence encodes polyHis (such as hexaHis), or
other "tag" such as FLAG, HA (hemaglutinin Influenza virus) or myc
for which commercially available antibodies exist. This tag is
typically fused to the polypeptide upon expression of the
polypeptide, and can serve as a means for affinity purification of
the .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer from the
host cell. Affinity purification can be accomplished, for example,
by column chromatography using antibodies against the tag as an
affinity matrix. Optionally, the tag can subsequently be removed
from the purified .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer by various means such as using certain peptidases for
cleavage.
[0172] Flanking sequences may be homologous (i.e., from the same
species and/or strain as the host cell), heterologous (i.e., from a
species other than the host cell species or strain), hybrid (i.e.,
a combination of flanking sequences from more than one source) or
synthetic, or the flanking sequences may be native sequences which
normally function to regulate .beta.10 polypeptide expression. As
such, the source of a flanking sequence may be any prokaryotic or
eukaryotic organism, any vertebrate or invertebrate organism, or
any plant, provided that the flanking sequence is functional in,
and can be activated by, the host cell machinery.
[0173] The flanking sequences useful in the vectors of this
invention may be obtained by any of several methods well known in
the art. Typically, flanking sequences useful herein other than the
.beta.10 gene flanking sequences will have been previously
identified by mapping and/or by restriction endonuclease digestion
and can thus be isolated from the proper tissue source using the
appropriate restriction endonucleases. In some cases, the full
nucleotide sequence of a flanking sequence may be known. Here, the
flanking sequence may be synthesized using the methods described
herein for nucleic acid synthesis or cloning.
[0174] Where all or only a portion of the flanking sequence is
known, it may be obtained using PCR and/or by screening a genomic
library with suitable oligonucleotide and/or flanking sequence
fragments from the same or another species. Where the flanking
sequence is not known, a fragment of DNA containing a flanking
sequence may be isolated from a larger piece of DNA that may
contain, for example, a coding sequence or even another gene or
genes. Isolation may be accomplished by restriction endonuclease
digestion to produce the proper DNA fragment followed by isolation
using agarose gel purification, Qiagen.RTM. column chromatography
(Chatsworth, Calif.), or other methods known to the skilled
artisan. The selection of suitable enzymes to accomplish this
purpose will be readily apparent to one of ordinary skill in the
art.
[0175] An origin of replication is typically a part of those
prokaryotic expression vectors purchased commercially, and the
origin aids in the amplification of the vector in a host cell.
Amplification of the vector to a certain copy number can, in some
cases, be important for the optimal expression of .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer. If the vector of
choice does not contain an origin of replication site, one may be
chemically synthesized based on a known sequence, and ligated into
the vector. For example, the origin of replication from the plasmid
pBR322 (Product No. 303-3s, New England Biolabs, Beverly, Mass.) is
suitable for most Gram-negative bacteria and various origins (e.g.,
SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV) or
papillomaviruses such as HPV or BPV) are useful for cloning vectors
in mammalian cells. Generally, the origin of replication component
is not needed for mammalian expression vectors (for example, the
SV40 origin is often used only because it contains the early
promoter).
[0176] A transcription termination sequence is typically located 3'
of the end of a polypeptide coding region and serves to terminate
transcription. Usually, a transcription termination sequence in
prokaryotic cells is a G-C rich fragment followed by a poly T
sequence. While the sequence is easily cloned from a library or
even purchased commercially as part of a vector, it can also be
readily synthesized using methods for nucleic acid synthesis such
as those described herein.
[0177] A selectable marker gene element encodes a protein necessary
for the survival and growth of a host cell grown in a selective
culture medium. Typical selection marker genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, tetracycline, or kanamycin for prokaryotic host cells,
(b) complement auxotrophic deficiencies of the cell; or (c) supply
critical nutrients not available from complex media. Preferred
selectable markers are the kanamycin resistance gene, the
ampicillin resistance gene, and the tetracycline resistance gene. A
neomycin resistance gene may also be used for selection in
prokaryotic and eukaryotic host cells.
[0178] Other selection genes may be used to amplify the gene which
will be expressed. Amplification is the process wherein genes which
are in greater demand for the production of a protein critical for
growth are reiterated in tandem within the chromosomes of
successive generations of recombinant cells. Examples of suitable
selectable markers for mammalian cells include dihydrofolate
reductase (DHFR) and thymidine kinase. The mammalian cell
transformants are placed under selection pressure which only the
transformants are uniquely adapted to survive by virtue of the
selection gene present in the vector. Selection pressure is imposed
by culturing the transformed cells under conditions in which the
concentration of selection agent in the medium is successively
changed, thereby leading to the amplification of both the selection
gene and the DNA that encodes a .beta.10 polypeptide of this
invention. As a result, increased quantities of the .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer are synthesized from
the amplified DNA.
[0179] A ribosome binding site is usually necessary for translation
initiation of mRNA and is characterized by a Shine-Dalgarno
sequence (prokaryotes) or a Kozak sequence (eukaryotes). The
element is typically located 3' to the promoter and 5' to the
coding sequence of the .beta.10 polypeptide to be expressed. The
Shine-Dalgarno sequence is varied but is typically a polypurine
(i.e., having a high A-G content). Many Shine-Dalgarno sequences
have been identified, each of which can be readily synthesized
using methods set forth herein and used in a prokaryotic
vector.
[0180] A leader, or signal, sequence may be used to direct the
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer out of the
host cell. Typically, a nucleotide sequence encoding the signal
sequence is positioned in the coding region of a .beta.10 nucleic
acid molecule, or directly at the 5' end of a .beta.10 polypeptide
coding region. Many signal sequences have been identified, and any
of those that are functional in the selected host cell may be used
in conjunction with a .beta.10 nucleic acid molecule. Therefore, a
signal sequence may be homologous (naturally occurring) or
heterologous to a .beta.10 gene or cDNA. Additionally, a signal
sequence may be chemically synthesized using methods described
herein. In most cases, the secretion of a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer from the host cell via the presence
of a signal peptide will result in the removal of the signal
peptide from the secreted .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer. The signal sequence may be a component of the vector,
or it may be a part of a .beta.10 nucleic acid molecule that is
inserted into the vector.
[0181] Included within the scope of this invention is the use of
either a nucleotide sequence encoding a native .beta.10 polypeptide
signal sequence joined to a .beta.10 polypeptide coding region or a
nucleotide sequence encoding a heterologous signal sequence joined
to an .beta.10 polypeptide coding region. The heterologous signal
sequence selected should be one that is recognized and processed,
i.e., cleaved by a signal peptidase, by the host cell. For
prokaryotic host cells that do not recognize and process the native
.beta.10 polypeptide signal sequence, the signal sequence is
substituted by a prokaryotic signal sequence selected, for example,
from the group of the alkaline phosphatase, penicillinase, or
heat-stable enterotoxin II leaders. For yeast secretion, the native
.beta.10 polypeptide signal sequence may be substituted by the
yeast invertase, alpha factor, or acid phosphatase leaders. In
mammalian cell expression the native signal sequence is
satisfactory, although other mammalian signal sequences may be
suitable.
[0182] In some cases, such as where glycosylation is desired in a
eukaryotic host cell expression system, one may manipulate the
various presequences to improve glycosylation or yield. For
example, one may alter the peptidase cleavage site of a particular
signal peptide, or add presequences, which also may affect
glycosylation. The final protein product may have, in the-1
position (relative to the first amino acid of the mature protein)
one or more additional amino acids incident to expression, which
may not have been totally removed. For example, the final protein
product may have one or two amino acid residues found in the
peptidase cleavage site, attached to the N-terminus. Alternatively,
use of some enzyme cleavage sites may result in a slightly
truncated form of the desired .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer if the enzyme cuts at such area
within the mature polypeptide.
[0183] In many cases, transcription of a nucleic acid molecule is
increased by the presence of one or more introns in the vector;
this is particularly true where a polypeptide is produced in
eukaryotic host cells, especially mammalian host cells. The introns
used may be naturally occurring within the .beta.10 gene,
especially where the gene used is a full length genomic sequence or
a fragment thereof. Where the intron is not naturally occurring
within the gene (as for most cDNAs), the intron(s) may be obtained
from another source. The position of the intron with respect to
flanking sequences and the .beta.10 gene is generally important, as
the intron must be transcribed to be effective. Thus, when a
.beta.10 cDNA molecule is being transcribed, the preferred position
for the intron is 3' to the transcription start site, and 5' to the
polyA transcription termination sequence. Preferably, the intron or
introns will be located on one side or the other (i.e., 5' or 3')
of the cDNA such that it does not interrupt the coding sequence.
Any intron from any source, including any viral, prokaryotic and
eukaryotic (plant or animal) organisms, may be used to practice
this invention, provided that it is compatible with the host
cell(s) into which it is inserted. Also included herein are
synthetic introns. Optionally, more than one intron may be used in
the vector.
[0184] The expression and cloning vectors of the present invention
will each typically contain a promoter that is recognized by the
host organism and operably linked to the molecule encoding a
.beta.10 polypeptide. Promoters are untranscribed sequences located
upstream (5') to the start codon of a structural gene (generally
within about 100 to 1000 base pairs) that control the transcription
of the structural gene. Promoters are conventionally grouped into
one of two classes, inducible promoters and constitutive promoters.
Inducible promoters initiate increased levels of transcription from
DNA under their control in response to some change in culture
conditions, such as the presence or absence of a nutrient or a
change in temperature. Constitutive promoters, on the other hand,
initiate continual gene product production; that is, there is
little or no control over gene expression. A large number of
promoters, recognized by a variety of potential host cells, are
well known. A suitable promoter is operably linked to the DNA
encoding a .beta.10 polypeptide by removing the promoter from the
source DNA by restriction enzyme digestion and inserting the
desired promoter sequence into the vector. The native .beta.10 gene
promoter sequence may be used to direct amplification and/or
expression of a .beta.10 nucleic acid molecule. A heterologous
promoter is preferred, however, if it permits greater transcription
and higher yields of the expressed protein as compared to the
native promoter, and if it is compatible with the host cell system
that has been selected for use.
[0185] Promoters suitable for use with prokaryotic hosts include
the beta-lactamase and lactose promoter systems; alkaline
phosphatase, a tryptophan (trp) promoter system; and hybrid
promoters such as the tac promoter. Other known bacterial promoters
are also suitable. Their sequences have been published, thereby
enabling one skilled in the art to ligate them to the desired DNA
sequence(s), using linkers or adapters as needed to supply any
useful restriction sites.
[0186] Suitable promoters for use with yeast hosts are also well
known in the art. Yeast enhancers are advantageously used with
yeast promoters. Suitable promoters for use with mammalian host
cells are well known and include, but are not limited to, those
obtained from the genomes of viruses such as polyoma virus, fowlpox
virus, adenovirus (such as Adenovirus 2), bovine papilloma virus,
avian sarcoma virus, cytomegalovirus (CMV), a retrovirus,
hepatitis-B virus and most preferably Simian Virus 40 (SV40). Other
suitable mammalian promoters include heterologous mammalian
promoters, e.g., heat-shock promoters and the actin promoter.
[0187] Additional promoters which may be of interest in controlling
.beta.10 gene transcription include, but are not limited to: the
SV40 early promoter region (Bernoist and Chambon, Nature,
290:304-310, 1981); the CMV promoter; the promoter contained in the
3' long terminal repeat of Rous sarcoma virus (Yamamoto et al.,
Cell, 22:787-797, 1980); the herpes thymidine kinase promoter
(Wagner et al., Proc. Natl. Acad. Sci. USA, 78:144-1445, 1981); the
regulatory sequences of the metallothionine gene (Brinster et al.,
Nature, 296:39-42, 1982); prokaryotic expression vectors such as
the beta-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl.
Acad. Sci. USA, 75:3727-3731, 1978); or the tac promoter (DeBoer,
et al., Proc. Natl. Acad. Sci. USA, 80:21-25, 1983). Also of
interest are the following animal transcriptional control regions,
which exhibit tissue specificity and have been utilized in
transgenic animals: the elastase I gene control region which is
active in pancreatic acinar cells (Swift et al., Cell, 38:639-646,
1984; Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.,
50:399-409 (1986); MacDonald, Hepatology, 7:425-515, 1987); the
insulin gene control region which is active in pancreatic beta
cells (Hanahan, Nature, 315:115-122, 1985); the immunoglobulin gene
control region which is active in lymphoid cells (Grosschedl et
al., Cell, 38:647-658 (1984); Adames et al., Nature, 318:533-538
(1985); Alexander et al., Mol. Cell. Biol., 7:1436-1444, 1987); the
mouse mammary tumor virus control region which is active in
testicular, breast, lymphoid and mast cells (Leder et al., Cell,
45:485-495, 1986); the albumin gene control region which is active
in liver (Pinkert et al., Genes and Devel., 1:268-276, 1987); the
alphafetoprotein gene control region which is active in liver
(Krumlauf et al., Mol. Cell. Biol., 5:1639-1648, 1985; Hammer et
al., Science, 235:53-58, 1987); the alpha 1-antitrypsin gene
control region which is active in the liver (Kelsey et al., Genes
and Devel., 1:161-171, 1987); the beta-globin gene control region
which is active in myeloid cells (Mogram et al., Nature,
315:338-340, 1985; Kollias et al., Cell, 46:89-94, 1986); the
myelin basic protein gene control region which is active in
oligodendrocyte cells in the brain (Readhead et al., Cell,
48:703-712, 1987); the myosin light chain-2 gene control region
which is active in skeletal muscle (Sani, Nature, 314:283-286,
1985); and the gonadotropic releasing hormone gene control region
which is active in the hypothalamus (Mason et al., Science,
234:1372-1378, 1986).
[0188] An enhancer sequence may be inserted into the vector to
increase the transcription of a DNA encoding a .beta.10 polypeptide
of the present invention by higher eukaryotes. Enhancers are
cis-acting elements of DNA, usually about 10-300 bp in length, that
act on the promoter to increase transcription. Enhancers are
relatively orientation and position independent. They have been
found 5' and 3' to the transcription unit. Several enhancer
sequences available from mammalian genes are known (e.g., globin,
elastase, albumin, alpha-feto-protein and insulin). Typically,
however, an enhancer from a virus will be used. The SV40 enhancer,
the cytomegalovirus early promoter enhancer, the polyoma enhancer,
and adenovirus enhancers are exemplary enhancing elements for the
activation of eukaryotic promoters. While an enhancer may be
spliced into the vector at a position 5' or 3' to a .beta.10
nucleic acid molecule, it is typically located at a site 5' from
the promoter.
[0189] Expression vectors of the invention may be constructed from
a starting vector such as a commercially available vector. Such
vectors may or may not contain all of the desired flanking
sequences. Where one or more of the desired flanking sequences are
not already present in the vector, they may be individually
obtained and ligated into the vector. Methods used for obtaining
each of the flanking sequences are well known to one skilled in the
art.
[0190] Preferred vectors for practicing this invention are those
which are compatible with bacterial, insect, and mammalian host
cells. Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1
(Invitrogen Company, Carlsbad, Calif.), pBSII (Stratagene Company,
La Jolla, Calif.), pET15 (Novagen, Madison, Wis.), pGEX (Pharmacia
Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.),
pETL (BlueBacII; Invitrogen), pDSR-alpha (PCT Publication No.
WO90/14363) and pFastBacDual (Gibco/BRL, Grand Island, N.Y.).
[0191] Additional suitable vectors include, but are not limited to,
cosmids, plasmids or modified viruses, but it will be appreciated
that the vector system must be compatible with the selected host
cell. Such vectors include, but are not limited to plasmids such as
Bluescript.RTM. plasmid derivatives (a high copy number ColEl-based
phagemid, Stratagene Cloning Systems Inc., La Jolla Calif.), PCR
cloning plasmids designed for cloning Taq-amplified PCR products
(e.g., TOPO.TM. TA Cloning.RTM. Kit, PCR2.1.RTM. plasmid
derivatives, Invitrogen, Carlsbad, Calif.), and mammalian, yeast,
or virus vectors such as a baculovirus expression system (pBacPAK
plasmid derivatives, Clontech, Palo Alto, Calif.).
[0192] After the vector has been constructed and a nucleic acid
molecule encoding a .beta.10 polypeptide has been inserted into the
proper site of the vector, the completed vector may be inserted
into a suitable host cell for amplification and/or polypeptide
expression. The transformation of an expression vector for a
.beta.10 polypeptide into a selected host cell may be accomplished
by well known methods including methods such as transfection,
infection, calcium chloride, electroporation, microinjection,
lipofection or the DEAE-dextran method or other known techniques.
The method selected will in part be a function of the type of host
cell to be used. These methods and other suitable methods are well
known to the skilled artisan, and are set forth, for example, in
Sambrook et al., supra.
[0193] Host cells may be prokaryotic host cells (such as E. coli)
or eukaryotic host cells (such as a yeast cell, an insect cell or a
vertebrate cell). The host cell, when cultured under appropriate
conditions, synthesizes a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer which can subsequently be collected from the culture
medium (if the host cell secretes it into the medium) or directly
from the host cell producing it (if it is not secreted). The
selection of an appropriate host cell will depend upon various
factors, such as desired expression levels, polypeptide
modifications that are desirable or necessary for activity, such as
glycosylation or phosphorylation, and ease of folding into a
biologically active molecule.
[0194] A number of suitable host cells are known in the art and
many are available from the American Type Culture Collection
(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209.
Examples include, but are not limited to, mammalian cells, such as
Chinese hamster ovary cells (CHO) (ATCC No. CCL61) CHO DHFR-cells
(Urlaub et al., Proc. Natl. Acad. Sci. USA, 97:4216-4220 (1980)),
human embryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573),
or 3T3 cells (ATCC No. CCL92). The selection of suitable mammalian
host cells and methods for transformation, culture, amplification,
screening and product production and purification are known in the
art. Other suitable mammalian cell lines, are the monkey COS-1
(ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651), and the
CV-1 cell line (ATCC No. CCL70). Further exemplary mammalian host
cells include primate cell lines and rodent cell lines, including
transformed cell lines. Normal diploid cells, cell strains derived
from in vitro culture of primary tissue, as well as primary
explants, are also suitable. Candidate cells may be genotypically
deficient in the selection gene, or may contain a dominantly acting
selection gene. Other suitable mammalian cell lines include but are
not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929
cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK
hamster cell lines, which are available from the ATCC. Each of
these cell lines is known by and available to those skilled in the
art of protein expression.
[0195] Similarly useful as host cells suitable for the present
invention are bacterial cells. For example, the various strains of
E. coli (e.g., HB101, (ATCC No. 33694) DH5.alpha., DH10, and MC1061
(ATCC No. 53338)) are well-known as host cells in the field of
biotechnology. Various strains of B. subtilis, Pseudomonas spp.,
other Bacillus spp., Streptomyces spp., and the like may also be
employed in this method.
[0196] Many strains of yeast cells known to those skilled in the
art are also available as host cells for the expression of the
.beta.10 polypeptides or .alpha.2/.beta.10 heterodimers of the
present invention. Preferred yeast cells include, for example,
Saccharomyces cerivisae and Pichia pastoris.
[0197] Additionally, where desired, insect cell systems may be
utilized in the methods of the present invention. Such systems are
described for example in Kitts et al., Biotechniques, 14:810-817
(1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and
Lucklow et al. (J. Virol., 67:4566-4579 (1993). Preferred insect
cells are Sf-9 and Hi5 (Invitrogen, Carlsbad, Calif.).
[0198] One may also use transgenic animals to express glycosylated
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer. For example,
one may use a transgenic milk-producing animal (a cow or goat, for
example) and obtain glycosylated .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer in the animal milk. One may also use
plants to produce .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer however, in general, the glycosylation occurring in
plants is different from that produced in mammalian cells, and may
result in a glycosylated product which is not suitable for human
therapeutic use.
