U.S. patent application number 11/636370 was filed with the patent office on 2008-10-23 for edg: modulators of lymphocyte activation and migration.
This patent application is currently assigned to Rigel Pharmaceuticals, Inc.. Invention is credited to Peter Chu, Ying-Ping Jiang, Congfen Li, X. Charlene Liao, Esteban Masuda, Jorge Pardo, Collin Spencer, Haoran Zhao.
Application Number | 20080260719 11/636370 |
Document ID | / |
Family ID | 26962795 |
Filed Date | 2008-10-23 |
United States Patent
Application |
20080260719 |
Kind Code |
A1 |
Liao; X. Charlene ; et
al. |
October 23, 2008 |
EDG: Modulators of lymphocyte activation and migration
Abstract
The present invention relates to regulation of lymphocyte
activation and migration. More particularly, the present invention
is directed to nucleic acids encoding EDG family GPCR proteins,
e.g., EDG-1, 2, 3, 4, 5, 6, 7, or 8, which are involved in
modulation of lymphocyte activation and migration. The invention
further relates to methods for identifying and using agents,
including small organic molecules, antibodies, peptides, cyclic
peptides, nucleic acids, antisense nucleic acids, sphingolipid
analogs, and ribozymes, that modulate lymphocyte activation or
migration via modulation of EDG GPCRs and EDG related signal
transduction; as well as to the use of expression profiles and
compositions in diagnosis and therapy related to lymphocyte
activation and suppression, and lymphocyte migration.
Inventors: |
Liao; X. Charlene; (Palo
Alto, CA) ; Masuda; Esteban; (Menlo Park, CA)
; Chu; Peter; (San Francisco, CA) ; Pardo;
Jorge; (San Francisco, CA) ; Li; Congfen;
(Davis, CA) ; Zhao; Haoran; (Foster City, CA)
; Jiang; Ying-Ping; (Lafayette, CA) ; Spencer;
Collin; (San Francisco, CA) |
Correspondence
Address: |
Klarquist Sparkman, LLP
121 SW Salmon St, Floor 16
Portland
OR
97204
US
|
Assignee: |
Rigel Pharmaceuticals, Inc.
South San Francisco
CA
|
Family ID: |
26962795 |
Appl. No.: |
11/636370 |
Filed: |
December 6, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09971228 |
Oct 3, 2001 |
|
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11636370 |
|
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60284763 |
Apr 18, 2001 |
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Current U.S.
Class: |
424/130.1 ;
435/29; 506/10; 514/1.1; 514/625 |
Current CPC
Class: |
G01N 33/56972 20130101;
G01N 2500/10 20130101; G01N 2333/4703 20130101; C07K 14/705
20130101; A61P 37/00 20180101 |
Class at
Publication: |
424/130.1 ;
435/29; 514/625; 514/12; 506/10 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/02 20060101 C12Q001/02; A61K 31/164 20060101
A61K031/164; A61K 38/16 20060101 A61K038/16; C40B 30/06 20060101
C40B030/06; A61P 37/00 20060101 A61P037/00 |
Claims
1. A method for identifying a compound that modulates lymphocyte
activation, the method comprising the steps of: (i) contacting a
cell comprising an EDG polypeptide or fragment thereof with the
compound, the EDG polypeptide or fragment thereof encoded by a
nucleic acid that hybridizes under stringent conditions to a
nucleic acid comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5,
6, 7, or 8; and (ii) determining the chemical or phenotypic effect
of the compound upon the cell comprising the EDG polypeptide or
fragment thereof, thereby identifying a compound that modulates
lymphocyte activation.
2. The method of claim 1, wherein the EDG polypeptide or fragment
thereof is encoded by a nucleic acid that hybridizes under
stringent conditions to a nucleic acid comprising a sequence of SEQ
ID NO:1-4.
3. The method of claim 1, wherein the host cell is a B
lymphocyte.
4. The method of claim 3, wherein the host cell is a cultured B
lymphocyte.
5. The method of claim 4, wherein the host cell is a BJAB cell.
6. The method of claim 1, wherein the host cell is a T
lymphocyte.
7. The method of claim 6, wherein the host cell is primary T
lymphocyte.