Polypeptide Production
[0199] Host cells comprising a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer expression vector may be cultured
using standard media well known to the skilled artisan. The media
will usually contain all nutrients necessary for the growth and
survival of the cells. Suitable media for culturing E. coli cells
include, for example, Luria Broth (LB) and/or Terrific Broth (TB).
Suitable media for culturing eukaryotic cells include Roswell Park
Memorial Institute medium 1640 (RPMI 1640), Minimal Essential
Medium (MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of
which may be supplemented with serum and/or growth factors as
indicated by the particular cell line being cultured. A suitable
medium for insect cultures is Grace's medium supplemented with
yeastolate, lactalbumin hydrolysate and/or fetal calf serum, as
necessary.
[0200] Typically, an antibiotic or other compound useful for
selective growth of transformed cells is added as a supplement to
the media. The compound to be used will be dictated by the
selectable marker element present on the plasmid with which the
host cell was transformed. For example, where the selectable marker
element is kanamycin resistance, the compound added to the culture
medium will be kanamycin. Other compounds for selective growth
include ampicillin, tetracycline, and neomycin.
[0201] The amount of .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer produced by a host cell can be evaluated using standard
methods known in the art. Such methods include, without limitation,
Western blot analysis, SDS-polyacrylamide gel electrophoresis,
non-denaturing gel electrophoresis, HPLC separation,
immunoprecipitation, and/or activity assays such as DNA binding gel
shift assays.
[0202] If a .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
has been designed to be secreted from the host cells, the majority
of .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer may be
found in the cell culture medium. If however, the .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer is not secreted from
the host cells, it will be present in the cytoplasm and/or the
nucleus (for eukaryotic host cells) or in the cytosol (for
bacterial host cells).
[0203] For .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
situated in the host cell cytoplasm and/or the nucleus (for
eukaryotic host cells) or in the cytosol (for bacterial host
cells), intracellular material (including inclusion bodies for
gram-negative bacteria) can be extracted from the host cell using
any standard technique known to the skilled artisan. For example,
the host cells can be lysed to release the contents of the
periplasm/cytoplasm by French press, homogenization, and/or
sonication followed by centrifugation.
[0204] If the .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
has formed inclusion bodies in the cytosol, the inclusion bodies
can often bind to the inner and/or outer cellular membranes and
thus will be found primarily in the pellet material after
centrifugation. The pellet material can then be treated at pH
extremes or with a chaotropic agent such as a detergent, guanidine,
guanidine derivatives, urea, or urea derivatives in the presence of
a reducing agent such as dithiothreitol at alkaline pH or tris
carboxyethyl phosphine at acid pH to release, break apart, and
solubilize the inclusion bodies. The .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer in its now soluble form can then be
analyzed using gel electrophoresis, immuno-precipitation or the
like. If it is desired to isolate the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer, isolation may be accomplished using
standard methods such as those described herein and in Marston et
al., Meth. Enz., 182:264-275 (1990).
[0205] In some cases, the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer may not be biologically active upon isolation. Various
methods for "refolding" or converting the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer to its tertiary structure and
generating disulfide linkages can be used to restore biological
activity. Such methods include exposing the solubilized .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer to a pH usually above
7 and in the presence of a particular concentration of a chaotrope.
The selection of chaotrope is very similar to the choices used for
inclusion body solubilization, but usually the chaotrope is used at
a lower concentration and is not necessarily the same as chaotropes
used for the solubilization. In most cases the refolding/oxidation
solution will also contain a reducing agent or the reducing agent
plus its oxidized form in a specific ratio to generate a particular
redox potential allowing for disulfide shuffling to occur in the
formation of the protein's cysteine bridge(s). Some of the commonly
used redox couples include cysteine/cystamine, glutathione
(GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane
DTT, and 2-2mercaptoethanol(bME)/dithio-b(ME). A cosolvent may be
used to increase the efficiency of the refolding, and the more
common reagents used for this purpose include glycerol,
polyethylene glycol of various molecular weights, arginine and the
like.
[0206] If inclusion bodies are not formed to a significant degree
upon expression of .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer then the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer will be found primarily in the supernatant after
centrifugation of the cell homogenate. The .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer may be further isolated from the
supernatant using methods such as those described herein.
[0207] The purification of .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer from solution can be accomplished
using a variety of techniques. If the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer has been synthesized such that it
contains a tag such as Hexahistidine (.beta.10 polypeptide-hexaHis,
.alpha.2/.beta.10-hexaHis heterodimer) or other small peptide such
as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen,
Carlsbad, Calif.) at either its carboxyl or amino terminus, it may
be purified in a one-step process by passing the solution through
an affinity column where the column matrix has a high affinity for
the tag.
[0208] For example, polyhistidine binds with great affinity and
specificity to nickel, thus an affinity column of nickel (such as
the Qiagen.RTM. nickel columns) can be used for purification of
.beta.10 polypeptide-polyHis or .alpha.2/.beta.10-hexaHis
heterodimer. See for example, Ausubel et al., eds., Current
Protocols in Molecular Biology, Section 10.11.8, John Wiley &
Sons, New York (1993).
[0209] Additionally, the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer may be purified through the use of a monoclonal
antibody which is capable of specifically recognizing and binding
to the .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer.
[0210] Suitable procedures for purification thus include, without
limitation, affinity chromatography, immunoaffinity chromatography,
ion exchange chromatography, molecular sieve chromatography, High
Performance Liquid Chromatography (HPLC), electrophoresis
(including native gel electrophoresis) followed by gel elution, and
preparative isoelectric focusing ("Isoprime" machine/technique,
Hoefer Scientific, San Francisco, Calif.). In some cases, two or
more purification techniques may be combined to achieve increased
purity.
[0211] .beta.10 polypeptides or .alpha.2/.beta.10 heterodimers may
also be prepared by chemical synthesis methods (such as solid phase
peptide synthesis) using techniques known in the art, such as those
set forth by Merrifield et al., J. Am. Chem. Soc., 85:2149 (1963),
Houghten et al., Proc Natl Acad. Sci. USA, 82:5132 (1985), and
Stewart and Young, Solid Phase Peptide Synthesis, Pierce Chemical
Co., Rockford, Ill. (1984). Such .beta.10 polypeptides or
.alpha.2/.beta.10 heterodimers may be synthesized with or without a
methionine on the amino terminus. Chemically synthesized .beta.10
polypeptides or .alpha.2/.beta.10 heterodimers may be oxidized
using methods set forth in these references to form disulfide
bridges. Chemically synthesized .beta.10 polypeptides or
.alpha.2/.beta.10 heterodimers are expected to have comparable
biological activity to the corresponding .beta.10 polypeptides
(when heterodimerized to .alpha.2) or .alpha.2/.beta.10
heterodimers produced recombinantly or purified from natural
sources, and thus may be used interchangeably with a recombinant or
natural .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer.
[0212] Another means of obtaining a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer according to this invention is via
purification from biological samples such as source tissues and/or
fluids in which the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer is naturally found. Such purification can be conducted
using methods for protein purification as described herein. The
presence of the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer during purification may be monitored using, for
example, a corresponding antibody prepared against recombinantly
produced .beta.10 polypeptide or peptide fragment thereof, or
prepared against recombinantly produced .alpha.2/.beta.10
heterodimer or peptide fragment.
[0213] A number of additional methods for producing nucleic acids
and polypeptides are known in the art, and can be used to produce
polypeptides having specificity for .beta.10 polypeptides or
.alpha.2/.beta.10 heterodimers of this invention. See for example,
Roberts, et al., Proc. Natl. Acad. Sci., 94:12297-12303 (1997),
which describes the production of fusion proteins between an mRNA
and its encoded peptide. See also U.S. Pat. No. 5,824,469, which
describes methods of obtaining oligonucleotides capable of carrying
out a specific biological function. The procedure involves
generating a heterogeneous pool of oligonucleotides, each having a
5' randomized sequence, a central preselected sequence, and a 3'
randomized sequence. The resulting heterogeneous pool is introduced
into a population of cells that do not exhibit the desired
biological function. Subpopulations of the cells are then screened
for those which exhibit a predetermined biological function. From
that subpopulation, oligonucleotides capable of carrying out the
desired biological function are isolated.
[0214] U.S. Pat. Nos. 5,763,192, 5,814,476, 5,723,323, and
5,817,483 describe processes for producing peptides or
polypeptides. This is done by producing stochastic genes or
fragments thereof, and then introducing these genes into host cells
which produce one or more proteins encoded by the stochastic genes.
The host cells are then screened to identify those clones producing
peptides or polypeptides having the desired activity.
Chemical Derivatives
[0215] Chemically modified derivatives of the .beta.10 polypeptides
or .alpha.2/.beta.10 heterodimers of this invention may be prepared
by one skilled in the art, given the disclosures set forth
hereinbelow. Such .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer derivatives are modified in a manner that is different,
either in the type or location of the molecules naturally attached
to the .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer.
Derivatives may include molecules formed by the deletion of one or
more naturally-attached chemical groups. The polypeptide comprising
the amino acid sequence of SEQ ID NO: 3, a .beta.10-like
polypeptide variant thereof or a .alpha.2/.beta.10 heterodimer may
be modified by the covalent attachment of one or more polymers. For
example, the polymer selected is typically water soluble so that
the protein to which it is attached does not precipitate in an
aqueous environment, such as a physiological environment. Included
within the scope of suitable polymers is a mixture of polymers.
Preferably, for therapeutic use of the end-product preparation, the
polymer will be pharmaceutically acceptable.
[0216] The polymers each may be of any molecular weight and may be
branched or unbranched. The polymers each typically have an average
molecular weight of between about 2 kDa to about 100 kDa (the term
"about" indicating that in preparations of a water soluble polymer,
some molecules will weigh more, some less, than the stated
molecular weight). The average molecular weight of each polymer
preferably is between about 5 kDa and about 50 kDa, more preferably
between about 12 kDa and about 40 kDa and most preferably between
about 20 kDa and about 35 kDa.
[0217] Suitable water soluble polymers or mixtures thereof include,
but are not limited to, N-linked or O-linked carbohydrates, sugars,
phosphates, polyethylene glycol (PEG) (including the forms of PEG
that have been used to derivatize proteins, including
mono-(C.sub.1-C.sub.10) alkoxy- or aryloxy-polyethylene glycol),
monomethoxy-polyethylene glycol, dextran (such as low molecular
weight dextran, of, for example about 6 kD), cellulose, or other
carbohydrate based polymers, poly-(N-vinyl pyrrolidone)
polyethylene glycol, propylene glycol homopolymers, a polypropylene
oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g.,
glycerol) and polyvinyl alcohol. Also encompassed by the present
invention are bifunctional crosslinking molecules which may be used
to prepare covalently attached multimers of the polypeptide
comprising the amino acid sequence of SEQ ID NO: 3, a polypeptide
variant thereof or a .alpha.2/.beta.10 heterodimer.
[0218] In general, chemical derivatization may be performed under
any suitable condition used to react a protein with an activated
polymer molecule. Methods for preparing chemical derivatives of
polypeptides or heterodimers will generally comprise the steps of
(a) reacting the polypeptide or heterodimer with the activated
polymer molecule (such as a reactive ester or aldehyde derivative
of the polymer molecule) under conditions whereby the polypeptide
comprising the amino acid sequence of SEQ ID NO: 3, or a
polypeptide variant thereof or an .alpha.2/.beta.10 heterodimer
becomes attached to one or more polymer molecules, and (b)
obtaining the reaction product(s). The optimal reaction conditions
will be determined based on known parameters and the desired
result. For example, the larger the ratio of polymer
molecules:protein, the greater the percentage of attached polymer
molecule. In one embodiment, the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer derivative may have a single polymer
molecule moiety at the amino terminus. See, for example, U.S. Pat.
No. 5,234,784.
[0219] The pegylation of the polypeptide or heterodimer
specifically may be carried out by any of the pegylation reactions
known in the art, as described for example in the following
references: Francis et al., Focus on Growth Factors, 3:4-10 (1992);
EP 0154316; EP 0401384 and U.S. Pat. No. 4,179,337. For example,
pegylation may be carried out via an acylation reaction or an
alkylation reaction with a reactive polyethylene glycol molecule
(or an analogous reactive water-soluble polymer) as described
herein. For the acylation reactions, the polymer(s) selected should
have a single reactive ester group. For reductive alkylation, the
polymer(s) selected should have a single reactive aldehyde group. A
reactive aldehyde is, for example, polyethylene glycol
propionaldehyde, which is water stable, or mono C.sub.1-C.sub.10
alkoxy or aryloxy derivatives thereof (see U.S. Pat. No.
5,252,714).
[0220] In another embodiment, a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer may be chemically coupled to biotin,
and the biotin-.beta.10 polypeptide or biotin-.alpha.2/.beta.10
heterodimer molecules which are conjugated are then allowed to bind
to avidin, resulting in tetravalent avidin/biotin/.beta.10
polypeptide molecules or avidin/biotin/.alpha.2/.b- eta.10
heterodimer molecules. .beta.10 polypeptides or .alpha.2/.beta.10
heterodimers may also be covalently coupled to dinitrophenol (DNP)
or trinitrophenol (TNP) and the resulting conjugates precipitated
with anti-DNP or anti-TNP-IgM to form decameric conjugates with a
valency of 10.
[0221] Generally, conditions which may be alleviated or modulated
by the administration of the present .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer derivatives include those described
herein for .beta.10 polypeptides or .alpha.2/.beta.10 heterodimers.
However, the .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
derivatives disclosed herein may have additional activities,
enhanced or reduced biological activity, or other characteristics,
such as increased or decreased half-life, as compared to the
non-derivatized molecules.
Genetically Engineered Non-Human Animals
[0222] Additionally included within the scope of the present
invention are non-human animals such as mice, rats, or other
rodents, rabbits, goats, or sheep, or other farm animals, in which
the gene (or genes) encoding the native .beta.10 polypeptide has
(have) been disrupted ("knocked out") such that the level of
expression of this gene or genes is (are) significantly decreased
or completely abolished. Such animals may be prepared using
techniques and methods such as those described in U.S. Pat. No.
5,557,032.
[0223] The present invention further includes non-human animals
such as mice, rats, or other rodents, rabbits, goats, sheep, or
other farm animals, in which either the native form of the .beta.10
gene(s) for that animal or a heterologous .beta.10 gene(s) is (are)
over-expressed by the animal, thereby creating a "transgenic"
animal. Such transgenic animals may be prepared using well known
methods such as those described in U.S. Pat. No 5,489,743 and PCT
application No. WO94/28122.
[0224] The present invention further includes non-human animals in
which the promoter for one or more of the .beta.10 polypeptides
is/are either activated or inactivated (e.g., by using homologous
recombination methods) to alter the level of expression of native
.beta.10 polypeptide.
[0225] These non-human animals may be used for drug candidate
screening. In such screening, the impact of a drug candidate on the
animal may be measured. For example, drug candidates may decrease
or increase the expression of the .beta.10 gene. In certain
embodiments, the amount of .beta.10 polypeptide that is produced
may be measured after the exposure of the animal to the drug
candidate. Additionally, in certain embodiments, one may detect the
actual impact of the drug candidate on the animal. For example, the
overexpression of a particular gene may result in, or be associated
with, a disease or pathological condition. In such cases, one may
test a drug candidate's ability to decrease expression of the gene
or its ability to prevent or inhibit a pathological condition. In
other examples, the production of a particular metabolic product
such as a fragment of a polypeptide, may result in, or be
associated with, a disease or pathological condition. In such
cases, one may test a drug candidate's ability to decrease the
production of such a metabolic product or its ability to prevent or
inhibit a pathological condition.
Microarray
[0226] It will be appreciated that DNA microarray technology can be
utilized in accordance with the present invention. DNA microarrays
are miniature, high density arrays of nucleic acids positioned on a
solid support, such as glass. Each cell or element within the array
has numerous copies of a single species of DNA which acts as a
target for hybridization for its cognate mRNA. In expression
profiling using DNA microarray technology, mRNA is first extracted
from a cell or tissue sample and then converted enzymatically to
fluorescently labeled cDNA. This material is hybridized to the
microarray and unbound cDNA is removed by washing. The expression
of discrete genes represented on the array is then visualized by
quantitating the amount of labeled cDNA which is specifically bound
to each target DNA. In this way, the expression of thousands of
genes can be quantitated in a high throughput, parallel manner from
a single sample of biological material.
[0227] This high throughput expression profiling has a broad range
of applications with respect to the .beta.10 molecules of the
invention, including, but not limited to: the identification and
validation of .beta.10 disease-related genes as targets for
therapeutics; molecular toxicology of .beta.10 molecules and
inhibitors thereof; stratification of populations and generation of
surrogate markers for clinical trials; and enhancing .beta.10
related small molecule drug discovery by aiding in the
identification of selective compounds in high throughput screens
(HTS).
Selective Binding Agents
[0228] As used herein, the term "selective binding agent" refers to
a molecule which has specificity for one or more .beta.10
polypeptides or .alpha.2/.beta.10 heterodimers. Suitable selective
binding agents include, but are not limited to, antibodies and
derivatives thereof, polypeptides, and small molecules. Suitable
selective binding agents may be prepared using methods known in the
art. An exemplary .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer selective binding agent of the present invention is
capable of binding a certain portion of the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer thereby inhibiting the binding of the
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer to the
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
receptor(s).
[0229] Selective binding agents such as antibodies and antibody
fragments that bind .beta.10 polypeptides or .alpha.2/.beta.10
heterodimers are within the scope of the present invention. The
antibodies may be polyclonal including monospecific polyclonal,
monoclonal (MAbs), recombinant, chimeric, humanized such as
CDR-grafted, human, single chain, and/or bispecific, as well as
fragments, variants or derivatives thereof. Antibody fragments
include those portions of the antibody which bind to an epitope on
the .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer. Examples
of such fragments include Fab and F(ab') fragments generated by
enzymatic cleavage of full-length antibodies. Other binding
fragments include those generated by recombinant DNA techniques,
such as the expression of recombinant plasmids containing nucleic
acid sequences encoding antibody variable regions.
[0230] Polyclonal antibodies directed toward .beta.10 polypeptide
or .alpha.2/.beta.10 heterodimer generally are produced in animals
(e.g., rabbits or mice) by means of multiple subcutaneous or
intraperitoneal injections of .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer and an adjuvant. It may be useful to
conjugate a .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
to a carrier protein that is immunogenic in the species to be
immunized, such as keyhole limpet heocyanin, serum, albumin, bovine
thyroglobulin, or soybean trypsin inhibitor. Also, aggregating
agents such as alum are used to enhance the immune response. After
immunization, the animals are bled and the serum is assayed for
anti-.beta.10 polypeptide or anti-.alpha.2/.beta.10 heterodimer
antibody titer.
[0231] Monoclonal antibodies directed toward .beta.10 polypeptide
or .alpha.2/.beta.10 heterodimer are produced using any method
which provides for the production of antibody molecules by
continuous cell lines in culture. Examples of suitable methods for
preparing monoclonal antibodies include the hybridoma methods of
Kohler et al., Nature, 256:495-497 (1975) and the human B-cell
hybridoma method, Kozbor, J. Immunol., 133:3001 (1984); Brodeur et
al., Monoclonal Antibody Production Techniques and Applications,
pp. 51-63 (Marcel Dekker, Inc., New York, 1987). Also provided by
the invention are hybridoma cell lines which produce monoclonal
antibodies reactive with .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer of this invention.
[0232] Monoclonal antibodies of the invention may be modified for
use as therapeutics. One embodiment is a "chimeric" antibody in
which a portion of the heavy and/or light chain is identical with
or homologous to a corresponding sequence in antibodies derived
from a particular species or belonging to a particular antibody
class or subclass, while the remainder of the chain(s) is identical
with or homologous to a corresponding sequence in antibodies
derived from another species or belonging to another antibody class
or subclass. Also included are fragments of such antibodies, so
long as they exhibit the desired biological activity. See, U.S.
Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci.,
81:6851-6855 (1985).
[0233] In another embodiment, a monoclonal antibody of the
invention is a "humanized" antibody. Methods for humanizing
non-human antibodies are well known in the art. See U.S. Pat. Nos.
5,585,089, and 5,693,762. Generally, a humanized antibody has one
or more amino acid residues introduced into it from a source which
is non-human. Humanization can be performed, for example, using
methods described in the art (Jones et al., Nature 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science 239:1534-1536 (1988)), by substituting at least a
portion of a rodent complementarity-determining region (CDR) for
the corresponding regions of a human antibody.
[0234] Also encompassed by the invention are human antibodies which
bind .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer. Using
transgenic animals (e.g., mice) that are capable of producing a
repertoire of human antibodies in the absence of endogenous
immunoglobulin production such antibodies are produced by
immunization with a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer antigen (i.e., having at least 6 contiguous amino
acids), optionally conjugated to a carrier. See, for example,
Jakobovits et al., Proc. Natl. Acad. Sci., 90:2551-2555 (1993);
Jakobovits et al., Nature 362:255-258 (1993); Bruggermann et al.,
Year in Immuno., 7:33 (1993). In one method, such transgenic
animals are produced by incapacitating the endogenous loci encoding
the heavy and light immunoglobulin chains therein, and inserting
loci encoding human heavy and light chain proteins into the genome
thereof. Partially modified animals, that is those having less than
the full complement of modifications, are then cross-bred to obtain
an animal having all of the desired immune system modifications.
When administered an immunogen, these transgenic animals produce
antibodies with human (rather than e.g., murine) amino acid
sequences, including variable regions which are immunospecific for
these antigens. See PCT application nos. PCT/US96/05928 and
PCT/US93/06926. Additional methods are described in U.S. Pat. No.
5,545,807, PCT application nos. PCT/US91/245, PCT/GB89/01207, and
in EP 546073B1 and EP 546073A1. Human antibodies may also be
produced by the expression of recombinant DNA in host cells or by
expression in hybridoma cells as described herein.
[0235] In an alternative embodiment, human antibodies can be
produced from phage-display libraries (Hoogenboom et al., J. Mol.
Biol. 227:381 (1991); Marks et al., J. Mol. Biol. 222:581 (1991).
These processes mimic immune selection through the display of
antibody repertoires on the surface of filamentous bacteriophage,
and subsequent selection of phage by their binding to an antigen of
choice. One such technique is described in PCT Application no.
PCT/US98/17364, which describes the isolation of high affinity and
functional agonistic antibodies for MPL- and msk-receptors using
such an approach.
[0236] Chimeric, CDR grafted, and humanized antibodies are
typically produced by recombinant methods. Nucleic acids encoding
the antibodies are introduced into host cells and expressed using
materials and procedures described herein. In a preferred
embodiment, the antibodies are produced in mammalian host cells,
such as CHO cells. Monoclonal (e.g., human) antibodies may be
produced by the expression of recombinant DNA in host cells or by
expression in hybridoma cells as described herein.
[0237] The anti-.beta.10 polypeptide antibodies or
anti-.alpha.2/.beta.10 heterodimer antibodies of the invention may
be employed in any known assay method, such as competitive binding
assays, direct and indirect sandwich assays, and
immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual
of Techniques, pp. 147-158, CRC Press, Inc., 1987) for the
detection and quantitation of .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer. The antibodies will bind the
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer with an
affinity which is appropriate for the assay method being
employed.
[0238] For diagnostic applications, in certain embodiments,
anti-.beta.10 polypeptide antibodies or anti-.alpha.2/.beta.10
heterodimer antibodies may be labeled with a detectable moiety. The
detectable moiety can be any one which is capable of producing,
either directly or indirectly, a detectable signal. For example,
the detectable moiety may be a radioisotope, such as .sup.3H,
.sup.14C, .sup.32p, .sup.35S, or .sup.125I, a fluorescent or
chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin; or an enzyme, such as alkaline
phosphatase, .alpha.-galactosidase, or horseradish peroxidase
(Bayer et al., Meth. Enz., 184:138-163 (1990).
[0239] Competitive binding assays rely on the ability of a labeled
standard (e.g., a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer or an immunologically reactive portion thereof) to
compete with the test sample analyte (a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer) for binding with a limited amount of
anti-.beta.10 polypeptide antibody or anti-.alpha.2/.beta.10
heterodimer antibody. The amount of .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer in the test sample is inversely
proportional to the amount of standard that becomes bound to the
antibodies. To facilitate determining the amount of standard that
becomes bound, the antibodies typically are insolubilized before or
after the competition, so that the standard and analyte that are
bound to the antibodies may conveniently be separated from the
standard and analyte which remain unbound.
[0240] Sandwich assays typically involve the use of two antibodies,
each capable of binding to a different immunogenic portion, or
epitope, of the protein to be detected and/or quantitated. In a
sandwich assay, the test sample analyte is typically bound by a
first antibody which is immobilized on a solid support, and
thereafter a second antibody binds to the analyte, thus forming an
insoluble three part complex. See, e.g., U.S. Pat. No. 4,376,110.
The second antibody may itself be labeled with a detectable moiety
(direct sandwich assays) or may be measured using an
anti-immunoglobulin antibody that is labeled with a detectable
moiety (indirect sandwich assays). For example, one type of
sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in
which case the detectable moiety is an enzyme.
[0241] The selective binding agents, including anti-.beta.10
polypeptide antibodies or anti-.alpha.2/.beta.10 heterodimer
antibodies, also are useful for in vivo imaging. An antibody
labeled with a detectable moiety may be administered to an animal,
preferably into the bloodstream, and the presence and location of
the labeled antibody in the host is assayed. The antibody may be
labeled with any moiety that is detectable in an animal, whether by
nuclear magnetic resonance, radiology, or other detection means
known in the art.
[0242] Selective binding agents of the invention, including
antibodies, may be used as therapeutics. These therapeutic agents
are generally agonists or antagonists, in that they either enhance
or reduce, respectively, at least one of the biological activities
of a .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
according to this invention. In one embodiment, antagonist
antibodies of the invention are antibodies or binding fragments
thereof which are capable of specifically binding to a .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer and which are capable
of inhibiting or eliminating the functional activity of a .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer in vivo or in vitro.
In preferred embodiments, the selective binding agent, e.g., an
antagonist antibody, will inhibit the functional activity of a
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer by at least
about 50%, and preferably by at least about 80%. In another
embodiment, the selective binding agent may be an antibody that is
capable of interacting with a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer binding partner (a ligand or
receptor) thereby inhibiting or eliminating .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer activity in vitro or in vivo.
Selective binding agents, including agonist and antagonist
anti-.beta.10 polypeptide antibodies or anti-.alpha.2/.beta.10
heterodimer antibodies, are identified by screening assays which
are well known in the art.
[0243] The invention also relates to a kit comprising .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer selective binding
agents (such as antibodies) and other reagents useful for detecting
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer levels in
biological samples. Such reagents may include, a detectable label,
blocking serum, positive and negative control samples, and
detection reagents.
[0244] .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer of
this invention can also be used to clone .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer receptor(s), using an "expression
cloning" strategy. Radiolabeled (125-Iodine) .beta.10 polypeptide
or .alpha.2/.beta.10 heterodimer or "affinity/activity-tagged"
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer (such as an
Fc fusion or an alkaline phosphatase fusion) can be used in binding
assays to identify a cell type or cell line or tissue that
expresses .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
receptor(s). RNA isolated from such cells or tissues would be
converted to cDNA, cloned into a mammalian expression vector, and
transfected into mammalian cells (for example, COS, 293) to create
an expression library. Radiolabeled or tagged .beta.10 polypeptide
or .alpha.2/.beta.10 heterodimer would then be used as an affinity
ligand to identify and isolate the subset of cells in this library
expressing the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer receptor(s) on their surface. DNA would be isolated
from these cells and transfected into mammalian cells to create a
secondary expression library in which the fraction of cells
expressing .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
receptor(s) would be many-fold higher than in the original library.
This enrichment process would be repeated iteratively until a
single recombinant clone containing a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer receptor is isolated. Isolation of
the .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
receptor(s) would be very useful in terms of being able to identify
or develop novel agonists and antagonists of the .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer signaling pathway(s).
Such agonists and antagonists would include soluble .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer receptor(s),
anti-.beta.10 polypeptide-receptor(s) antibodies or
anti-.alpha.2/.beta.10 heterodimer(s)-receptor(s) antibodies, small
molecules or antisense oligonucleotides, and they could be used in
the diagnosis and/or treatment of one or more of the
diseases/disorders listed below.
Assaying for Other Modulators of .beta.10 Polypeptide or
.alpha.2/.beta.10 Heterodimer Activity
[0245] In some situations, it may be desirable to identify
molecules that are modulators, i.e., agonists or antagonists, of
the activity of a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer of this invention. Natural or synthetic molecules that
modulate the .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
may be identified using one or more screening assays, such as those
described herein. Such molecules may be administered either in an
ex vivo manner, or in an in vivo manner by injection, or by oral
delivery, implantation device, or the like.
[0246] "Test molecule(s)" refers to the molecule(s) that is/are
under evaluation for the ability to modulate (i.e., increase or
decrease) the activity of a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer of this invention. Most commonly, a
test molecule will interact directly with the polypeptide or
heterodimer. However, it is also contemplated that a test molecule
may also modulate .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer activity indirectly, such as by affecting .beta.10 gene
expression, or by binding to a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer binding partner (e.g., receptor or
ligand). In one embodiment, a test molecule will bind to a .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer with an affinity
constant of at least about 10.sup.-6 M, preferably about 10.sup.-8
M, more preferably about 10.sup.-9 M, and even more preferably
about 10.sup.-10 M.
[0247] Methods for identifying compounds which interact with
.beta.10 polypeptides or .alpha.2/.beta.10 heterodimers of this
invention are encompassed by the present invention. In certain
embodiments, a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer is incubated with a test molecule under conditions
which permit the interaction of the test molecule with the
polypeptide, and the extent of the interaction can be measured. The
test molecule(s) can be screened in a substantially purified form
or in a crude mixture.
[0248] In certain embodiments, a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer agonist or antagonist may be a
protein, peptide, carbohydrate, lipid, or small molecular weight
molecule which interacts with the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer to regulate its activity. Molecules
which regulate .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer expression include nucleic acids which are
complementary to nucleic acids encoding a .beta.10 polypeptide of
this invention, or are complementary to nucleic acids sequences
which direct, control or influence the expression of the .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer and which act as
anti-sense regulators of expression.
[0249] Once a set of test molecules has been identified as
interacting with a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer the molecules may be further evaluated for their
ability to increase or decrease .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer activity. The measurement of the
interaction of test molecules with .beta.10 polypeptides or
.alpha.2/.beta.10 heterodimers may be carried out in several
formats, including cell-based binding assays, membrane binding
assays, solution-phase assays and immunoassays. In general, test
molecules are incubated with a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer for a specified period of time, and
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer activity is
determined by one or more assays for measuring biological
activity.
[0250] The interaction of test molecules with .beta.10 polypeptides
or .alpha.2/.beta.10 heterodimers according to this invention may
also be assayed directly using polyclonal or monoclonal antibodies
in an immunoassay. Alternatively, modified forms of .beta.10
polypeptides or .alpha.2/.beta.10 heterodimers containing epitope
tags as described herein may be used in immunoassays.
[0251] In the event that .beta.10 polypeptides or .alpha.2/.beta.10
heterodimers display biological activity through an interaction
with a binding partner (e.g., a receptor or a ligand), a variety of
in vitro assays may be used to measure the binding of a .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer to the corresponding
binding partner (such as a selective binding agent, receptor, or
ligand). These assays may be used to screen test molecules for
their ability to increase or decrease the rate and/or the extent of
binding of a .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
to its binding partner. In one assay, a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer is immobilized in the wells of a
microtiter plate. Radiolabeled .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer binding partner (for example,
iodinated .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
binding partner) and the test molecule(s) can then be added either
one at a time (in either order) or simultaneously to the wells.
After incubation, the wells can be washed and counted, using a
scintillation counter, for radioactivity to determine the extent to
which the binding partner bound to the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer. Typically, the molecules will be
tested over a range of concentrations, and a series of control
wells lacking one or more elements of the test assays can be used
for accuracy in the evaluation of the results. An alternative to
this method involves reversing the "positions" of the proteins,
i.e., immobilizing .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer binding partner to the microtiter plate wells,
incubating with the test molecule and radiolabeled .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer and determining the
extent of .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
binding. See, for example, chapter 18, Current Protocols in
Molecular Biology, Ausubel et al., eds., John Wiley & Sons, New
York, N.Y. (1995).
[0252] As an alternative to radiolabeling, a .beta.10 polypeptide
or .alpha.2/.beta.10 heterodimer or its respective binding partner
may be conjugated to biotin and the presence of biotinylated
protein can then be detected using streptavidin linked to an
enzyme, such as horseradish peroxidase (HRP) or alkaline
phosphatase (AP), that can be detected colorometrically, or by
fluorescent tagging of streptavidin. An antibody directed to a
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer or to a
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer binding
partner and conjugated to biotin may also be used and can be
detected after incubation with enzyme-linked streptavidin linked to
AP or HRP.
[0253] A .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer or a
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer binding
partner can also be immobilized by attachment to agarose beads,
acrylic beads or other types of such inert solid phase substrates.
The substrate-protein complex can be placed in a solution
containing the complementary protein and the test compound. After
incubation, the beads can be precipitated by centrifugation, and
the amount of binding between a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer and its respective binding partner
can be assessed using the methods described herein. Alternatively,
the substrate-protein complex can be immobilized in a column, and
the test molecule and complementary protein are passed through the
column. The formation of a complex between a .beta.10 polypeptide
or .alpha.2/.beta.10 heterodimer and its respective binding partner
can then be assessed using any of the techniques set forth herein,
i.e., radiolabeling, antibody binding, or the like.
[0254] Another in vitro assay that is useful for identifying a test
molecule which increases or decreases the formation of a complex
between a .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer and
a corresponding .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer binding partner is a surface plasmon resonance detector
system such as the BIAcore assay system (Pharmacia, Piscataway,
N.J.). The BlAcore system may be carried out using the
manufacturer's protocol. This assay essentially involves the
covalent binding of either a .beta.10 polypeptide,
.alpha.2/.beta.10 heterodimer, .beta.10 polypeptide binding partner
or .alpha.2/.beta.10 heterodimer binding partner to a
dextran-coated sensor chip which is located in a detector. The test
compound and the other complementary protein can then be injected,
either simultaneously or sequentially, into the chamber containing
the sensor chip. The amount of complementary protein that binds can
be assessed based on the change in molecular mass which is
physically associated with the dextran-coated side of the sensor
chip; the change in molecular mass can be measured by the detector
system.
[0255] In some cases, it may be desirable to evaluate two or more
test compounds together for their ability to increase or decrease
the formation of a complex between .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer and a corresponding 10 polypeptide or
.alpha.2/.beta.10 heterodimer binding partner. In these cases, the
assays set forth herein can be readily modified by adding such
additional test compound(s) either simultaneous with, or subsequent
to, the first test compound. The remainder of the steps in the
assay are as set forth herein.
[0256] In vitro assays such as those described herein may be used
advantageously to screen large numbers of compounds for effects on
complex formation by the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer and a corresponding .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer binding partner. The assays may be
automated to screen compounds generated in phage display, synthetic
peptide, and chemical synthesis libraries.
[0257] Compounds which increase or decrease the formation of a
complex between a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer and a corresponding .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer binding partner may also be screened
in cell culture using cells and cell lines expressing either
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer and a
corresponding .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
binding partner. Cells and cell lines may be obtained from any
mammal, but preferably will be from human or other primate, canine,
or rodent sources. The binding of a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer to cells expressing the corresponding
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer binding
partner at the surface is evaluated in the presence or absence of
test molecules, and the extent of binding may be determined by, for
example, flow cytometry using a biotinylated antibody to a .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer binding partner. Cell
culture assays can be used advantageously to further evaluate
compounds that score positive in protein binding assays described
herein.
[0258] Cell cultures can also be used to screen the impact of a
drug candidate. For example, drug candidates may decrease or
increase the expression of the .beta.10 gene. In certain
embodiments, the amount of .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer that is produced may be measured
after exposure of the cell culture to the drug candidate. In
certain embodiments, one may detect the actual impact of the drug
candidate on the cell culture. For example, the overexpression of a
particular gene may have a particular impact on the cell culture.
In such cases, one may test a drug candidate's ability to increase
or decrease the expression of the gene or its ability to prevent or
inhibit a particular impact on the cell culture. In other examples,
the production of a particular metabolic product such as a fragment
of a polypeptide, may result in, or be associated with, a disease
or pathological condition. In such cases, one may test a drug
candidate's ability to decrease the production of such a metabolic
product in a cell culture.
Therapeutic/Diagnostic Applications of .beta.10 Polypeptides,
.alpha.2/.beta.10 Heterodimers and Nucleic Acids
[0259] Biological function is anticipated for a .beta.10
polypeptide or an .alpha.2/.beta.10 heterodimer similar to that of
the glycoprotein hormones FAS, TSH, FSH, LH and CG, which, among
other things, are known to act as growth factors in promoting the
development (proliferation, differentiation) of prolactin producing
cells, the thyroid gland and the gonads. These glycoproteins also
act as endocrine hormones in their role as regulators of placental,
thyroidal and gonadal function. FAS plays a role in stimulating
prolactin secretion from decidual cells in the placenta, TSH plays
a major role in the regulation of basal metabolism via the thyroid
gland, and FSH, LH and CG play critical roles in male and female
fertility, as well as in pregnancy. As such, a .beta.10 polypeptide
or an .alpha.2/.beta.10 heterodimer may also play roles in the
regulation of basal metabolism, the development/function of the
gonads, fertility and pregnancy.
[0260] As shown in the example further below, .beta.10 polypeptide
is expressed in brain, liver, fetal liver, stomach, pituitary,
colon, small intestine, thyroid gland, adrenal gland, pancreas,
skin, peripheral blood leucocytes, spleen, testis and placenta. The
fact that .beta.10 is expressed in many of the organs and tissues
that make up the endocrine system suggests an important role for a
.beta.10 polypeptide or an .alpha.2/.beta.10 heterodimer in the
regulation and coordination of one or more endocrine system
functions. The endocrine system is known to exert major control
over metabolism, physiological responses to stress, and the
development and function of reproductive organs.
[0261] The expression of .beta.10 in pituitary, pancreas, adrenal
gland, thyroid gland, stomach, small intestine, colon and liver
indicates a possible role for a .beta.10 polypeptide or an
.alpha.2/.beta.10 heterodimer in the common function of these
organs or tissues, namely, metabolism and energy/nutritional
homeostasis (i.e., energy balance, basal metabolic rate, digestion,
glucose homeostasis, distribution of body fat, general growth).
[0262] The expression of .beta.10 in the pituitary and adrenal
glands indicates a possible role for a .beta.10 polypeptide or an
.alpha.2/.beta.10 heterodimer in one of the critical functions
subserved by these two important organs, namely, the body's ability
to cope with a variety of environmental and physiological stresses
(for example, infection, fever, inflammation, fasting, high and low
blood pressure, anxiety, shock). Consistent with these possible
functions for a .beta.10 polypeptide or an .alpha.2/.beta.10
heterodimer is the expression of .beta.10 in cells and organs known
to be important components of the immune system (peripheral blood
leucocytes, spleen, small intestine).