8. The method of claim 6, wherein the host cell is a cultured T
lymphocyte.
9. The method of claim 8, wherein the host cell is a Jurkat
cell.
10. The method of claim 1, wherein the chemical or phenotypic
effect is determined by measuring CD69 expression, IL-2 production,
intracellular Ca.sup.2+ mobilization, or lymphocyte
proliferation.
11. The method of claim 1, wherein modulation is inhibition of
lymphocyte activation.
12. The method of claim 11, wherein modulation is inhibition of T
lymphocyte activation.
13. The method of claim 11, wherein modulation is inhibition of B
lymphocyte activation.
14. The method of claim 1, wherein the polypeptide is
recombinant.
15. The method of claim 1, wherein the EDG polypeptide is selected
from the group consisting of EDG-1, 3, 5, 6, 7, or 8.
16. The method of claim 1, wherein the EDG polypeptide is an EDG-1
polypeptide encoded by a nucleic acid selected from the group
consisting of SEQ ID NOS:1, 2, 3, and 4.
17. The method of claim 1, wherein the fragment of an EDG
polypeptide is a polypeptide that comprises an extracellular domain
of an EDG polypeptide.
18. The method of claim 1, wherein the fragment of an EDG
polypeptide is a polypeptide that comprises a cytoplasmic domain of
an EDG polypeptide.
19. The method of claim 17, wherein the polypeptide further
comprises at least one transmembrane domain of an EDG
polypeptide.
20. The method of claim 1, wherein the EDG polypeptide or fragment
thereof has GPCR activity.
21. The method of claim 1, wherein the compound is an antibody.
22. The method of claim 1, wherein the compound is an antisense
molecule.
23. The method of claim 1, wherein the compound is a small organic
molecule.
24. The method of claim 1, wherein the compound is a
sphingolipid.
25. The method of claim 1, wherein the compound is a sphingolipid
analog.
26. The method of claim 25, wherein the compound is a synthetic
sphingolipid analog.
27. The method of claim 25, wherein the compound is a naturally
occurring sphingolipid analog.
28. A method for identifying a compound that modulates lymphocyte
activation, the method comprising the steps of: (i) contacting the
compound with an EDG polypeptide or a fragment thereof, the EDG
polypeptide or fragment thereof encoded by a nucleic acid that
hybridizes under stringent conditions to a nucleic acid comprising
a nucleotide sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8; (ii)
determining the physical effect of the compound upon the EDG
polypeptide; and (iii) determining the chemical or phenotypic
effect of the compound upon a cell comprising an EDG polypeptide or
fragment thereof, thereby identifying a compound that modulates
lymphocyte activation.
29. A method of modulating lymphocyte activation in a subject, the
method comprising the step of administering to the subject a
therapeutically effective amount of a compound identified using the
method of claim 1.
30. The method of claim 29, wherein the lymphocyte is a T
lymphocyte.
31. The method of claim 29, wherein the lymphocyte is a B
lymphocyte.
32. The method of claim 29, wherein the subject is a human.
33. The method of claim 29, wherein the compound is an
antibody.
34. The method of claim 29, wherein the compound is an antisense
molecule.
35. The method of claim 29, wherein the compound is a small organic
molecule.
36. The method of claim 29, wherein the compound is a
sphingolipid.
37. The method of claim 29, wherein the compound is a sphingolipid
analog.
38. The method of claim 37, wherein the compound is a
naturally-occurring sphingolipid analog.
39. The method of claim 37, wherein the compound is a synthetic
sphingolipid analog.
40. The method of claim 29, wherein the compound inhibits
lymphocyte activation.
41. The method of claim 29, wherein the compound inhibits T
lymphocyte activation.
42. The method of claim 29, wherein the compound inhibits B
lymphocyte activation.
43. A method for identifying a compound that modulates lymphocyte
migration, the method comprising the steps of: (i) contacting a
cell comprising an EDG polypeptide or fragment thereof with the
compound, the EDG polypeptide or fragment thereof encoded by a
nucleic acid that hybridizes under stringent conditions to a
nucleic acid comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5,
6, 7, or 8; and (ii) determining the chemical or phenotypic effect
of the compound upon the cell comprising the EDG polypeptide or
fragment thereof, thereby identifying a compound that modulates
lymphocyte migration.