[0263] In addition, the expression of .beta.10 in pituitary, testis
and placenta indicates a possible role for a .beta.10 polypeptide
or an .alpha.2/.beta.10 heterodimer in the shared function of these
organs, specifically, fertility and pregnancy.
[0264] .beta.10 polypeptide or an .alpha.2/.beta.10 heterodimer may
also act as a growth factor involved in the regeneration
(proliferation and differentiation) of tissues or specialized cell
types present in brain, liver, stomach, pituitary, colon, small
intestine, thyroid gland, adrenal gland, pancreas, skin, peripheral
blood leucocytes, spleen, testis and placenta.
[0265] Consistent with the three major areas of potential .beta.10
polypeptide or an .alpha.2/.beta.10 heterodimer function, i.e., (1)
metabolism and energy/nutritional homeostasis, (2) physiological
responses to stress (including immune system function) and (3)
fertility and pregnancy, is the fact that .alpha.2, which forms a
heterodimer with .beta.10, is expressed (see Example 2 below) in
many of the same organs/tissues (anterior pituitary, placenta,
pancreas, adrenal cortex, intestinal crypts and gall bladder
mucosa) that play important roles in these 3 major areas.
[0266] Based on the above described potential functions, .beta.10
polypeptide or an .alpha.2/.beta.10 heterodimer may be useful for
the treatment and/or diagnosis of metabolic or energy/nutritional
homeostasic disorders. Examples of such disorders include, but are
not limited to, obesity, wasting syndromes (for example, cancer
associated cachexia), myopathies, gastrointestinal disorders,
diabetes, growth failure, hypercholesterolemia, atherosclerosis and
aging. Other diseases involving metabolic or energy/nutritional
homeostasic disorders are encompassed within the therapeutic and
diagnostic utilities that are part of the invention.
[0267] Based on the above described potential functions, .beta.10
polypeptide or an .alpha.2/.beta.10 heterodimer may be useful for
the treatment and/or diagnosis of disorders related to
physiological responses to stress (including immune system
functions). Examples of such disorders include, but are not limited
to, hypertension, immune system dysfunction (for example, excessive
inflammation, autoimmune disease, susceptibility to infection such
as AIDS, poor wound healing, psoriasis, asthma, arthritis and
allergies), shock, anxiety, and high or low blood pressure. Other
diseases involving physiological responses to stress, including,
but not limited to, immune system functions, are also encompassed
within the therapeutic and diagnostic utilities that are part of
the invention.
[0268] Based on the above described potential functions, .beta.10
polypeptide or an .alpha.2/.beta.10 heterodimer may be useful for
the treatment and/or diagnosis of disorders related to pregnancy
and/or the development and function of reproductive organs.
Examples of such disorders include, but are not limited to,
infertility, fertility (contraception), impotence, endometriosis,
menopause, miscarriage, pre-term labor and delivery. Other diseases
involving pregnancy and/or the development and function of
reproductive organs are also encompassed within the therapeutic and
diagnostic utilities that are part of the invention.
[0269] Based on the fact that the .beta.10 polypeptide or an
.alpha.2/.beta.10 heterodimer is likely to have
hormone/growth-factor activities, .beta.10 polypeptide or an
.alpha.2/.beta.10 heterodimer may be useful for the treatment
and/or diagnosis of disorders that could be treated by increasing
cell proliferation and/or differentiation. Examples of such
disorders include, but are not limited to, tissue
damage/degeneration (such as caused by cancer treatments,
infections, autoimmune diseases), aging and wound healing. Other
diseases that could be treated by increasing cell proliferation
and/or differentiation are also encompassed within the therapeutic
and diagnostic utilities that are part of the invention.
[0270] Based on the fact that the .beta.10 polypeptide or an
.alpha.2/.beta.10 heterodimer is likely to have
hormone/growth-factor activities, .beta.10 polypeptide or an
.alpha.2/.beta.10 heterodimer may be useful for the treatment
and/or diagnosis of disorders that could be treated by decreasing
cell proliferation and/or differentiation. Examples of such
disorders include, but are not limited to, cancers, hyperplasias
and hypertrophies. Other diseases that could be treated by
decreasing cell proliferation and/or differentiation are also
encompassed within the therapeutic and diagnostic utilities that
are part of the invention.
[0271] Other diseases caused or mediated by undesirable levels of
.beta.10 polypeptide or an .alpha.2/.beta.10 heterodimer are
encompassed within the therapeutic and diagnostic utilities that
are part of the invention. By way of illustration, such undesirable
levels include excessively elevated levels and sub-normal
levels.
[0272] Transgenic mice were made that overexpressed mouse .alpha.2
alone, mouse .beta.10 alone or the mouse .alpha.2/.beta.10
heterodimer (see example 6). Only those transgenics over expressing
the .alpha.2/.beta.10 heterodimer showed distinct phenotypic
differences as compared to control mice. The .alpha.2/.beta.10
overexpressor transgenic mice exhibited a phenotype characterized
by bilateral thyroid enlargement with multiple follicular papillary
adenomas and resulting hyperthyroidism, as indicated by elevated
serum T4 levels. Other phenotypic changes were felt to be related
to the systemic hyperthyroid state, and included moderate
hepatomegaly, hepatocellular hyperplasia, and slightly decreased
serum cholesterol levels, bilateral renal hypertrophy, and a mild
to moderate leukocytosis with a predominance of lymphocytes (see
example 6). Thus in a normal mouse setting .alpha.2/.beta.10
clearly has a thyroid stimulating hormone (TSH) like activity. Due
to the high level of amino acid conservation between mouse .alpha.2
and human .alpha.2 [88.5% identity and 90.4% similarity for the
predicted mature forms (ie. without signal peptide)], the high
level of amino acid conservation between mouse .beta.10 and human
.beta.10 [93.4% identity and 97.2% similarity for the predicted
mature forms (ie. without signal peptide)], and the very high level
of similarity between mouse thyroid gland biology and human thyroid
gland biology, it is anticipated that human .alpha.2/.beta.10
heterodimer has the same thyroid stimulating hormone (TSH) like
activity as that found for the mouse .alpha.2/.beta.10 heterodimer.
In addition to TSH-like activity, .alpha.2/.beta.10 may have other,
distinct, biological effects in different physiological settings
(i.e., disease conditions), as described in greater detail further
herein.
[0273] TSH influences basal metabolism by regulating the production
of thyroid hormones and is used clinically for enhancing the
detection and treatment of thyroid carcinoma; see McEvoy, G.(ed.),
AHFS Drug Information, pp. 2041-2042, American Society of
Health-System Pharmacists, Inc., Bethesda, Md. (1998). In addition,
diagnostic tests for measuring TSH levels in the blood are commonly
used for determining the functional status of the thyroid gland
when thyroid gland disorder is suspected. It is likely that human
.alpha.2/.beta.10 will have similar clinical utilities as TSH and
will be useful for the treatment and diagnosis of thyroid gland
related diseases and disorders. In addition, human
.alpha.2/.beta.10 may have other therapeutic and diagnostic uses
which are described herein. It is reasonable to surmise that human
.alpha.2/.beta.10 selective binding agents, for example,
antibodies, will have similar clinical utilities to TSH selective
binding agents and will therefore be useful for the treatment and
diagnosis of thyroid gland related diseases and disorders. In
addition, human .alpha.2/.beta.10 selective binding agents may have
other therapeutic and diagnostic uses as described herein.
Compositions and Administration
[0274] Therapeutic compositions are within the scope of the present
invention. Such pharmaceutical compositions may comprise a
therapeutically effective amount of a .beta.10 polypeptide,
.alpha.2/.beta.10 heterodimer or a .beta.10 nucleic acid molecule
in admixture with a pharmaceutically or physiologically acceptable
formulation agent selected for suitability with the mode of
administration. Pharmaceutical compositions may comprise a
therapeutically effective amount of one or more .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer selective binding
agents in admixture with a pharmaceutically or physiologically
acceptable formulation agent selected for suitability with the mode
of administration.
[0275] Acceptable formulation materials preferably are nontoxic to
recipients at the dosages and concentrations employed.
[0276] The pharmaceutical composition may contain formulation
materials for modifying, maintaining or preserving, for example,
the pH, osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption or
penetration of the composition. Suitable formulation materials
include, but are not limited to, amino acids (such as glycine,
glutamine, asparagine, arginine or lysine), antimicrobials,
antioxidants (such as ascorbic acid, sodium sulfite or sodium
hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl,
citrates, phosphates, other organic acids), bulking agents (such as
mannitol or glycine), chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)), complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides,
disaccharides, and other carbohydrates (such as glucose, mannose,
or dextrins), proteins (such as serum albumin, gelatin or
immunoglobulins), coloring, flavoring and diluting agents,
emulsifying agents, hydrophilic polymers (such as
polyvinylpyrrolidone), low molecular weight polypeptides,
salt-forming counterions (such as sodium), preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide), solvents (such as glycerin,
propylene glycol or polyethylene glycol), sugar alcohols (such as
mannitol or sorbitol), suspending agents, surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin,
cholesterol, tyloxapal), stability enhancing agents (sucrose or
sorbitol), tonicity enhancing agents (such as alkali metal halides
(preferably sodium or potassium chloride), mannitol sorbitol),
delivery vehicles, diluents, excipients and/or pharmaceutical
adjuvants. (Remington's Pharmaceutical Sciences, 18.sup.th Edition,
A. R. Gennaro, ed., Mack Publishing Company [1990]).
[0277] The optimal pharmaceutical composition will be determined by
one skilled in the art depending upon, for example, the intended
route of administration, delivery format, and desired dosage. See
for example, Remington's Pharmaceutical Sciences, supra. Such
compositions may influence the physical state, stability, rate of
in vivo release, and rate of in vivo clearance of .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer molecule.
[0278] The primary vehicle or carrier in a pharmaceutical
composition may be either aqueous or non-aqueous in nature. For
example, a suitable vehicle or carrier may be water for injection,
physiological saline solution, or artificial cerebrospinal fluid,
possibly supplemented with other materials common in compositions
for parenteral administration. Neutral buffered saline or saline
mixed with serum albumin are further exemplary vehicles. Other
exemplary pharmaceutical compositions comprise Tris buffer of about
pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may
further include sorbitol or a suitable substitute therefor. In one
embodiment of the present invention, .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer compositions may be prepared for
storage by mixing the selected composition having the desired
degree of purity with optional formulation agents (Remington's
Pharmaceutical Sciences, supra) in the form of a lyophilized cake
or an aqueous solution. Further, the .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer product may be formulated as a
lyophilizate using appropriate excipients such as sucrose.
[0279] The pharmaceutical compositions of this invention can be
selected for parenteral delivery. Alternatively, the compositions
may be selected for inhalation or for delivery through the
digestive tract, such as orally. The preparation of such
pharmaceutically acceptable compositions is within the skill of the
art.
[0280] The formulation components are present in concentrations
that are acceptable to the site of administration. For example,
buffers are used to maintain the composition at physiological pH or
at slightly lower pH, typically within a pH range of from about 5
to about 8.
[0281] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention may be in the
form of a pyrogen-free, parenterally acceptable aqueous solution
comprising the desired .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer molecule in a pharmaceutically acceptable vehicle. A
particularly suitable vehicle for parenteral injection is sterile
distilled water in which a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer molecule is formulated as a sterile,
isotonic solution, properly preserved. Yet another preparation can
involve the formulation of the desired molecule with an agent, such
as injectable microspheres, bio-erodible particles, polymeric
compounds (polylactic acid, polyglycolic acid), or beads, or
liposomes, that provides for the controlled or sustained release of
the product which may then be delivered as a depot injection.
Hyaluronic acid may also be used, and this may have the effect of
promoting sustained duration in the circulation. Other suitable
means for the introduction of the desired molecule include
implantable drug delivery devices.
[0282] In one embodiment, a pharmaceutical composition may be
formulated for inhalation. For example, a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer molecule may be formulated as a dry
powder for inhalation. .beta.10 polypeptide, .alpha.2/.beta.10
heterodimer or .beta.10 nucleic acid molecule inhalation solutions
may also be formulated with a propellant for aerosol delivery. In
yet another embodiment, solutions may be nebulized. Pulmonary
administration is further described in PCT application no.
PCT/US94/001875, which describes pulmonary delivery of chemically
modified proteins.
[0283] It is also contemplated that certain formulations may be
administered orally. In one embodiment of the present invention,
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer molecules
which are administered in this fashion can be formulated with or
without those carriers customarily used in the compounding of solid
dosage forms such as tablets and capsules. For example, a capsule
may be designed to release the active portion of the formulation at
the point in the gastrointestinal tract when bioavailability is
maximized and pre-systemic degradation is minimized. Additional
agents can be included to facilitate absorption of the .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer molecule. Diluents,
flavorings, low melting point waxes, vegetable oils, lubricants,
suspending agents, tablet disintegrating agents, and binders may
also be employed.
[0284] Another pharmaceutical composition may involve an effective
quantity of .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer
molecules in a mixture with non-toxic excipients which are suitable
for the manufacture of tablets. By dissolving the tablets in
sterile water, or other appropriate vehicle, solutions can be
prepared in unit dose form. Suitable excipients include, but are
not limited to, inert diluents, such as calcium carbonate, sodium
carbonate or bicarbonate, lactose, or calcium phosphate; or binding
agents, such as starch, gelatin, or acacia; or lubricating agents
such as magnesium stearate, stearic acid, or talc.
[0285] Additional pharmaceutical compositions will be evident to
those skilled in the art, including formulations involving .beta.10
polypeptides or .alpha.2/.beta.10 heterodimers in sustained- or
controlled-delivery formulations. Techniques for formulating a
variety of other sustained- or controlled-delivery means, such as
liposome carriers, bio-erodible microparticles or porous beads and
depot injections, are also known to those skilled in the art. See
for example, PCT/US93/00829 which describes controlled release of
porous polymeric microparticles for the delivery of pharmaceutical
compositions. Additional examples of sustained-release preparations
include semipermeable polymer matrices in the form of shaped
articles, e.g. films, or microcapsules. Sustained release matrices
may include polyesters, hydrogels, polylactides (U.S. Pat. No.
3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., Biopolymers, 22:547-556 (1983)),
poly (2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed.
Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105
(1982)), ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (EP 133,988). Sustained-release
compositions also may include liposomes, which can be prepared by
any of several methods known in the art. See e.g., Eppstein et al.,
Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 36,676; EP
88,046: EP 143,949.
[0286] The pharmaceutical composition to be used for in vivo
administration typically must be sterile. This may be accomplished
by filtration through sterile filtration membranes. Where the
composition is lyophilized, sterilization using these methods may
be conducted either prior to, or following, lyophilization and
reconstitution. The composition for parenteral administration may
be stored in lyophilized form or in solution. In addition,
parenteral compositions generally are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having a stopper pierceable by a hypodermic injection
needle.
[0287] Once the pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to
administration.
[0288] In a specific embodiment, the present invention is directed
to kits for producing a single-dose administration unit. The kits
may each contain both a first container having a dried protein and
a second container having an aqueous formulation. Also included
within the scope of this invention are kits containing single and
multi-chambered pre-filled syringes (e.g., liquid syringes and
lyosyringes).
[0289] An effective amount of a pharmaceutical composition to be
employed therapeutically will depend, for example, upon the
therapeutic context and objectives. One skilled in the art will
appreciate that the appropriate dosage levels for treatment will
thus vary depending, in part, upon the molecule delivered, the
indication for which the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer molecule is being used, the route of administration,
and the size (body weight, body surface or organ size) and
condition (the age and general health) of the patient. Accordingly,
the clinician may titer the dosage and modify the route of
administration to obtain the optimal therapeutic effect. A typical
dosage may range from about 0.1 .mu.g/kg to up to about 100 mg/kg
or more, depending on the factors mentioned above. In other
embodiments, the dosage may range from 0.1 .mu.g/kg up to about 100
mg/kg; or 1 .mu.g/kg up to about 100 mg/kg; or 5 .mu.g/kg up to
about 100 mg/kg.
[0290] The frequency of dosing will depend upon the pharmacokinetic
parameters of the .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer molecule in the formulation used. Typically, a
clinician will administer the composition until a dosage is reached
that achieves the desired effect. The composition may therefore be
administered as a single dose, or as two or more doses (which may
or may not contain the same amount of the desired molecule) over
time, or as a continuous infusion via implantation device or
catheter. Further refinement of the appropriate dosage is routinely
made by those of ordinary skill in the art and is within the ambit
of tasks routinely performed by them. Appropriate dosages may be
ascertained through use of appropriate dose-response data.
[0291] The route of administration of the pharmaceutical
composition is in accord with known methods, e.g. oral, injection
by intravenous, intraperitoneal, intracerebral (intra-parenchymal),
intracerebroventricular, intramuscular, intra-ocular,
intraarterial, intraportal, or intralesional routes, or by
sustained release systems or implantation device. Where desired,
the compositions may be administered by bolus injection or
continuously by infusion, or by implantation device.
[0292] Alternatively or additionally, the composition may be
administered locally via implantation of a membrane, sponge, or
other appropriate material on to which the desired molecule has
been absorbed or encapsulated. Where an implantation device is
used, the device may be implanted into any suitable tissue or
organ, and delivery of the desired molecule may be via diffusion,
timed release bolus, or continuous administration.
[0293] In some cases, it may be desirable to use pharmaceutical
compositions according to this invention in an ex vivo manner. In
such instances, cells, tissues, or organs that have been removed
from the patient are exposed to pharmaceutical compositions after
which the cells, tissues and/or organs are subsequently implanted
back into the patient.
[0294] In other cases, a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer of this invention can be delivered by implanting
certain cells that have been genetically engineered, using methods
such as those described herein, to express and secrete the
polypeptide or heterodimer. Such cells may be animal or human
cells, and may be autologous, heterologous, or xenogeneic.
Optionally, the cells may be immortalized. In order to decrease the
chance of an immunological response, the cells may be encapsulated
to avoid infiltration of surrounding tissues. The encapsulation
materials are typically biocompatible, semi-permeable polymeric
enclosures or membranes that allow the release of the protein
product(s) but prevent the destruction of the cells by the
patient's immune system or by other detrimental factors from the
surrounding tissues.
[0295] Additional embodiments of the present invention relate to
cells and methods (e.g., homologous recombination and/or other
recombinant production methods) for both the in vitro production of
therapeutic polypeptides and for the production and delivery of
therapeutic polypeptides by gene therapy or cell therapy.
Homologous and other recombination methods may be used to modify a
cell that contains a normally transcriptionally silent .beta.10
gene, or an under expressed gene, and thereby produce a cell which
expresses therapeutically efficacious amounts of .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer.
[0296] Homologous recombination is a technique originally developed
for targeting genes to induce or correct mutations in
transcriptionally active genes (Kucherlapati, Prog. in Nucl. Acid
Res. & Mol. Biol., 36:301, 1989). The basic technique was
developed as a method for introducing specific mutations into
specific regions of the mammalian genome (Thomas et al., Cell,
44:419-428, 1986; Thomas and Capecchi, Cell, 51:503-512, 1987;
Doetschman et al., Proc. Natl. Acad. Sci., 85:8583-8587, 1988) or
to correct specific mutations within defective genes (Doetschman et
al., Nature, 330:576-578, 1987). Exemplary homologous recombination
techniques are described in U.S. Pat. No. 5,272,071 (EP 9193051, EP
Publication No. 505500; PCT/US90/07642, International Publication
No. WO 91/09955).
[0297] Through homologous recombination, the DNA sequence to be
inserted into the genome can be directed to a specific region of
the gene of interest by attaching it to targeting DNA. The
targeting DNA is a nucleotide sequence that is complementary
(homologous) to a region of the genomic DNA. Small pieces of
targeting DNA that are complementary to a specific region of the
genome are put in contact with the parental strand during the DNA
replication process. It is a general property of DNA that has been
inserted into a cell to hybridize, and therefore, recombine with
other pieces of endogenous DNA through shared homologous regions.
If this complementary strand is attached to an oligonucleotide that
contains a mutation or a different sequence or an additional
nucleotide, it too is incorporated into the newly synthesized
strand as a result of the recombination. As a result of the
proofreading function, it is possible for the new sequence of DNA
to serve as the template. Thus, the transferred DNA is incorporated
into the genome.
[0298] Attached to these pieces of targeting DNA are regions of DNA
which may interact with or control the expression of a .beta.10
polypeptide, e.g., flanking sequences. For example, a
promoter/enhancer element, a suppresser, or an exogenous
transcription modulatory element is inserted in the genome of the
intended host cell in proximity and orientation sufficient to
influence the transcription of DNA encoding the desired .beta.10
polypeptide. The control element controls a portion of the DNA
present in the host cell genome. Thus, the expression of the
desired .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer may
be achieved not by transfection of DNA that encodes the .beta.10
gene itself, but rather by the use of targeting DNA (containing
regions of homology with the endogenous gene of interest) coupled
with DNA regulatory segments that provide the endogenous gene
sequence with recognizable signals for transcription of the
.beta.10 gene.
[0299] In an exemplary method, the expression of a desired targeted
gene in a cell (i.e., a desired endogenous cellular gene) is
altered via homologous recombination into the cellular genome at a
preselected site, by the introduction of DNA which includes at
least a regulatory sequence, an exon and a splice donor site. These
components are introduced into the chromosomal (genomic) DNA in
such a manner that this, in effect, results in the production of a
new transcription unit (in which the regulatory sequence, the exon
and the splice donor site present in the DNA construct are
operatively linked to the endogenous gene). As a result of the
introduction of these components into the chromosomal DNA, the
expression of the desired endogenous gene is altered.
[0300] Altered gene expression, as described herein, encompasses
activating (or causing to be expressed) a gene which is normally
silent (unexpressed) in the cell as obtained, as well as increasing
the expression of a gene which is not expressed at physiologically
significant levels in the cell as obtained. The embodiments further
encompass changing the pattern of regulation or induction such that
it is different from the pattern of regulation or induction that
occurs in the cell as obtained, and reducing (including
eliminating) the expression of a gene which is expressed in the
cell as obtained.
[0301] One method by which homologous recombination can be used to
increase, or cause, .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer production from a cell's endogenous .beta.10 gene
involves first using homologous recombination to place a
recombination sequence from a site-specific recombination system
(e.g., Cre/loxP, FLP/FRT) (Sauer, Current Opinion In Biotechnology,
5:521-527, 1994; Sauer, Methods In Enzymology, 225:890-900, 1993)
upstream (that is, 5' to) of the cell's endogenous genomic .beta.10
polypeptide coding region. A plasmid containing a recombination
site homologous to the site that was placed just upstream of the
genomic .beta.10 polypeptide coding region is introduced into the
modified cell line along with the appropriate recombinase enzyme.
This recombinase causes the plasmid to integrate, via the plasmid's
recombination site, into the recombination site located just
upstream of the genomic .beta.10 polypeptide coding region in the
cell line (Baubonis and Sauer, Nucleic Acids Res., 21:2025-2029,
1993; O'Gorman et al., Science, 251:1351-1355, 1991). Any flanking
sequences known to increase transcription (e.g., enhancer/promoter,
intron, translational enhancer), if properly positioned in this
plasmid, would integrate in such a manner as to create a new or
modified transcriptional unit resulting in de novo or increased
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer production
from the cell's endogenous .beta.10 gene.
[0302] A further method to use the cell line in which the site
specific recombination sequence had been placed just upstream of
the cell's endogenous genomic .beta.10 polypeptide coding region is
to use homologous recombination to introduce a second recombination
site elsewhere in the cell line's genome. The appropriate
recombinase enzyme is then introduced into the
two-recombination-site cell line, causing a recombination event
(deletion, inversion, translocation) (Sauer, Current Opinion In
Biotechnology, supra, 1994; Sauer, Methods In Enzymology, supra,
1993) that would create a new or modified transcriptional unit
resulting in de novo or increased .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer production from the cell's endogenous
.beta.10 gene.
[0303] An additional approach for increasing, or causing, the
expression of the .beta.10 polypeptide from a cell's endogenous
.beta.10 gene involves increasing, or causing, the expression of a
gene or genes (e.g., transcription factors) and/or decreasing the
expression of a gene or genes (e.g., transcriptional repressors) in
a manner which results in de novo or increased .beta.10 polypeptide
production from the cell's endogenous .beta.10 gene. This method
includes the introduction of a non-naturally occurring polypeptide
(e.g., a polypeptide comprising a site specific DNA binding domain
fused to a transcriptional factor domain) into the cell such that
de novo or increased .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer production from the cell's endogenous .beta.10 gene
results.
[0304] The present invention further relates to DNA constructs
useful in the method of altering expression of a target gene. In
certain embodiments, the exemplary DNA constructs comprise: (a) one
or more targeting sequences; (b) a regulatory sequence; (c) an
exon; and (d) an unpaired splice-donor site. The targeting sequence
in the DNA construct directs the integration of elements (a)-(d)
into a target gene in a cell such that the elements (b)-(d) are
operatively linked to sequences of the endogenous target gene. In
another embodiment, the DNA constructs comprise: (a) one or more
targeting sequences, (b) a regulatory sequence, (c) an exon, (d) a
splice-donor site, (e) an intron, and (f) a splice-acceptor site,
wherein the targeting sequence directs the integration of elements
(a)-(f) such that the elements of (b)-(f) are operatively linked to
the endogenous gene. The targeting sequence is homologous to the
preselected site in the cellular chromosomal DNA with which
homologous recombination is to occur. In the construct, the exon is
generally 3' of the regulatory sequence and the splice-donor site
is 3' of the exon.
[0305] If the sequence of a particular gene is known, such as the
nucleic acid sequence of the .beta.10 gene presented herein, a
piece of DNA that is complementary to a selected region of the gene
can be synthesized or otherwise obtained, such as by appropriate
restriction of the native DNA at specific recognition sites
bounding the region of interest. This piece serves as a targeting
sequence(s) upon insertion into the cell and will hybridize to its
homologous region within the genome. If this hybridization occurs
during DNA replication, this piece of DNA, and any additional
sequence attached thereto, will act as an Okazaki fragment and will
be incorporated into the newly synthesized daughter strand of DNA.
The present invention, therefore, includes nucleotides encoding a
.beta.10 polypeptide, which nucleotides may be used as targeting
sequences.
[0306] .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer cell
therapy, e.g., the implantation of cells producing .beta.10
polypeptides or .alpha.2/.beta.10 heterodimers is also
contemplated. This embodiment involves implanting cells capable of
synthesizing and secreting a biologically active form of the
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer. Such
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer-producing
cells can be cells that are natural producers of .beta.10
polypeptides or .alpha.2/.beta.10 heterodimers or may be
recombinant cells whose ability to produce .beta.10 polypeptides or
.alpha.2/.beta.10 heterodimers has been augmented by transformation
with a gene encoding the desired .beta.10 polypeptide or with a
gene augmenting the expression of .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer. Such a modification may be
accomplished by means of a vector suitable for delivering the gene
as well as promoting its expression and secretion. In order to
minimize a potential immunological reaction in patients being
administered a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer as may occur with the administration of a polypeptide
of a foreign species, it is preferred that the natural cells
producing .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer be
of human origin and produce human .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer. Likewise, it is preferred that the
recombinant cells producing .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer be transformed with an expression
vector containing a gene encoding a human .beta.10 polypeptide.
[0307] Implanted cells may be encapsulated to avoid the
infiltration of surrounding tissue. Human or non-human animal cells
may be implanted in patients in biocompatible, semipermeable
polymeric enclosures or membranes that allow the release of
.beta.10 polypeptide or .alpha.2/.beta.10 heterodimer but that
prevent the destruction of the cells by the patient's immune system
or by other detrimental factors from the surrounding tissue.
Alternatively, the patient's own cells, transformed to produce
.beta.10 polypeptides or .alpha.2/.beta.10 heterodimers ex vivo,
may be implanted directly into the patient without such
encapsulation.
[0308] Techniques for the encapsulation of living cells are known
in the art, and the preparation of the encapsulated cells and their
implantation in patients may be routinely accomplished. For
example, Baetge et al. (WO95/05452; PCT/US94/09299) describe
membrane capsules containing genetically engineered cells for the
effective delivery of biologically active molecules. The capsules
are biocompatible and are easily retrievable. The capsules
encapsulate cells transfected with recombinant DNA molecules
comprising DNA sequences coding for biologically active molecules
operatively linked to promoters that are not subject to down
regulation in vivo upon implantation into a mammalian host. The
devices provide for the delivery of the molecules from living cells
to specific sites within a recipient. In addition, see U.S. Pat.
Nos. 4,892,538, 5,011,472, and 5,106,627. A system for
encapsulating living cells is described in PCT Application no.
PCT/US91/00157 of Aebischer et al. See also, PCT Application no.
PCT/US91/00155 of Aebischer et al., Winn et al., Exper. Neurol.,
113:322-329 (1991), Aebischer et al., Exper. Neurol., 111:269-275
(1991); and Tresco et al., ASAIO, 38:17-23 (1992).
[0309] In vivo and in vitro gene therapy delivery of .beta.10
polypeptides or .beta.2/.beta.10 heterodimers is also envisioned.
One example of a gene therapy technique is to use the .beta.10 gene
(either genomic DNA, cDNA, and/or synthetic DNA) encoding a
.beta.10 polypeptide which may be operably linked to a constitutive
or inducible promoter to form a "gene therapy DNA construct". The
promoter may be homologous or heterologous to the endogenous gene,
provided that it is active in the cell or tissue type into which
the construct will be inserted. Other components of the gene
therapy DNA construct may optionally include, DNA molecules
designed for site-specific integration (e.g., endogenous sequences
useful for homologous recombination), tissue-specific promoter,
enhancer(s) or silencer(s), DNA molecules capable of providing a
selective advantage over the parent cell, DNA molecules useful as
labels to identify transformed cells, negative selection systems,
cell specific binding agents (as, for example, for cell targeting),
cell-specific internalization factors, and transcription factors to
enhance expression by a vector as well as factors to enable vector
manufacture.
[0310] A gene therapy DNA construct can then be introduced into
cells (either ex vivo or in vivo) using viral or non-viral vectors.
One means for introducing the gene therapy DNA construct is by
means of viral vectors as described herein. Certain vectors, such
as retroviral vectors, will deliver the DNA construct to the
chromosomal DNA of the cells, and the gene can integrate into the
chromosomal DNA. Other vectors will function as episomes, and the
gene therapy DNA construct will remain in the cytoplasm.
[0311] In yet other embodiments, regulatory elements can be
included for the controlled expression of the .beta.10 gene in the
target cell. Such elements are turned on in response to an
appropriate effector. In this way, a therapeutic polypeptide can be
expressed when desired. One conventional control means involves the
use of small molecule dimerizers or rapalogs (as described in
WO9641865 (PCT/US96/099486); WO9731898 (PCT/US97/03137) and
WO9731899 (PCT/US95/03157) used to dimerize chimeric proteins which
contain a small molecule-binding domain and a domain capable of
initiating biological process, such as a DNA-binding protein or
transcriptional activation protein. The dimerization of the
proteins can be used to initiate transcription of the
transgene.
[0312] An alternative regulation technology uses a method of
storing proteins expressed from the gene of interest inside the
cell as an aggregate or cluster. The gene of interest is expressed
as a fusion protein that includes a conditional aggregation domain
which results in the retention of the aggregated protein in the
endoplasmic reticulum. The stored proteins are stable and inactive
inside the cell. The proteins can be released, however, by
administering a drug (e.g., small molecule ligand) that removes the
conditional aggregation domain and thereby specifically breaks
apart the aggregates or clusters so that the proteins may be
secreted from the cell. See, Science 287:816-817, and 826-830
(2000).
[0313] Other suitable control means or gene switches include, but
are not limited to, the following systems. Mifepristone (RU486) is
used as a progesterone antagonist. The binding of a modified
progesterone receptor ligand-binding domain to the progesterone
antagonist activates transcription by forming a dimer of two
transcription factors which then pass into the nucleus to bind DNA.
The ligand binding domain is modified to eliminate the ability of
the receptor to bind to the natural ligand. The modified steroid
hormone receptor system is further described in U.S. Pat. No.
5,364,791; WO9640911, and WO9710337.
[0314] Yet another control system uses ecdysone (a fruit fly
steroid hormone) which binds to and activates an ecdysone receptor
(cytoplasmic receptor). The receptor then translocates to the
nucleus to bind a specific DNA response element (promoter from
ecdysone-responsive gene). The ecdysone receptor includes a
transactivation domain/DNA-binding domain/ligand-binding domain to
initiate transcription. The ecdysone system is further described in
U.S. Pat. No. 5,514,578; WO9738117; WO9637609; and WO9303162.
[0315] Another control means uses a positive
tetracycline-controllable transactivator. This system involves a
mutated tet repressor protein DNA-binding domain (mutated tet R-4
amino acid changes which resulted in a reverse
tetracycline-regulated transactivator protein, i.e., it binds to a
tet operator in the presence of tetracycline) linked to a
polypeptide which activates transcription. Such systems are
described in U.S. Pat. Nos. 5,464,758; 5,650,298 and 5,654,168.
[0316] Additional expression control systems and nucleic acid
constructs are described in U.S. Pat. Nos. 5,741,679 and 5,834,186,
to Innovir Laboratories Inc.
[0317] In vivo gene therapy may be accomplished by introducing the
gene encoding a .beta.10 polypeptide into cells via local injection
of a .beta.10 nucleic acid molecule or by other appropriate viral
or non-viral delivery vectors. Hefti, Neurobiology, 25:1418-1435
(1994). For example, a nucleic acid molecule encoding a .beta.10
polypeptide of this invention may be contained in an
adeno-associated virus (AAV) vector for delivery to the targeted
cells (e.g., Johnson, International Publication No. WO95/34670;
International Application No. PCT/US95/07178). The recombinant AAV
genome typically contains AAV inverted terminal repeats flanking a
DNA sequence encoding a .beta.10 polypeptide operably linked to
functional promoter and polyadenylation sequences.
[0318] Alternative suitable viral vectors include, but are not
limited to, retrovirus, adenovirus, herpes simplex virus,
lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus,
alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma
virus vectors. U.S. Pat. No. 5,672,344 describes an in vivo
viral-mediated gene transfer system involving a recombinant
neurotrophic HSV-1 vector. U.S. Pat. No. 5,399,346 provides
examples of a process for providing a patient with a therapeutic
protein by the delivery of human cells which have been treated in
vitro to insert a DNA segment encoding a therapeutic protein.
Additional methods and materials for the practice of gene therapy
techniques are described in U.S. Pat. No. 5,631,236 involving
adenoviral vectors; U.S. Pat. No. 5,672,510 involving retroviral
vectors; and U.S. Pat. No. 5,635,399 involving retroviral vectors
expressing cytokines.
[0319] Nonviral delivery methods include, but are not limited to,
liposome-mediated transfer, naked DNA delivery (direct injection),
receptor-mediated transfer (ligand-DNA complex), electroporation,
calcium phosphate precipitation, and microparticle bombardment
(e.g., gene gun). Gene therapy materials and methods may also
include the use of inducible promoters, tissue-specific
enhancer-promoters, DNA sequences designed for site-specific
integration, DNA sequences capable of providing a selective
advantage over the parent cell, labels to identify transformed
cells, negative selection systems and expression control systems
(safety measures), cell-specific binding agents (for cell
targeting), cell-specific internalization factors, and
transcription factors to enhance expression by a vector as well as
methods of vector manufacture. Such additional methods and
materials for the practice of gene therapy techniques are described
in U.S. Pat. No. 4,970,154 involving electroporation techniques;
WO96/40958 involving nuclear ligands; U.S. Pat. No. 5,679,559
describing a lipoprotein-containing system for gene delivery; U.S.
Pat. No. 5,593,875 concerning methods for calcium phosphate
transfection; and U.S. Pat. No. 4,945,050 wherein biologically
active particles are propelled at cells at a speed whereby the
particles penetrate the surface of the cells and become
incorporated into the interior of the cells.
[0320] It is also contemplated that .beta.10 gene therapy or cell
therapy can further include the delivery of one or more additional
polypeptide(s) in the same or a different cell(s). Such cells may
be separately introduced into the patient, or the cells may be
contained in a single implantable device, such as the encapsulating
membrane described above, or the cells may be separately modified
by means of viral vectors.
[0321] A means to increase endogenous .beta.10 polypeptide
expression in a cell via gene therapy is to insert one or more
enhancer elements into the .beta.10 polypeptide promoter, where the
enhancer element(s) can serve to increase transcriptional activity
of the .beta.10 gene. The enhancer element(s) used will be selected
based on the tissue in which one desires to activate the gene(s);
enhancer elements known to confer promoter activation in that
tissue will be selected. For example, if a gene encoding a .beta.10
polypeptide is to be "turned on" in T-cells, the lck promoter
enhancer element may be used. Here, the functional portion of the
transcriptional element to be added may be inserted into a fragment
of DNA containing the .beta.10 polypeptide promoter (and
optionally, inserted into a vector and/or 5' and/or 3' flanking
sequence(s), etc.) using standard cloning techniques. This
construct, known as a "homologous recombination construct", can
then be introduced into the desired cells either ex vivo or in
vivo.
[0322] Gene therapy can also be used to decrease .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer expression by
modifying the nucleotide sequence of the endogenous promoter(s).
Such modification is typically accomplished via homologous
recombination methods. For example, a DNA molecule containing all
or a portion of the promoter of the .beta.10 gene(s) selected for
inactivation can be engineered to remove and/or replace pieces of
the promoter that regulate transcription. For example the TATA box
and/or the binding site of a transcriptional activator of the
promoter may be deleted using standard molecular biology
techniques; such deletion can inhibit promoter activity thereby
repressing the transcription of the corresponding .beta.10 gene.
The deletion of the TATA box or the transcription activator binding
site in the promoter may be accomplished by generating a DNA
construct comprising all or the relevant portion of the .beta.10
polypeptide promoter(s) (from the same or a related species as the
.beta.10 gene(s) to be regulated) in which one or more of the TATA
box and/or transcriptional activator binding site nucleotides are
mutated via substitution, deletion and/or insertion of one or more
nucleotides. As a result, the TATA box and/or activator binding
site has decreased activity or is rendered completely inactive. The
construct will typically contain at least about 500 bases of DNA
that correspond to the native (endogenous) 5' and 3' DNA sequences
adjacent to the promoter segment that has been modified. The
construct may be introduced into the appropriate cells (either ex
vivo or in vivo) either directly or via a viral vector as described
herein. Typically, the integration of the construct into the
genomic DNA of the cells will be via homologous recombination,
where the 5' and 3' DNA sequences in the promoter construct can
serve to help integrate the modified promoter region via
hybridization to the endogenous chromosomal DNA.
Other Uses of the Nucleic Acids and Polypeptides of this
Invention
[0323] Nucleic acid molecules of the present invention (including
those that do not themselves encode biologically active
polypeptides) may be used to map the locations of the .beta.10 gene
and related genes on chromosomes. Mapping may be done by techniques
known in the art, such as PCR amplification and in situ
hybridization.
[0324] .beta.10 nucleic acid molecules (including those that do not
themselves encode biologically active polypeptides), may be useful
as hybridization probes in diagnostic assays to test, either
qualitatively or quantitatively, for the presence of a .beta.10 DNA
or corresponding RNA in mammalian tissue or bodily fluid
samples.