44. The method of claim 43, wherein the EDG polypeptide or fragment
thereof is encoded by a nucleic acid that hybridizes under
stringent conditions to a nucleic acid comprising a sequence of SEQ
ID NO:1-4.
45. The method of claim 43, wherein the host cell is a B
lymphocyte.
46. The method of claim 46, wherein the host cell is a primary B
lymphocyte.
47. The method of claim 46, wherein the host cell is a cultured B
lymphocyte.
48. The method of claim 47, wherein the host cell is a BJAB
cell.
49. The method of claim 43, wherein the host cell is a T
lymphocyte.
50. The method of claim 49, wherein the host cell is primary T
lymphocyte.
51. The method of claim 49, wherein the host cell is a cultured T
lymphocyte.
52. The method of claim 51, wherein the host cell is a Jurkat
cell.
53. The method of claim 43, wherein the chemical or phenotypic
effect is determined by measuring lymphocyte migration in vitro
toward an EDG ligand.
54. The method of claim 53, wherein the EDG ligand is SPP or
LPA.
55. The method of claim 43, wherein modulation is inhibition of
lymphocyte migration.
56. The method of claim 43, wherein modulation is inhibition of T
lymphocyte migration.
57. The method of claim 43, wherein modulation is inhibition of B
lymphocyte migration.
58. The method of claim 43, wherein the polypeptide is
recombinant.
59. The method of claim 43, wherein the EDG polypeptide is selected
from the group consisting of EDG-1, 3, 5, 6, 7, or 8.
60. The method of claim 43, wherein the EDG polypeptide is an EDG-1
polypeptide encoded by a nucleic acid selected from the group
consisting of SEQ ID NOS:1, 2, 3, and 4.
61. The method of claim 43, wherein the fragment of an EDG
polypeptide is a polypeptide that comprises an extracellular domain
of an EDG polypeptide.
62. The method of claim 61, wherein the polypeptide further
comprises at least one transmembrane domain of an EDG
polypeptide.
63. The method of claim 43, wherein the EDG polypeptide or fragment
thereof has GPCR activity.
64. The method of claim 43, wherein the compound is an
antibody.
65. The method of claim 43, wherein the compound is an antisense
molecule.
66. The method of claim 43, wherein the compound is a small organic
molecule.
67. The method of claim 43, wherein the compound is a
sphingolipid.
68. The method of claim 43, wherein the compound is a sphingolipid
analog.
69. The method of claim 68, wherein the compound is a synthetic
sphingolipid analog.
70. The method of claim 68, wherein the compound is a naturally
occurring sphingolipid analog.
71. A method for identifying a compound that modulates lymphocyte
migration, the method comprising the steps of: (i) contacting the
compound with an EDG polypeptide or a fragment thereof, the EDG
polypeptide or fragment thereof encoded by a nucleic acid that
hybridizes under stringent conditions to a nucleic acid comprising
a nucleotide sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8; (ii)
determining the physical effect of the compound upon the EDG
polypeptide; and (iii) determining the chemical or phenotypic
effect of the compound upon a cell comprising an EDG polypeptide or
fragment thereof, thereby identifying a compound that modulates T
lymphocyte migration.
72. A method of modulating lymphocyte migration in a subject, the
method comprising the step of administering to the subject a
therapeutically effective amount of a compound identified using the
method of claim 43.
73. The method of claim 72, wherein the subject is a human.
74. The method of claim 72, wherein the lymphocyte is a T
lymphocyte.
75. The method of claim 72, wherein the lymphocyte is a B
lymphocyte.
76. The method of claim 72, wherein the compound is an
antibody.
77. The method of claim 72, wherein the compound is an antisense
molecule.
78. The method of claim 72, wherein the compound is a small organic
molecule.
79. The method of claim 72, wherein the compound is a
sphingolipid.
80. The method of claim 72, wherein the compound is a sphingolipid
analog.
81. The method of claim 80, wherein the compound is a
naturally-occurring sphingolipid analog.