[0325] The .beta.10 polypeptides or .alpha.2/.beta.10 heterodimers
may be used (simultaneously or sequentially) in combination with
one or more cytokines, growth factors, antibiotics,
anti-inflammatories, and/or chemotherapeutic agents as is
appropriate for the indication being treated.
[0326] Other methods may also be employed where it is desirable to
inhibit the activity of one or more .beta.10 polypeptides or
.alpha.2/.beta.10 heterodimers of this invention. Such inhibition
may be effected by nucleic acid molecules which are complementary
to and hybridize to expression control sequences (triple helix
formation) or to .beta.10 mRNA. For example, antisense DNA or RNA
molecules, which have a sequence that is complementary to at least
a portion of the selected .beta.10 gene(s) can be introduced into
the cell. Anti-sense probes may be designed by available techniques
using the sequence of the .beta.10 polypeptide disclosed herein.
Typically, each such antisense molecule will be complementary to
the start site (5' end) of each selected .beta.10 gene. When the
antisense molecule then hybridizes to the corresponding .beta.10
mRNA, translation of this mRNA is prevented or reduced. Anti-sense
inhibitors provide information relating to the decrease or absence
of a .beta.10 polypeptide or .alpha.2/.beta.10 heterodimer in a
cell or organism.
[0327] Alternatively, gene therapy may be employed to create a
dominant-negative inhibitor of one or more .beta.10 polypeptides or
.alpha.2/.beta.10 heterodimers. In this situation, the DNA encoding
a mutant polypeptide of each selected .beta.10 polypeptide can be
prepared and introduced into the cells of a patient using either
viral or non-viral methods as described herein. Each such mutant is
typically designed to compete with endogenous polypeptide or
heterodimer in its biological role.
[0328] In addition, a .beta.10 polypeptide or .alpha.2/.beta.10
heterodimer of this invention, whether biologically active or not,
may be used as an immunogen, that is, the polypeptide contains at
least one epitope to which antibodies may be raised. Selective
binding agents that bind to a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer (as described herein) may be used for
in vivo and in vitro diagnostic purposes, including, but not
limited to, use in labeled form to detect the presence of .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer in a body fluid or
cell sample. The antibodies may also be used to prevent, treat, or
diagnose a number of diseases and disorders, including those
recited herein. The antibodies may bind to a .beta.10 polypeptide
or .alpha.2/.beta.10 heterodimer so as to diminish or block at
least one activity characteristic of a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer, or may bind to a polypeptide to
increase at least one activity characteristic of a .beta.10
polypeptide or .alpha.2/.beta.10 heterodimer (including by
increasing the pharmacokinetics of a .beta.10 polypeptide or
.alpha.2/.beta.10 heterodimer).
[0329] cDNA encoding human .beta.10 polypeptide in E. coli was
deposited with the American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, Va. 20110-2209, on Dec. 28, 1999,
under accession number PTA-1210.
[0330] The following examples are intended for illustration
purposes only, and should not be construed as limiting the scope of
the invention in any way.
EXAMPLE 1
DNA Encoding Human Beta-10
[0331] The amino acid sequence of CG-(chorionic
gonadotropin)-.beta.-subun- it was Blasted against an, in house
generated, Virtual Protein database derived from public human
genomic sequences present in GenBank. A virtual protein containing
a 45 amino acid region with significant homology to the carboxy
half of CG-.beta. was identified. The short (135 base pair) region
of human genomic sequence that encoded the 45 amino acid stretch
came from an over 160 kilobase pair GenBank human genomic DNA
sequence (accession # AL049871). By analyzing an 8 kilobase pair
stretch of genomic sequence just 5' of the 135 base pair sequence,
a region having significant homology (and containing a frameshift)
to the N-terminal half of CG-.beta. was identified. The nucleotide
sequence of this novel gene was compiled from the genomic
sequences. The amino acid sequence of this compiled gene had
significant homology to the four known human glycoprotein hormone
.beta.-subunit polypeptides and had an N-terminal predicted signal
peptide, consistent with this novel human gene being a new
.beta.-like member of the glycoprotein hormone family. There was a
4.5-kb intron located between the two putative N-terminal half and
C-terminal half coding exons. Intron spanning PCR (see Tissue
Expression, Beta-10 section below) of cDNAs from various tissues
sources revealed that .beta.10 was expressed in numerous tissues
including pituitary.
[0332] The full coding region (ATG to TGA stop codon) and some of
the 3'UTR (untranslated region) of .beta.10 was cloned as one
fragment by PCR using the following reaction mix and PCR
conditions:
[0333] Template: ten microliters of Human Pituitary Marathon Ready
cDNA (Clontech Laboratories, Inc., Palo Alto, Calif.; catalog no.
7424-1).
3 Forward primer: 5'-ATGAAGCTGGCATTCCTCTTCCTT-3'. (SEQ ID NO: 4)
Reverse primer: 5'-GCATGTGCTGCTCACACAGGT-3'. (SEQ ID NO: 5)
[0334] Final concentration of each primer: 1.0 micromolar.
[0335] Final concentration of dNTPs: 200 micromolar.
[0336] Five units of Pfu polymerase (Stratagene, La Jolla,
Calif.).
[0337] Ten microliters of 10.times. Pfu reaction buffer
(Stratagene, La Jolla, Calif.).
[0338] Ten microliters of GC melt (Clontech Laboratories, Inc.,
Palo Alto, Calif.; Advantage GC cDNA PCR kit; catalog no.
K1907-1).
[0339] Final reaction volume: 100 microliters.
[0340] Cycling conditions: 94.degree. C. for sixty seconds followed
by 45 cycles of 94.degree. C. (ten seconds), 60.degree. C. (twenty
seconds), 72.degree. C. (ninety seconds), and then at the end of
the 45th cycle an incubation at 72.degree. C. for seven minutes,
then an additional 10 cycles of 94.degree. C. (ten seconds),
60.degree. C. (twenty seconds), 72.degree. C. (ninety seconds), and
then at the end of the additional 10th cycle an incubation at
72.degree. C. for seven minutes.
[0341] The PCR reaction was run on an agarose gel, and a single
band was seen. This DNA band was cloned into pPCR-Script AMP
(Stratagene). The sequence of the insert in one of the resulting
clones is that of SEQ ID NO: 2 which contains the full coding
region (ATG to TGA stop codon) and some of the 3'UTR (untranslated
region) of .beta.10.
[0342] The following is a list and description of .beta.10
sequences from publicly available databases:
[0343] GenBank Accession # AL049871: 170 kilobase pairs of human
genomic sequence. No exons, genes or homologies are identified in
this record and the full coding region sequence of .beta.10 is
broken up by an intronic sequence.
[0344] GenBank Accession # AL118555: 126 kilobase pairs of human
genomic sequence. No exons, genes or homologies are identified in
this record and the full coding region sequence of .beta.10 is
broken up by an intronic sequence.
EXAMPLE 2
Tissue Expression, Beta-10
[0345] Using a PCR fragment as a probe, it was not possible to
obtain a hybridization signal on various Human Multiple Tissue
Northern Blots (Clontech Inc., Palo Alto, Calif.). Intron spanning
PCR was used to determine the expression pattern of beta-10 as
described below.
[0346] For the Human Sure-RACE Panels (OriGene Technologies, Inc.,
Rockville, Md.; catalog no. HRAA-101) the cDNA samples represented
brain, heart, kidney, spleen, liver, colon, lung, small intestine,
muscle, stomach, testis, placenta, pituitary, thyroid gland,
adrenal gland, pancreas, ovary, uterus, prostate, peripheral blood
leucocytes, fetal brain, fetal liver, fat and mammary gland. Each
cDNA sample was in a separate tube in the form of a dried down
pellet of DNA. The reaction mixture was composed as follows:
4 Forward primer: 5'-CTGCAGGTGCCTTCGGATC-3'; (SEQ ID NO: 6) Reverse
primer: 5'-GCATGTGCTGCTCACACAGGT- -3'; (SEQ ID NO: 5)
[0347] Amount of each primer: 0.5 picomoles;
[0348] Final concentration of dNTPs: 200 micromolar;
[0349] 2.5 microliters of GC melt (Clontech Laboratories, Inc.,
Palo Alto, Calif.; Advantage GC cDNA PCR kit; catalog no.
K1907-1);
[0350] 2.5 units of Taq (Boehringer Mannheim, Indianapolis, Ind.;
PCR Core Kit; catalog no. 1578 553);
[0351] 2.5 microliters of 10.times.PCR-reaction buffer (Boehringer
Mannheim, Indianapolis, Ind.; PCR Core Kit; catalog no. 1578
553);
[0352] For each cDNA sample the above reaction mixture was made up
to a volume of 25 microliters, and 20 microliters of this mixture
was added to the dried down cDNA pellet. The PCR conditions were as
follows: 94.degree. C. for sixty seconds, followed by 5 cycles of
94.degree. C. (ten seconds) and 72.degree. C. (forty seconds),
followed by 5 cycles of 94.degree. C. (ten seconds)and 70.degree.
C. (forty seconds), followed by 35 cycles of 94.degree. C. (ten
seconds)and 68.degree. C. (forty seconds), and then followed by
68.degree. C. for seven minutes.
[0353] PCR products were then analyzed by agarose gel
electrophoresis. The correct size PCR product of 293 base pairs,
indicating expression of .beta.10, was found in colon, small
intestine, testis, pituitary and fetal liver.
[0354] For the Human Rapid-Scan Plate (OriGene Technologies, Inc.,
Rockville Md.; catalog no. HSCA-101) the cDNA samples represented
brain, heart, kidney, spleen, liver, colon, lung, small intestine,
muscle, stomach, testis, placenta, salivary gland, thyroid gland,
adrenal gland, pancreas, ovary, uterus, prostate, skin, peripheral
blood leucocytes, bone marrow, fetal brain and fetal liver. Each
cDNA sample was in a separate tube in the form of a dried down
pellet of DNA.
[0355] The reaction mixture that was utilized was as follows:
5 Forward primer: 5'-CTGCAGGTGCCTTCGGATC-3'; (SEQ ID NO: 6) Reverse
primer: 5'-GCATGTGCTGCTCACACAGGT- -3'; (SEQ ID NO: 5)
[0356] Amount of each primer: 0.5 picomoles;
[0357] Final concentration of dNTPs: 200 micromolar;
[0358] 2.5 microliters of GC melt (Clontech Laboratories, Inc.,
Palo Alto, Calif.; Advantage GC cDNA PCR kit; catalog no.
K1907-1);
[0359] 2.5 units of Taq (Boehringer Mannheim, Indianapolis, Ind.;
PCR Core Kit; catalog no. 1578 553);
[0360] 2.5 microliters of 10.times. PCR-reaction buffer (Boehringer
Mannheim, Indianapolis, Ind.; PCR Core Kit; catalog no. 1578
553);
[0361] For each cDNA sample the above reaction mixture was made up
to a volume of 25 microliters, and then 20 microliters of this
mixture was added to the dried down cDNA pellet. The PCR conditions
were as follows: 94.degree. C. for sixty seconds followed by 5
cycles of 94.degree. C. (ten seconds), 72.degree. C. (forty
seconds) and then followed by 5 cycles of 94.degree. C. (ten
seconds), 70.degree. C. (forty seconds) and then followed by 35
cycles of 94.degree. C. (ten seconds), 68.degree. C. (forty
seconds) and then followed by 68.degree. C. for seven minutes.
[0362] PCR products were then analyzed by agarose gel
electrophoresis. The correct size PCR product of 293 base pairs,
indicating expression of .beta.10, was found in brain, spleen,
liver, colon, stomach, placenta, thyroid gland, adrenal gland,
pancreas, skin and peripheral blood leucocytes.
[0363] Combining the expression results from the Human Sure-RACE
Panels and the Human Rapid-Scan Plate indicated that .beta.10 is
expressed in brain, liver, fetal liver, stomach, pituitary, colon,
small intestine, thyroid gland, adrenal gland, pancreas, skin,
peripheral blood leucocytes, spleen, testis and placenta.
EXAMPLE 3
Tissue Expression, .alpha.2
[0364] Northern analysis was carried out to determine the
expression pattern of alpha-2. The probe for the Northerns was a
390-base pair PCR product (corresponding to nucleotides 56-445 of
SEQ ID NO: 1 from WO99/41377). This PCR product was generated via a
466-base pair PCR intermediate as follows:
[0365] PCR was first used to clone a 466-base pair fragment of
alpha-2 from human testis cDNA using the following reaction mixture
and PCR conditions:
[0366] Template: ten microliters of Human Testis Marathon Ready
cDNA (Clontech Laboratories, Inc., Palo Alto, Calif.; catalog no.
7414-1);
6 Forward primer: 5'-GAGACATCTCCCCACTGTGTTT-3'; (SEQ ID NO: 7)
Reverse primer: 5'-GTTTCCCCCAACAGAATGTCA- A-3' ; (SEQ ID NO: 8)
[0367] Final concentration of each primer: 1.0 micromolar;
[0368] Final concentration of dNTPs: 200 micromolar;
[0369] Five units of Pfu polymerase;
[0370] Final reaction volume: 100 microliters;
[0371] Cycling conditions: 94.degree. C. for sixty seconds followed
by 35 cycles of 94.degree. C. (ten seconds), 60.degree. C. (thirty
seconds), 72.degree. C. (sixty seconds), and then at the end of the
35th cycle an incubation at 72.degree. C. for five minutes.
[0372] The PCR reaction was run on an agarose gel, and four
distinct bands were seen. The multiple bands arose from PCR
amplification of contaminating human genomic DNA present in the
Human Testis Marathon Ready cDNA. The 466-base pair-PCR product was
isolated from the agarose gel and cloned. A plasmid clone
containing the 466-base pair sequence was used as a template for
generating the 390-base pair PCR fragment using the following
reaction mix and PCR conditions:
[0373] Template: ten picograms of the plasmid clone containing the
above mentioned 466-base pair sequence;
7 Forward primer: 5'-ATGCCTATGGCGTCCCCTCAAAC-3'; (SEQ ID NO: 9)
Reverse primer: 5'-CTAGTAGCGAGAGAGGCGACACATGTCA-3'; (SEQ ID NO:
10)
[0374] Final concentration of each primer: 1.0 micromolar.
[0375] Final concentration of dNTPs: 200 micromolar;
[0376] Ten units of Taq polymerase;
[0377] Final reaction volume: 100 microliters;
[0378] Cycling conditions: 94.degree. C. for sixty seconds,
followed by 35 cycles of 94.degree. C. (ten seconds), 68.degree. C.
(sixty seconds), and then at the end of the 35th cycle an
incubation at 68.degree. C. for six minutes.
[0379] The 390-base pair PCR product was then purified by agarose
gel electrophoresis. This PCR fragment was labeled with .sup.32P
and hybridized to various Clontech Human Multiple Tissue Northern
Blots (tissues/cells represented were: pancreas, adrenal medulla,
thyroid, adrenal cortex, testis, thymus, small intestine, stomach,
spleen, prostate, ovary, colon, peripheral blood leucocytes, brain,
heart, skeletal muscle, kidney, liver, placenta and lung) and to a
Northern blot made with pituitary mRNA using high stringency
conditions as follows:
[0380] Hybridization was for one hour at 68.degree. C. using
Clontech "ExpressHyb Hybridization Solution". The blots were washed
in 2.times.SSC, 0.1% SDS at room temperature twice, for twenty
minutes each time. The blots were then washed in 0.1.times.SSC,
0.1% SDS at 50.degree. C. for ten minutes, and then exposed to
film.
[0381] A strong signal representing a single band was obtained in
the pancreas mRNA lane and the pituitary mRNA lane. A significantly
weaker signal was seen in the placenta mRNA lane.
[0382] In situ hybridization was done to further determine sites of
.alpha.2 gene expression. A panel of normal embryonic (E10.5
through E18.5) and adult mouse tissues and adult rhesus monkey
tissues were fixed in 4% paraformaldehyde, embedded in paraffin,
and sectioned at 5 micrometers. Prior to in situ hybridization,
tissues were permeabilized with 0.2M HCL, followed by digestion
with Proteinase K and acetylation with triethanolamine and acetic
anhydride. Sections were hybridized overnight at 55.degree. C. with
a .sup.33p-labeled antisense RNA probe complementary to either the
mouse or human (for rhesus tissues) .alpha.2 sequence and with
sense (control) probes. The antisense and sense .sup.33p-labeled
RNA probes were obtained by in vitro transcription of plasmid DNAs
containing either the mouse .alpha.2 cDNA (bacterial clone no.
1224990 from the public WashU-HHMI Mouse EST Project) or the human
.alpha.2 cDNA (plasmid clone containing the above described PCR
generated 390-base pair human .alpha.2 coding region sequence).
[0383] Following hybridization, sections were washed in buffer,
treated with RNaseA to remove unhybridized probe, and then
subjected to a high stringency wash in 0.1.times.SSC at 55.degree.
C. Slides were dipped in Kodak NTB2 emulsion, exposed at 4.degree.
C. for two-three weeks, developed, and then counterstained.
Sections were examined with darkfield and standard illumination to
allow simultaneous evaluation of tissue morphology and
hybridization signal. The following tissues were then examined:
[0384] Mouse tissues: Brain (1 sagittal, 2 coronal sections); GI
tract(esophagus, stomach, duodenum, jejunum, ileum, proximal &
distal colon); pituitary; liver; lung; heart; spleen; thymus; lymph
nodes; kidney; adrenal; bladder; pancreas; salivary gland; male and
female reproductive organs (ovary, oviduct and uterus in the
female; testis, epididymus, prostate, seminal vesicle and vas
deferens in the male); BAT & WAT (subcutaneous, peri-renal);
bone (femur); skin; breast; and skeletal muscle.
[0385] Rhesus tissues: adrenal gland; liver; gall bladder;
intestine; pancreas; and salivary gland.
[0386] Both mouse and human antisense probes produced positive
signal detectable above a very low level of background seen with
the sense strand controls. In the embryonic mouse, no signal was
observed in any major organs from E8.5 through E18.5. At E15.5 and
E18.5, signal was present over scattered cells adjacent to some of
the developing bones of the head and teeth. In the adult mouse, a
moderate level of signal was present in the adrenal cortex. A lower
level of signal was detectable in the anterior and intermediate
lobes of the pituitary as well as in intestinal epithelium at the
level of the crypts. In addition, grain density was slightly above
background in developing sperm within the seminiferous tubules of
the testis and in granulosa cells surrounding developing follicles
in the ovary.
[0387] In rhesus tissues, moderate signal was noted in the adrenal
cortex, gall bladder epithelium, and in the intestinal epithelium
primarily at the level of the crypts.
[0388] Combining the expression results from the .alpha.2 Northerns
and the .alpha.2 in situ analysis indicates that .alpha.2 is
expressed in anterior pituitary, placenta, pancreas, adrenal
cortex, intestinal crypts and gall bladder mucosa.
EXAMPLE 4
Antibodies Against .alpha.2
[0389] Rabbit polyclonal antibodies were generated against .alpha.2
by immunizing rabbits with peptide CSPRYSVLVASGYRHN (SEQ ID NO: 28)
that had been conjugated to Keyhole Limpet Hemocyanin (cat# 77605
Pierce Inc., Rockford, Ill.). The peptide was synthesized with a
C-terminal amide so rather than the C-terminus being COOH the
C-terminus was CONH.sub.2. The RYSVLVASGYRHN portion of the peptide
sequence is totally conserved between human and mouse .alpha.2.
This region was chosen so that the antibodies would be able to bind
to human .alpha.2 and mouse .alpha.2. The antibodies were affinity
purified from rabbit serum over a column (SulfoLink Kit, cat#
44895, Pierce Inc., Rockford, Ill.) to which the peptide antigen
(SEQ ID NO: 28) had been attached. Western blot analysis of
conditioned media harvested from 293 cells that had been
transfected with either a human .alpha.2-polyHis-tag mammalian
expression vector or a human alpha-subunit-polyHis-tag mammalian
expression vector demonstrated that these affinity purified
polyclonal antibodies had high specificity for .alpha.2 polypeptide
and did not cross react with alpha-subunit.
EXAMPLE 5
DNA Encoding Mouse Beta-10
[0390] Various human Beta-10 cDNA probes were used to probe a mouse
genomic 129SvJ BAC library arrayed on high density filters
(catalog#FBAC-4431, Genome Systems, St. Louis, Mo.). The mouse BAC
clone in plate#218-well#P22 was obtained (catalog# FBAC-4432,
Genome Systems, St. Louis, Mo.). A 10-kb HindIII sub-fragment from
this BAC clone hybridized strongly to a human Beta-10 cDNA probe.
This 10-kb HindIII fragment was subcloned into pBluescriptII-KS(-)
and fully sequenced. Computational analysis of this 10-kb mouse
genomic sequence was used to identify two exons encoding the mouse
ortholog of human Beta-10.
[0391] Primers were designed from this electronic sequence to clone
the mouse Beta-10 cDNA as follows:
[0392] Template: twenty microliters of mouse testis Marathon Ready
cDNA (Clontech Laboratories, Inc., Palo Alto, Calif.; catalog no.
7455-1).
8 Forward primer: 5'-ATTACTAGTTCCACCATGAAGTTGGTATACCTTGTCCTT-3';
(SEQ ID NO: 14) Reverse primer: 5'-TTAATAATCGATCGTCAGATG-
GTCTCACACTCAGTG-3'; (SEQ ID NO: 15)
[0393] Final concentration of each primer: 1.0 micromolar.
[0394] PCR kit: Expand High Fidelity PCR System (catalog# 1732641,
Boehringer Mannheim Corporation, Indianapolis, Ind.).
[0395] Final reaction volume: 50 microliters;
[0396] Cycling conditions: 94.degree. C. for sixty seconds,
followed by 55 cycles of 94.degree. C. (ten seconds), 65.degree. C.
(twenty seconds), 72.degree. C. (forty seconds), and then at the
end of the 55th cycle an incubation at 72.degree. C. for seven
minutes.
[0397] The 0.4-kb PCR product was then purified by agarose gel
electrophoresis and cloned into pCR2.1 (Invitrogen Inc., Carlsbad,
Calif.). A clone containing the full coding region cDNA of mouse
Beta-10 (SEQ ID NO: 12) was identified by sequencing.
EXAMPLE 6
Production and Analysis of Transgenic Mice Over Expressing .alpha.2
Alone, .beta.10 Alone and Co-Expressinq .alpha.2 and .beta.10
[0398] Transgenic mice over expressing .alpha.2 alone, .beta.10
alone and co-expressing .alpha.2 and .beta.10 from the human
apolipoprotein E promoter were generated essentially as previously
described (Simonet et al, 1997, Cell vol 89 p309-319). This human
apoE promoter vector directs high level, liver specific, gene
expression in transgenic mice, and has been previously used to
generate transgenic mice having high levels of transgene encoded
secreted protein in their circulation. For all phenotypic analyses
described below the non-transgenic controls were mice that were
produced during the same series of microinjections as the
transgenic expressors in question.
[0399] Genomic (ie. containing .alpha.2 and .beta.10 exons and
introns) transgenes were used for generating transgenics to
maximize expression. Additionally, in all cases a Kozak site
(CCACC) was engineered just 5' of the start site ATG.
[0400] The human apoE promoter expression vector contains the HCR
(liver specific enhancer element) followed by the apoE promoter and
first intron (in apoE 5'UTR) and then by the SV40
polyA-signal/terminator. There are unique SpeI and SfiI (compatible
with PvuI) sites for directional cloning of genes in between the
apoE promoter-first intron and the SV40 polyA-signal/terminator
regions of the vector.
[0401] The following procedure was used to generate the "mouse
genomic .alpha.2 human apoE expression vector transgene":
[0402] Pools of the BAC Mouse ES (129SvJ) Genomic Screening Kit
(catalog#BDTW-7460, Genome Systems, St. Louis, Mo.) were screened
by PCR using mouse .alpha.2 specific primers. The mouse BAC clone
in plate#44-well#H19 was obtained (catalog# FBAC-4432, Genome
Systems, St. Louis, Mo.). A 12-kb XbaI sub-fragment from this BAC
clone hybridized strongly to a mouse .alpha.2 cDNA probe. This
12-kb XbaI fragment was subcloned into
pBluescriptIII-KS(-)(Stratagene Inc, La Jolla, Calif.) and fully
sequenced. Computational analysis of this 12-kb mouse genomic
sequence was used to identify the three .alpha.2 coding exons.
[0403] Primers were designed to amplify the complete mouse genomic
.alpha.2 coding sequence (start ATG to stop TAG; SEQ ID NO: 18) as
follows:
[0404] Template: 5 nanograms of the 12-kb XbaI pBluescriptII-KS(-)
clone DNA.
9 Forward primer: 5'-CCGCACTAGTTCCACCATGCCCATGGCACCACGAGT-3'; (SEQ
ID NO: 16) Reverse primer: 5'-GCGGCGTTCGATCGCTAGTAGCGGGA-
GAAACGGCACATATC-3'; (SEQ ID NO: 17)
[0405] Final concentration of each primer: 1.0 micromolar.
[0406] PCR kit: Pfu DNA Polymerase kit (Stratagene, La Jolla,
Calif.).
[0407] Final reaction volume: 50 microliters;
[0408] Cycling conditions: 94.degree. C. for sixty seconds,
followed by 40 cycles of 94.degree. C. (ten seconds), 60.degree. C.
(twenty seconds), 72.degree. C. (180 seconds), and then at the end
of the 40th cycle an incubation at 72.degree. C. for four
minutes.
[0409] The 0.8-kb PCR product was purified by agarose gel
electrophoresis, cut with SpeI and PvuI and cloned into SpeI-SfiI
cut human apoE expression vector. A clone containing the accurate
full genomic coding region mouse .alpha.2 was identified by
sequencing. DNA from this "mouse genomic .alpha.2 human apoE
expression vector transgene" clone was digested with ClaI and
ApaLI; the 4-kb band was purified by gel electrophoresis and used
to generated transgenic mice as previously described (Simonet et
al, 1997, Cell vol 89 p309-319).
[0410] Transgenic mice were identified by PCR as follows:
[0411] Template: ear punch DNA.
10 Forward primer: 5'-GCCTCTAGAAAGAGCTGGGAC-3'; (SEQ ID NO: 19)
Reverse primer: 5'-CGCCGTGTTCCATTTATGAGC-3- '; (SEQ ID NO: 20)
[0412] Final concentration of each primer: 1.0 micromolar.
[0413] PCR kit: Ready-to-Go PCR Beads (Amersham Pharmacia Biotech
Inc., Piscataway, N.J.).
[0414] Final reaction volume: 25 microliters;
[0415] Cycling conditions: 30 cycles of 94.degree. C. (sixty
seconds), 62.degree. C. (twenty seconds), 72.degree. C. (30
seconds).
[0416] Upon electrophoresis of the PCR product the presence of the
0.37-kb band indicated that the particular mouse was
transgenic.
[0417] Transgenics over expressing .alpha.2 were identified by
Western blot of plasma (using the affinity purified anti-.alpha.2
polyclonal antibody described above in Example 4) obtained from the
various PCR identified transgenics. Six .alpha.2 overexpressors (#s
7, 8, 26, 28, 156 and 186) as well as six non-transgenic control
mice (#s 10, 11, 12, 18, 20, 21) were phenotypically analyzed. All
mice were injected with 50 mg/kg BrdU one hour prior to harvest,
radiographed, and sacrificed Mice were sacrificed at 12 weeks of
age. No significant findings were noted during necropsy.
[0418] For all mice, body and selected organ weights were taken,
blood was drawn for hematology and serum chemistries, and organs
were harvested for histologic analysis and BrdU labeling.
[0419] H&E stained sections of liver, gall bladder, spleen,
lung, brain, pituitary, heart, kidney, adrenal, stomach, small
intestine, pancreas, cecum, colon, mesenteric lymph node, skin,
mammary gland, trachea, esophagus, thyroid, parathyroid, salivary
gland, urinary bladder, ovary or testis, uterus or prostate and
seminal vesicle, bone, and bone marrow were examined.
[0420] There were no biologically meaningful differences in the
mean or individual animal organ weights, hematology values,
clinical chemistry values or histologic findings between the
.alpha.2 overexpressor transgenic mice and the non-transgenic
control mice. In other words the .alpha.2 overexpressor transgenic
mice did not have a phenotype.
[0421] The following procedure was used to generate the "mouse
genomic .beta.10 human apoE expression vector transgene".
[0422] Primers were designed to amplify the complete mouse genomic
.beta.10 coding sequence (start ATG to stop TGA; SEQ ID NO: 23) as
follows:
[0423] Template: 10 nanograms of the mouse genomic .beta.10 10-kb
HindIII pBluescriptII-KS(-) clone DNA described in example 5.
11 Forward primer: 5'-ATTACTAGTTCCACCATGAAGTTGGTATACCTTGTCCTT-3';
(SEQ ID NO: 21) Reverse primer: 5'-TTAATAATCGATCGTCAGATG-
GTCTCACACTCAGTG-3'; (SEQ ID NO: 22)
[0424] Final concentration of each primer: 1.0 micromolar.
[0425] PCR kit: PfuTurbo DNA Polymerase kit (Stratagene, La Jolla,
Calif.).
[0426] Final reaction volume: 50 microliters;
[0427] Cycling conditions: 92.degree. C. for sixty seconds,
followed by 15 cycles of 92.degree. C. (ten seconds), 65.degree. C.
(twenty seconds), 68.degree. C. (four minutes).
[0428] The 3-kb PCR product was purified by agarose gel
electrophoresis, cut with SpeI and PvuI and cloned into SpeI-SfiI
cut human apoE expression vector. A "mouse genomic .beta.10 human
apoE expression vector transgene" clone containing the accurate
full genomic coding region mouse .beta.10 was identified by
sequencing.
[0429] The following procedure was then used to generate the
combined "mouse genomic .beta.10 mouse genomic .alpha.2 human apoE
expression vector transgene".
[0430] "Mouse genomic .beta.10 human apoE expression vector
transgene" clone DNA was cut with HindIII and SacIII, and the ends
were made blunt with DNA polymerase I Large (Klenow) Fragment (New
England Biolabs, Beverly, Mass.). The 5.6-kb fragment was gel
purified and cloned into HincIII cut pBluescript II KS(-). A clone
with the 5.6-kb "mouse genomic .beta.10 human apoE expression
cassette" in the orientation that has the SV40
polyA-signal/terminator region of the cassette next to HindIII site
in the pBluescript II KS(-) polylinker was identified. DNA from
this clone was cut with HindIII and SacIII, and ligated to the
3.4-kb HindIII-SacIII "mouse genomic .alpha.2 human apoE expression
cassette" that had been isolated from the "mouse genomic .alpha.2
human apoE expression vector transgene" clone described above. The
final 11.8-kb "mouse genomic .beta.10 mouse genomic .alpha.2 human
apoE expression vector transgene" construct consists of the "mouse
genomic .beta.10 human apoE expression cassette" and the "mouse
genomic .alpha.2 human apoE expression cassette" cloned in tandem
(ie. both in the same transcriptional orientation) into pBluescript
II KS(-). In this construct .beta.10 and .alpha.2 each have their
own HCR/apoE promoter and SV40 polyA-signal/terminator for
expression purposes.
[0431] DNA from this "mouse genomic .beta.10 mouse genomic .alpha.2
human apoE expression vector transgene" clone was digested with
BssHII; the 9-kb band was purified by gel electrophoresis and used
to generated transgenic mice as previously described (Simonet et
al, 1997, Cell vol 89 p309-319).
[0432] Mice transgenic for the "mouse genomic .alpha.2 human apoE
expression cassette" were identified by PCR as follows:
[0433] Template: ear punch DNA.
12 Forward primer: 5'-CCAGTGTGATATGTGCCGTTTC-3'; (SEQ ID NO: 24)
Reverse primer: 5'-GAAGAGCGCAGAGCTCGGTA-3'; (SEQ ID NO: 25)
[0434] Final concentration of each primer: 1.0 micromolar.
[0435] PCR kit: Ready-to-Go PCR Beads (Amersham Pharmacia Biotech
Inc., Piscataway, N.J.).
[0436] Final reaction volume: 25 microliters;
[0437] Cycling conditions: 94.degree. C. for sixty seconds,
followed by 35 cycles of 94.degree. C. (ten seconds), 60.degree. C.
(twenty seconds), 72.degree. C. (forty seconds), and then at the
end of the 35th cycle an incubation at 72.degree. C. for seven
minutes.
[0438] The forward primer [5'-CCAGTGTGATATGTGCCGTTTC-3' (SEQ ID NO:
24)] for this PCR is located in the 3.sup.rd .alpha.2 coding exon
(this exon contains the stop codon).
[0439] The reverse primer [5'-GAAGAGCGCAGAGCTCGGTA-3' (SEQ ID NO:
25)] for this PCR is located in the SV40 polyA-signal/terminator
region.
[0440] Upon electrophoresis of the PCR product the presence of the
0.31-kb band indicated that the particular mouse was transgenic for
the "mouse genomic .alpha.2 human apoE expression cassette". Those
mouse numbers were: 25, 45, 53, 76, 94, 95, and 113.
[0441] Mice transgenic for the "mouse genomic .beta.10 human apoe
expression cassette" were identified by PCR as follows:
13 Forward primer: 5'-TGGAGTCGATCCTTTCTACACCTA-3'; (SEQ ID NO: 26)
Reverse primer: 5'-AGAGCGCAGAGCTCGGTAC-3'; (SEQ ID NO: 27)
[0442] Final concentration of each primer: 1.0 micromolar.
[0443] PCR kit: Ready-to-Go PCR Beads (Amersham Pharmacia Biotech
Inc., Piscataway, N.J.).
[0444] Final reaction volume: 25 microliters;
[0445] Cycling conditions: 94.degree. C. for sixty seconds,
followed by 35 cycles of 94.degree. C. (ten seconds), 60.degree. C.
(twenty seconds), 72.degree. C. (forty seconds), and then at the
end of the 35th cycle an incubation at 72.degree. C. for seven
minutes.
[0446] The forward primer [5'-TGGAGTCGATCCTTTCTACACCTA-3' (SEQ ID
NO: 26)] for this PCR is located in the 2nd .beta.10 coding exon
(this exon contains the stop codon).
[0447] The reverse primer [5'-AGAGCGCAGAGCTCGGTAC-3' (SEQ ID NO:
27)] for this PCR is located in the SV40 polyA-signal/terminator
region.
[0448] Upon electrophoresis of the PCR product the presence of the
0.37-kb band indicated that the particular mouse was transgenic for
the "mouse genomic .beta.10 human apoE expression cassette". Those
mouse numbers were: 25, 31, 45, 53, 76, 94, 95, and 113. Of note,
mouse #31 which was positive by PCR for the "mouse genomic .beta.10
human apoE expression cassette" was negative by PCR for the "mouse
genomic .alpha.2 human apoE expression cassette".
[0449] Mouse #76 and #113 died shortly after the PCR genotyping.
The remaining six transgenics [#s 25 (female), 31 (male), 45
(female), 53 (male), 94 (male), and 95 (male)] as well as five
non-transgenic control mice [#s 17 (male), 18 (female), 19
(female), 20 (male) and 21 (male)] were necropsied at 7 weeks of
age for subsequent phenotypical analysis. All mice were injected
with 50 mg/kg BrdU one hour prior to harvest, radiographed, and
sacrificed. Upon necropsy abnormally large thyroid glands were
found in some of the transgenic mice. As part of the necropsy, mice
were weighed, blood was drawn for hematology and serum chemistries,
and liver, spleen, kidney, heart, and thymus were weighed. Sections
of liver, gall bladder, spleen, lung, brain, pituitary, heart,
kidney, adrenal, thymus, stomach, small intestine, pancreas, cecum,
colon, mesenteric lymph node, skin, mammary gland, trachea,
esophagus, thyroid, parathyroid, salivary gland, urinary bladder,
ovary or testis, uterus or prostate and seminal vesicle, bone, and
bone marrow were harvested for histologic analysis.
[0450] Northern blot analysis was used to determine the levels of
.alpha.2 and .beta.10 mRNA in the livers of all of the transgenic
and non-transgenic control mice as described below.
[0451] Total RNA was isolated from liver samples, quantitated and
10 micrograms of total RNA for each mouse was electrophoresed in a
formaldehyde denaturation agarose gel and transferred to a Nylon
membrane. This was done in duplicate to generate 2 Northern blots
for probing. Ethidium Bromide staining of the agarose gels revealed
virtually equal loading of RNA across all wells and between both
gels. One Northern blot was probed with a random primed P32
labelled probe encompassing the full coding region of the .alpha.2
cDNA (from ATG to TAG) to assess .alpha.2 expression. The second
Northern blot was probed with a random primed P32 labelled probe
encompassing the full coding region of the .beta.10 cDNA (from ATG
to TGA) to assess .beta.10 expression.
[0452] Hybridization was for one hour at 65.degree. C. in
"ExpressHyb Hybridization Solution" (Clontech, Palo Alto, Calif.).
The blots were washed in 2.times.SSC, 0.1% SDS at room temperature
twice, for twenty minutes each time. The blots were then washed in
0.1.times.SSC, 0.1% SDS at 50.degree. C. for ten minutes, and then
exposed to film.
[0453] The results of the Northern analysis are as follows:
[0454] For the non-transgenic control mice no signal was found for
either .alpha.2 or .beta.10.
[0455] For transgenic mouse #94 (male) no signal was found for
either .alpha.2 or .beta.10.
[0456] For transgenic mice #s 25 (female) and 45 (female) a strong
signal was found for both .alpha.2 and .beta.10.
[0457] For transgenic mouse #95 (male) a moderate signal was found
for both .alpha.2 and .beta.10.
[0458] For transgenic mouse #53 (male) a moderate signal was found
for .alpha.2 and a weaker signal for .beta.10.
[0459] For transgenic mouse #31 (male) a moderate signal was found
for .beta.10 but no signal was found for .alpha.2, indicating that
mouse #31 overexpressed only .beta.10 and not .alpha.2. The level
of .beta.10 expression in mouse #31 was significantly greater than
that found in mouse #53. The expression results for transgenic
mouse #31 are consistent with the PCR genotyping described above
which for #31 was positive for the "mouse genomic .beta.10 human
apoE expression cassette" but negative for the "mouse genomic
.alpha.2 human apoE expression cassette". The data for mouse #31
indicates that the "mouse genomic .alpha.2 human apoE expression
cassette" region of the "mouse genomic .beta.10 mouse genomic
.alpha.2 human apoE expression vector transgene" DNA was truncated
at some point during the microinjection process resulting in a
mouse which overexpresses .beta.10 but not .alpha.2.
[0460] Shearing/truncation of transgene DNA during the process of
creating transgenic mice has been reported previously in the
transgenic literature.
[0461] H&E and BrdU stained sections of liver, gall bladder,
spleen, lung, brain, pituitary, heart, kidney, adrenal, thymus,
stomach, small intestine, pancreas, cecum, colon, mesenteric lymph
node, skin, mammary gland, trachea, esophagus, thyroid,
parathyroid, salivary gland, urinary bladder, ovary or testis,
uterus or prostate and seminal vesicle, bone, and bone marrow from
the .alpha.2/.beta.10 overexpressors (#s 25, 45, 53 and 95), the 5
non-transgenic control mice (#s 17, 18, 19, 20 and 21), and
transgenic mouse #31, which only overexpressed .beta.10 but not
.alpha.2, were examined.