82. The method of claim 80, wherein the compound is a synthetic
sphingolipid analog.
83. The method of claim 72, wherein the compound inhibits T
lymphocyte migration.
84. The method of claim 72, wherein the compound inhibits B
lymphocyte migration.
85. A method of modulating lymphocyte activation or migration in a
subject, the method comprising the step of administering to the
subject a therapeutically effective amount of an EDG polypeptide,
the polypeptide encoded by a nucleic acid that hybridizes under
stringent conditions to a nucleic acid comprising a nucleotide
sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8.
86. The method of claim 85, wherein the EDG polypeptide or fragment
thereof is encoded by a nucleic acid that hybridizes under
stringent conditions to a nucleic acid comprising a sequence of SEQ
ID NO:1-4.
87. The method of claim 85, wherein the EDG polypeptide is selected
from the group consisting of EDG-1, 3, 5, 6, 7, and 8.
88. The method of claim 85, wherein the EDG polypeptide is an EDG-1
polypeptide encoded by a nucleic acid selected from the group
consisting of SEQ ID NO:1, 2, 3, and 4.
89. A method of modulating lymphocyte activation or migration in a
subject, the method comprising the step of administering to the
subject a therapeutically effective amount of a nucleic acid
encoding an EDG polypeptide or fragment thereof, wherein the
nucleic acid hybridizes under stringent conditions to a nucleic
acid encoding a polypeptide comprising a nucleotide sequence of EDG
1, 2, 3, 4, 5, 6, 7, or 8.
90. The method of claim 89, wherein the EDG nucleic acid or
fragment thereof is encoded by a nucleic acid that hybridizes under
stringent conditions to a nucleic acid comprising a sequence of SEQ
ID NO:1-4.
91. The method of claim 89 wherein the EDG nucleic acid is selected
from the group consisting of EDG-1, 3, 5, 6, 7, and 8.
92. The method of claim 91, wherein the EDG nucleic acid is an
EDG-1 nucleic acid selected from the group consisting of SEQ ID
NO:1, 2, 3, and 4.
93. A method of modulating T lymphocyte migration and activation in
a subject, the method comprising the step of administering to the
subject a therapeutically effective amount of a compound identified
using the method of claim 43, and administering to the subject a
therapeutically effective amount of a compound identified using the
method of claim 1.
94. A method of screening for modulators of lymphocyte activation,
the methods comprising the steps of: (i) transfecting into
lymphocytes a cDNA library; (ii) stimulating T or B cell receptors
of the lymphocytes; (iii) screening for modulation of lymphocyte
activation by detecting the level of CD69 cell surface expression
via FACS; and (iv) rescuing cDNAs that modulate lymphocyte
activation.
95. The method of claim 94, wherein the lymphocytes are T
cells.
96. The method of claim 95, wherein the T cells are cultured T
cells.
97. The method of claim 95, wherein the T cells are Jurkat
cells.
98. The method of claim 94, wherein cDNAs of the library are
operably linked to an inducible promoter.
99. The method of claim 98, wherein the inducible promoter
comprises a thymidine kinase promoter and a tetracycline regulatory
element
100. The method of claim 94, wherein the library is transfected by
retroviral vectors.
101. The method of claim 94, wherein the cDNA library is from a
primary lymphocyte organ.
102. The method of claim 101, wherein the primary lymphocyte organ
is selected from the group consisting of thymus, spleen, lymph
node, and bone marrow.
103. The method of claim 94, wherein the modulation is inhibition
of lymphocyte activation.
104. The method of claim 103, wherein the modulation is inhibition
of T lymphocyte activation.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims priority to U.S. Ser. No.
60/284,763, filed Apr. 18, 2001, herein incorporated by reference
in its entirety.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] The present invention relates to regulation of lymphocyte
activation and migration. More particularly, the present invention
is directed to nucleic acids encoding EDG family GPCR proteins,
e.g., EDG-1, 2, 3, 4, 5, 6, 7, or 8, which are involved in
modulation of lymphocyte activation and migration. The invention
further relates to methods for identifying and using agents,
including small organic molecules, antibodies, peptides, cyclic
peptides, nucleic acids, antisense nucleic acids, sphingolipid
analogs, and ribozymes, that modulate lymphocyte activation or
migration via modulation of EDG GPCRs and EDG related signal
transduction; as well as to the use of expression profiles and
compositions in diagnosis and therapy related to lymphocyte
activation and suppression, and lymphocyte migration.