[0462] Immunohistochemical staining for BrdU was done on 4 .mu.m
thick paraffin embedded sections using an automated DPC Mark 5
Histochemical Staining System (Diagnostic Products Corp, Randolph,
N.J.). Deparaffinized tissue sections were digested with 0.1%
protease and then treated with 2N hydrochloric acid. Sections were
blocked with CAS BLOCK (Zymed Laboratories, San Francisco, Calif.),
incubated with a rat anti-BrdU monoclonal antibody (Accurate
Chemical and Scientific, Westbury, N.Y.). The primary antibody was
detected with a biotinylated rabbit anti-rat immunoglobulin
polyclonal antibody (Dako, Carpinteria, Calif.). Sections were then
quenched with 3% hydrogen peroxide, and reacted with an
avidin-biotin complex tertiary (Vector Laboratories). The staining
reaction was visualized with diaminobenzidine (DAB, Dako
Carpinteria, Calif.) and sections were counterstained with
hematoxylin.
[0463] All four .alpha.2/.beta.10 overexpressors exhibited
hepatomegaly (6.75.+-.0.68% BW vs. 4.98.+-.0.29% BW in
non-transgenic control mice, p=0.0011) and renal hypertrophy
(2.23.+-.0.21% BW vs. 1.75.+-.0.12% BW in non-transgenic control
mice, p=0.0033). .alpha.2/.beta.10 overexpressor mice also had a
slightly lower mean body weight than their non-transgenic control
counterparts; this difference was not statistically significant.
.alpha.2/.beta.10 overexpressor mice #s 45 and 53 also exhibited
moderate splenomegaly. Transgenic mouse #31, which only
overexpressed .beta.10 and not .alpha.2, had normal liver, kidney
and spleen weights.
[0464] All four .alpha.2/.beta.10 overexpressor mice had elevated
serum T4 levels (23.1.+-.5.4 micrograms/dl vs. 5.0.+-.0.7
micrograms/dl in non-transgenic control mice, p=0.0001) and
transgenic mice #s 25 and 45 had a modest circulating
lymphocytosis. Transgenic mouse #31, which only overexpressed
.beta.10 and not .alpha.2, had normal serum T4 levels and
lymphocyte counts. Individual serum T4 values for each mouse are as
follows: #17 (5.0 micrograms/dl), #18 (4.8 micrograms/dl), #19 (6.3
micrograms/dl), #20 (4.4 micrograms/dl), #21 (4.7 micrograms/dl),
#25 (28.5 micrograms/dl), #45 (26.9 micrograms/dl), #53 (18.2
micrograms/dl), #95 (18.7 micrograms/dl), and #31 (3.2
micrograms/dl).
[0465] H&E and BrdU stained sections of liver, gall bladder,
spleen, lung,-brain, pituitary, heart, kidney, adrenal, thymus,
stomach, small intestine, pancreas, cecum, colon, mesenteric lymph
node, skin, mammary gland, trachea, esophagus, thyroid,
parathyroid, salivary gland, urinary bladder, ovary or testis,
uterus or prostate and seminal vesicle, bone, and bone marrow from
the four .alpha.2/.beta.10 overexpressor mice, the 5 non-transgenic
control mice, and mouse #31, which only overexpressed .beta.10 and
not .alpha.2, were examined. Histologically, all four
.alpha.2/.beta.10 overexpressor mice exhibited bilaterally enlarged
thyroid glands containing multiple follicular papillary adenomas.
All four .alpha.2/.beta.10 overexpressor mice also exhibited mild
to moderate hepatocellular hyperplasia with an increase in
hepatocellular BrdU labeling vs. the non-transgenic mice.
Transgenic mouse #31 had none of these features.
[0466] In summary, the four .alpha.2/.beta.10 overexpressor
transgenic mice exhibited a phenotype characterized by bilateral
thyroid enlargement with multiple follicular papillary adenomas and
resulting hyperthyroidism, as indicated by elevated serum T4
levels. Other phenotypic changes were felt to be related to the
systemic hyperthyroid state, and included moderate hepatomegaly,
hepatocellular hyperplasia, and slightly decreased serum
cholesterol levels, bilateral renal hypertrophy, and a mild to
moderate leukocytosis with a predominance of lymphocytes.
[0467] Transgenic mice over expressing mouse .alpha.2 and not
.beta.10 (#s 7, 8, 26, 28, 156 and 186), described above, had no
phenotype and transgenic mouse #31 over expressing .beta.10 and not
.alpha.2 had no phenotype. This indicates that the hyperthyroid
phenotype found in all four .alpha.2/.beta.10 overexpressor
transgenic mice (#s 25, 45, 53 and 95) can be attributed to the
.alpha.2/.beta.10 heterodimeric hormone described in the present
invention.
Sequence CWU 1
1
28 1 130 PRT Homo sapiens 1 Met Lys Leu Ala Phe Leu Phe Leu Gly Pro
Met Ala Leu Leu Leu Leu 1 5 10 15 Ala Gly Tyr Gly Cys Val Leu Gly
Ala Ser Ser Gly Asn Leu Arg Thr 20 25 30 Phe Val Gly Cys Ala Val
Arg Glu Phe Thr Phe Leu Ala Lys Lys Pro 35 40 45 Gly Cys Arg Gly
Leu Arg Ile Thr Thr Asp Ala Cys Trp Gly Arg Cys 50 55 60 Glu Thr
Trp Glu Lys Pro Ile Leu Glu Pro Pro Tyr Ile Glu Ala His 65 70 75 80
His Arg Val Cys Thr Tyr Asn Glu Thr Lys Gln Val Thr Val Lys Leu 85
90 95 Pro Asn Cys Ala Pro Gly Val Asp Pro Phe Tyr Thr Tyr Pro Val
Ala 100 105 110 Ile Arg Cys Asp Cys Gly Ala Cys Ser Thr Ala Thr Thr
Glu Cys Glu 115 120 125 Thr Ile 130 2 390 DNA Homo sapiens 2
atgaagctgg cattcctctt ccttggcccc atggccctcc tccttctggc tggctatggc
60 tgtgtcctcg gtgcctccag tgggaacctg cgcacctttg tgggctgtgc
cgtgagggag 120 tttactttcc tggccaagaa gccaggctgc aggggccttc
ggatcaccac ggatgcctgc 180 tggggtcgct gtgagacctg ggagaaaccc
attctggaac ccccctatat tgaagcccat 240 catcgagtct gtacctacaa
cgagaccaaa caggtgactg tcaagctgcc caactgtgcc 300 ccgggagtcg
accccttcta cacctatccc gtggccatcc gctgtgactg cggagcctgc 360
tccactgcca ccacggagtg tgagaccatc 390 3 106 PRT Homo sapiens 3 Ala
Ser Ser Gly Asn Leu Arg Thr Phe Val Gly Cys Ala Val Arg Glu 1 5 10
15 Phe Thr Phe Leu Ala Lys Lys Pro Gly Cys Arg Gly Leu Arg Ile Thr
20 25 30 Thr Asp Ala Cys Trp Gly Arg Cys Glu Thr Trp Glu Lys Pro
Ile Leu 35 40 45 Glu Pro Pro Tyr Ile Glu Ala His His Arg Val Cys
Thr Tyr Asn Glu 50 55 60 Thr Lys Gln Val Thr Val Lys Leu Pro Asn
Cys Ala Pro Gly Val Asp 65 70 75 80 Pro Phe Tyr Thr Tyr Pro Val Ala
Ile Arg Cys Asp Cys Gly Ala Cys 85 90 95 Ser Thr Ala Thr Thr Glu
Cys Glu Thr Ile 100 105 4 24 DNA Homo sapiens 4 atgaagctgg
cattcctctt cctt 24 5 21 DNA Homo sapiens 5 gcatgtgctg ctcacacagg t
21 6 19 DNA Homo sapiens 6 ctgcaggtgc cttcggatc 19 7 22 DNA Homo
sapiens 7 gagacatctc cccactgtgt tt 22 8 22 DNA Homo sapiens 8
gtttccccca acagaatgtc aa 22 9 23 DNA Homo sapiens 9 atgcctatgg
cgtcccctca aac 23 10 28 DNA Homo sapiens 10 ctagtagcga gagaggcgac
acatgtca 28 11 130 PRT Mus musculus 11 Met Lys Leu Val Tyr Leu Val
Leu Gly Ala Val Ala Leu Leu Leu Leu 1 5 10 15 Gly Gly Pro Asp Ser
Val Leu Ser Ser Ser Ser Gly Asn Leu His Thr 20 25 30 Phe Val Gly
Cys Ala Val Arg Glu Phe Thr Phe Met Ala Lys Lys Pro 35 40 45 Gly
Cys Arg Gly Leu Arg Ile Thr Thr Asp Ala Cys Trp Gly Arg Cys 50 55
60 Glu Thr Trp Glu Lys Pro Ile Leu Glu Pro Pro Tyr Ile Glu Ala Tyr
65 70 75 80 His Arg Val Cys Thr Tyr Asn Glu Thr Arg Gln Val Thr Val
Lys Leu 85 90 95 Pro Asn Cys Ala Pro Gly Val Asp Pro Phe Tyr Thr
Tyr Pro Met Ala 100 105 110 Val Arg Cys Asp Cys Gly Ala Cys Ser Thr
Ala Thr Thr Glu Cys Glu 115 120 125 Thr Ile 130 12 393 DNA Mus
musculus 12 atgaagttgg tataccttgt ccttggtgca gtggccctcc ttctcctggg
tggccctgac 60 tctgtcctca gcagctccag tgggaacctg cacacttttg
tgggctgtgc tgtgagggaa 120 ttcactttca tggccaagaa gccaggctgc
aggggacttc ggatcaccac agatgcctgc 180 tggggccgct gcgagacctg
ggagaaaccc atcctggagc ctccctacat tgaagcctat 240 catcgagtgt
gtacatacaa tgagaccaga caggtgacag tgaagctgcc taactgtgcc 300
cctggagtcg atcctttcta cacctaccct atggctgtcc gatgtgactg tggggcgtgt
360 tccactgcca ccactgagtg tgagaccatc tga 393 13 106 PRT Mus
musculus 13 Ser Ser Ser Gly Asn Leu His Thr Phe Val Gly Cys Ala Val
Arg Glu 1 5 10 15 Phe Thr Phe Met Ala Lys Lys Pro Gly Cys Arg Gly
Leu Arg Ile Thr 20 25 30 Thr Asp Ala Cys Trp Gly Arg Cys Glu Thr
Trp Glu Lys Pro Ile Leu 35 40 45 Glu Pro Pro Tyr Ile Glu Ala Tyr
His Arg Val Cys Thr Tyr Asn Glu 50 55 60 Thr Arg Gln Val Thr Val
Lys Leu Pro Asn Cys Ala Pro Gly Val Asp 65 70 75 80 Pro Phe Tyr Thr
Tyr Pro Met Ala Val Arg Cys Asp Cys Gly Ala Cys 85 90 95 Ser Thr
Ala Thr Thr Glu Cys Glu Thr Ile 100 105 14 39 DNA Artificial
Sequence misc_feature Oligonucleotide 14 attactagtt ccaccatgaa
gttggtatac cttgtcctt 39 15 36 DNA Artificial Sequence misc_feature
Oligonucleotide 15 ttaataatcg atcgtcagat ggtctcacac tcagtg 36 16 36
DNA Artificial Sequence misc_feature Oligonucleotide 16 ccgcactagt
tccaccatgc ccatggcacc acgagt 36 17 41 DNA Artificial Sequence
misc_feature Oligonucleotide 17 gcggcgttcg atcgctagta gcgggagaaa
cggcacatat c 41 18 815 DNA Mus musculus 18 atgcccatgg caccacgagt
cttgctcctt tgcctgctgg gcctggcagt cactgaaggg 60 catagcccag
agacagccat cccaggctgc cacttgcacc gtgagtaact ctgcttgggg 120
agcggatgga cgggtaaccc ggccagcacg gccttcaccg gctgctccct tctctgcttc
180 cagccttcaa tgtgacggtg cgcagtgatc gcctcggcac ttgccagggc
tcccacgtgg 240 cacaggcctg tgtaggacac tgtgagtcta gtgctttccc
ttcccggtac tctgtgctgg 300 tggccagtgg ctatcggcac aacatcacct
cttcctccca gtgctgcacc atcagcagcc 360 tcagaaaggt aaggggcctg
agcctgatgg agcgtgaggg tggggaccca ggggcctgag 420 cctgatggag
cgtgagggtg gggacccagg ggtccgaacc tgacctggtg tgagggtggg 480
gacccaggag cccgaacctg accaggtatg agggtgggga cccaggggcc cgaacctgac
540 cggggtgtaa gggtggggtc ccccaggggc ccgaacctga ccgggccata
agggtgggga 600 cccccagggg cccgaacctg accaggtgtg agggtgagga
cccaggggtt cgaacctgat 660 gggggcgtga gggtggggtg gaatgggaac
aaacttgggt cctcctccaa caggtgaggg 720 tgtggctgca gtgcgtgggg
aaccagcgtg gggagcttga gatctttact gcaagggcct 780 gccagtgtga
tatgtgccgt ttctcccgct actag 815 19 21 DNA Homo sapiens 19
gcctctagaa agagctggga c 21 20 21 DNA Homo sapiens 20 cgccgtgttc
catttatgag c 21 21 39 DNA Artificial Sequence misc_feature
Oligonucleotide 21 attactagtt ccaccatgaa gttggtatac cttgtcctt 39 22
36 DNA Artificial Sequence misc_feature Oligonucleotide 22
ttaataatcg atcgtcagat ggtctcacac tcagtg 36 23 2985 DNA Mus musculus
23 atgaagttgg tataccttgt ccttggtgca gtggccctcc ttctcctggg
tggccctgac 60 tctgtcctca gcagctccag tgggaacctg cacacttttg
tgggctgtgc tgtgagggaa 120 ttcactttca tggccaagaa gccaggctgc
aggggacttc ggatcaccac agatgcctgc 180 tggggccgct gcgagacctg
ggaggtgagt agtccaaggg gcttgggtgg cagtgtcctc 240 ggggacaggg
ccttgatttc aagctcacag tttcacgatg gaggggaagc tggcatacct 300
gccctgccct ctgggctcta aaaagctgat cacacaatta tttggcttct tccatatggt
360 ctgaaaacca tgatgatggt tttttccaga ggactgttga ggttggtaat
gtaatttcca 420 tggttctgtt tgggagccat ccctaggagg gtgggtgcta
ctatttaccc attgtagaga 480 tcacaggaaa ggagtcgaga gggaatggct
tcgaagtcag agagagtcag tgcaaagttg 540 gaaatgaatt cttggtccaa
acctgtgacc acatctcctt cctgtatttt ctcagctgtg 600 aggaagtcag
gcagttaccc agaagaaccc ggaagctgca tgctgagaga ggcgtagtcc 660
caggctctgc cacgactgtt ttccctttgt cccagtccag tgtgagatct ggtctgtccc
720 tttataccca gttctgtctg gactttcatt tttagagtgg gtgcatacat
ctcaaagctg 780 gcttaactag aaagtgttcg tggtgtccag ctgagctgac
tcttgctgaa aatggtgacg 840 tctcagtgac ctgagcttca aagatggcag
atttagcaaa attaaagcca aaagaacctc 900 cccacaccga aatcaaccaa
ccaactcaaa acaccattaa cccccttcca cctcagaccc 960 tccccacaat
ctgaagtgaa gtgaaattaa aaaaaaaaaa gttaggggac tcagataaat 1020
ttgaattcag atcaataaca caattttttt ggcctaagcc caccccaaat attgcatgag
1080 acagatttat aaaataaaaa aattgaaata caaagttaat tgagtacgca
atttttctag 1140 aatcccagaa tgctgagagt cagaagacag aatggagaga
gaaacggaac ttctcctccc 1200 ggcccttgag aaggacaggt ctctgttttt
ataatattga agctggattt catcttgagc 1260 tggcttgcct gtcatctata
ggtgtacaca cacacacatg tgtatgtgtg tgtgcctatg 1320 cacatatgta
cttatgtatg tatatatgta tatctcttga ttctatgtac ctgcgtgcat 1380
tatctatata cgtgtatgcc tgtgcataca tctaagcacg tagctatgta tagatgtatg
1440 tatcatctat ctgatttccc tacttaacat tattattatt ttttggattg
gaacaaaggg 1500 actgttccct gaatgattat tgttattgat tcgttactac
atccttatac ttgcgctcat 1560 aagagccatt gactacttgt attgagccct
gacctttcgc cagggcttgt gcactgcaca 1620 catcacctca tctagctcca
tgacaatgct agcaaaggtt tttttttttt tttattcctt 1680 atagaagagg
gaacaagggc ttggaagagt taagagcttc ccctaggtct ccagcagcag 1740
taaagcaggc aggcatagtg gggctgactg cagactctgg gtctctctct actgatctct
1800 acgttctcta acagaatcat ctttgaagtc aaggtttata aaaggcaaag
ggaggaagtg 1860 aactaaccct ccagtcatta gagcagaata ttcaggaagc
tccctggccc tgctgtcttt 1920 tgtggattca gttacaagta gttcttgcag
aagtcctggg taccaggctg gctgggtact 1980 ggagaatagt ggctgaccta
acggagctcg gtctccacat taggagcaat gtcacacaaa 2040 gatacaggag
atggcatgtg gaaatggaga aacacagcaa accagcccct aaaccagaac 2100
cacacaggaa gggactaggg agcgccaggg cttggaggtg ggttgaagcg atttaaaata
2160 gcatcagaaa atgccgctct ggattgggtg agatttgaac agaatcctaa
gagcttggtg 2220 ataggtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt
gtgtgtgtgt gtggtgttct 2280 gggcagaggg aacagcagat acaaagaccc
tcagtttggg ttctgaagca gcatagagac 2340 cactgtgact ggagctggag
accgtgttgg caaggtcagg gccatagctg acatgtcaga 2400 agtaagagta
cgggcagaaa atacagggac ttgagaagaa tcctagtgtc tgtgtttacc 2460
ctgagggaga tggaaaacta ccggggtttg agcagcggtg agccaggact gacttgtatt
2520 ttaaaaggct cattcgtgct gtaaacattt tgtaggggta atggtaggag
aagggagacc 2580 agcatttact aaatatttac caagtgcatc ctgtgttctg
tgggctttcg tggaagctcg 2640 ggacatggta atgagcaaag taacttcctg
ctttcaggag tgtattcgta gtgggaggag 2700 tcagtacgta agtaaccagc
cagtgatgac tggcaccaag aacaggaagc ggatgctgta 2760 ttctaacatt
tttcctgttt tttacccttg ggatagaaac ccatcctgga gcctccctac 2820
attgaagcct atcatcgagt gtgtacatac aatgagacca gacaggtgac agtgaagctg
2880 cctaactgtg cccctggagt cgatcctttc tacacctacc ctatggctgt
ccgatgtgac 2940 tgtggggcgt gttccactgc caccactgag tgtgagacca tctga
2985 24 22 DNA Mus musculus 24 ccagtgtgat atgtgccgtt tc 22 25 20
DNA Simian virus 40 25 gaagagcgca gagctcggta 20 26 24 DNA Mus
musculus 26 tggagtcgat cctttctaca ccta 24 27 19 DNA Simian virus 40
27 agagcgcaga gctcggtac 19 28 16 PRT Artificial Sequence
misc_feature Oligopeptide 28 Cys Ser Pro Arg Tyr Ser Val Leu Val
Ala Ser Gly Tyr Arg His Asn 1 5 10 15
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