BACKGROUND OF THE INVENTION
[0004] The immune response includes both a cellular and a humoral
response. The cellular response is mediate largely by T lymphocytes
(alternatively and equivalently referred to herein as T cells),
while the humoral response is mediated by B lymphocytes
(alternatively and equivalently referred to herein as B cells).
Lymphocytes play a number of crucial roles in immune responses,
including direct killing of virus-infected cells, cytokine and
antibody production, and facilitation of B cell responses.
Lymphocytes are also involved in acute and chronic inflammatory
disease; asthma; allergies; autoimmune diseases such as
scleroderma, pernicious anemia, multiple sclerosis, myasthenia
gravis, IDDM, rheumatoid arthritis, systemic lupus erythematosus,
and Crohn's disease; and organ and tissue transplant disease, e.g.,
graft vs. host disease.
[0005] B lymphocytes produce and secrete antibodies in response to
the concerted presentation of antigen and MHC class II molecules on
the surface of antigen presenting cells. Antigen presentation
initiates B cell activation through the B cell receptor (BCR) at
the B cell surface. Signal transduction from the BCR leads to B
cell activation and changes in B cell gene expression, physiology,
and function, including secretion of antibodies.
[0006] T cells do not produce antibodies, but many subtypes of T
cells produce co-stimulatory molecules that augment antibody
production by B cells during the humoral immune response. In
addition, many T cells engulf and destroy cells or agents that are
recognized by cell surface receptors. Engagement of the cell
surface T cell receptor (TCR) initiates T cell activation. Signal
transduction from the TCR leads to T cell activation and changes in
T cell gene expression, physiology, and function, including the
secretion of cytokines.
[0007] Identifying ligands, receptors, and signaling proteins
downstream of TCR, as well as BCR, activation is important for
developing therapeutic regents to inhibit immune response in
inflammatory disease, autoimmune disease, and organ transplant, as
well as to activate immune response in immunocompromised subjects,
and in patients with infectious disease and cancer (see, e.g.,
Rogge et al., Nature Genetics 25:96-101 (2000)). In addition,
identification of molecules participating in lymphocyte migration
is important for developing therapeutic reagents, as described
above.
SUMMARY OF THE INVENTION
[0008] The present invention therefore provides nucleic acids
encoding EDG G-protein coupled receptors (GPCRs), e.g., EDG-1, 2,
3, 4, 5, 7, and 8, which are involved in modulation of lymphocyte
activation and migration. The invention therefore provides methods
of screening for compounds, e.g., SPP and LPA analogs, including
sphingolipid-like compounds, small organic molecules, antibodies,
peptides, lipids, peptides, cyclic peptides, nucleic acids,
antisense molecules, and ribozyme, that are capable of modulating
lymphocyte activation and lymphocyte migration, e.g., either
activating or inhibiting lymphocytes and their ability to migrate.
Therapeutic and diagnostic methods and reagents are also
provided.
[0009] In one aspect of the invention, nucleic acids encoding EDG
GPCRs, e.g., EDG-1, 2, 3, 4, 6, 7, and 8 protein, are provided. In
another aspect, the present invention provides nucleic acids, such
as probes, antisense oligonucleotides, and ribozymes, that
hybridize to a gene encoding an EDG protein, e.g., EDG-1, 2, 3, 4,
6, 7, or 8. In another aspect, the invention provides expression
vectors and host cells comprising EDG-encoding nucleic acids, e.g.,
EDG-1, 2, 3, 4, 6, 7, or 8. In another aspect, the present
invention provides EDG protein, e.g., EDG-1, 2, 3, 4, 6, 7, or 8,
and antibodies thereto.
[0010] In another aspect, the present invention provides a method
for identifying a compound that modulates lymphocyte activation or
lymphocyte migration, the method comprising the steps of: (i)
contacting a cell comprising an EDG polypeptide or fragment thereof
with the compound, the EDG polypeptide or fragment thereof encoded
by a nucleic acid that hybridizes under stringent conditions to a
nucleic acid comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5,
6, 7, or 8; and (ii) determining the chemical or phenotypic effect
of the compound upon the cell comprising the EDG polypeptide or
fragment thereof, thereby identifying a compound that modulates
lymphocyte activation or migration.
[0011] In another aspect, the present invention provides a method
for identifying a compound that modulates lymphocyte activation or
migration, the method comprising the steps of: (i) contacting the
compound with an EDG polypeptide or a fragment thereof, the EDG
polypeptide or fragment thereof encoded by a nucleic acid that
hybridizes under stringent conditions to a nucleic acid comprising
a nucleotide sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8; (ii)
determining the physical effect of the compound upon the EDG
polypeptide; and (iii) determining the chemical or phenotypic
effect of the compound upon a cell comprising an EDG polypeptide or
fragment thereof, thereby identifying a compound that modulates
lymphocyte activation or migration.
[0012] In one embodiment, the EDG polypeptide or fragment thereof
is encoded by a nucleic acid that hybridizes under stringent
conditions to a nucleic acid comprising a sequence of SEQ ID
NO:1-4.
[0013] In another embodiment, the host cell is a B lymphocyte or a
T lymphocyte. In another embodiment, the host cell is a primary or
cultured cell, e.g., a BJAB or Jurkat cell.
[0014] In one embodiment, the chemical or phenotypic effect is
determined by measuring CD69 expression, IL-2 production,
intracellular Ca2+ mobilization, or lymphocyte proliferation.
[0015] In another embodiment, modulation is inhibition of T or B
lymphocyte activation or migration.
[0016] In another embodiment, the polypeptide is recombinant.
[0017] In another embodiment, the EDG polypeptide is selected from
the group consisting of EDG-1, 3, 5, 6, 7, or 8. In another
embodiment, the EDG polypeptide is an EDG-1 polypeptide encoded by
a nucleic acid selected from the group consisting of SEQ ID NOS:1,
2, 3, and 4.
[0018] In one embodiment, the EDG-1 fragment is an extracellular
domain. In another embodiment, the EDG-1 fragment is a cytoplasmic
domain. In another embodiment, the polypeptide further comprises at
least one transmembrane domain of an EDG polypeptide.
[0019] In another embodiment, the EDG polypeptide or fragment
thereof has GPCR activity.
[0020] In another embodiment, the compound is an antibody, an
antisense molecule, a peptide, a circular peptide, a small organic
molecule, a sphingolipid, a sphingolipid analog, either naturally
occurring or synthetic, e.g.,
2-amino-2(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride or
an analog thereof.
[0021] In one embodiment, the chemical or phenotypic effect is
determined by measuring lymphocyte migration in vitro toward an EDG
ligand, e.g., SPP or LPA.
[0022] In one aspect, the present invention provides a method of
modulating lymphocyte activation or migration in a subject, the
method comprising the step of administering to the subject a
therapeutically effective amount of a compound identified using the
methods described above.
[0023] In one embodiment, the subject is a human.
[0024] In another aspect, the present invention provides a
composition comprising a therapeutically effective amount of an
analog of 2-amino-2(2-[4-octylphenyl]ethyl)-1,3-propanediol
hydrochloride and a physiologically acceptable carrier.
[0025] In one embodiment, the present invention provides method of
modulating lymphocyte activation or migration in a subject, the
method comprising the step of administering to the subject a
therapeutically effective amount of an EDG polypeptide, the
polypeptide encoded by a nucleic acid that hybridizes under
stringent conditions to a nucleic acid comprising a nucleotide
sequence of EDG 1, 2, 3, 4, 5, 6, 7, or 8.
[0026] In one embodiment, the EDG polypeptide or fragment thereof
is encoded by a nucleic acid that hybridizes under stringent
conditions to a nucleic acid comprising a sequence of SEQ ID
NO:1-4. In another embodiment, the EDG polypeptide is selected from
the group consisting of EDG-1, 3, 5, 6, 7, and 8. In another
embodiment, the EDG polypeptide is an EDG-1 polypeptide encoded by
a nucleic acid selected from the group consisting of SEQ ID NO:1,
2, 3, and 4.
[0027] In another aspect, the present invention provides a method
of modulating lymphocyte activation or migration in a subject, the
method comprising the step of administering to the subject a
therapeutically effective amount of a nucleic acid encoding an EDG
polypeptide or fragment thereof, wherein the nucleic acid
hybridizes under stringent conditions to a nucleic acid encoding a
polypeptide comprising a nucleotide sequence of EDG 1, 2, 3, 4, 5,
6, 7, or 8.
[0028] In one embodiment, the EDG nucleic acid or fragment thereof
is encoded by a nucleic acid that hybridizes under stringent
conditions to a nucleic acid comprising a sequence of SEQ ID
NO:1-4. In another embodiment, the EDG nucleic acid is selected
from the group consisting of EDG-1, 3, 5, 6, 7, and 8. In another
embodiment, the EDG nucleic acid is an EDG-1 nucleic acid selected
from the group consisting of SEQ ID NO:1, 2, 3, and 4.
[0029] In one aspect, the present invention provides a method of
modulating T lymphocyte migration and activation in a subject, the
method comprising the step of administering to the subject a
therapeutically effective amount of a first compound identified
using the methods described above, which first compound modulates
activation, and administering to the subject a therapeutically
effective amount of a second compound identified using the methods
described above, which second compound modulates migration.
[0030] In another aspect, the present invention provides a method
of modulating T lymphocyte migration and activation in a subject,
the method comprising the step of administering to the subject a
therapeutically effective amount of a compound identified using the
methods described above, which compounds modulates both activation
and migration.
[0031] In another aspect, the present invention provides a method
of screening for modulators of lymphocyte activation, the methods
comprising the steps of: (i) transfecting into lymphocytes a cDNA
library; (ii) stimulating T or B cell receptors of the lymphocytes;
(iii) screening for modulation of lymphocyte activation by
detecting the level of CD69 cell surface expression via FACS; and
(iv) rescuing cDNAs that modulate lymphocyte activation.
[0032] In one embodiment, the lymphocytes are T cells, e.g.,
cultured T cells, e.g., Jurkat cells.
[0033] In another embodiment, cDNAs of the library are operably
linked to an inducible promoter, e.g., a tetracycline regulatory
element and a thymidine kinase promoter.
[0034] In another embodiment, the library is transfected by
retroviral vectors. In another embodiment, the cDNA library is from
a primary lymphocyte organ, e.g., thymus, spleen, lymph node, and
bone marrow.
[0035] In another embodiment, the modulation is inhibition of T
lymphocyte activation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 provides the nucleotide and amino acid sequence of
four EDG-1 proteins. SEQ ID NO:1 is the nucleotide sequence of
wild-type EDG-1 (381 amino acids in length); SEQ ID NO:2 is the
nucleotide sequence of mutant #1 (341 amino acids in length); SEQ
ID NO:3 is the nucleotide sequence of mutant #2 (314 amino acids in
length); and SEQ ID NO:4 is the nucleotide sequence of mutant #3
(346 amino acids in length). Each of the mutant EDG-1 polypeptides
is truncated at the C-terminus relative to the wild-type EDG-1.
[0037] FIG. 2 shows a sequence comparison of EDG family
proteins.
[0038] FIG. 3 shows a schematic of identification of regulatory
proteins that affect T cell activation.
[0039] FIG. 4 shows a schematic of TCR activation-induced
expression of CD69.
[0040] FIG. 5 shows induction of endogenous CD69 by anti-TCR.
[0041] FIG. 6 shows phenotypic enrichment through sequential FACS
sorting.
[0042] FIG. 7 shows a schematic of the distinction between
cDNA-induced phenotypes and somatic mutations.
[0043] FIG. 8 shows dox-regulatable phenotypes in clones after TCR
stimulation.
[0044] FIG. 9 shows a schematic of phenotypic assays in Jurkat
cells.
[0045] FIG. 10 shows a schematic of cell specificity of potential
targets.
[0046] FIG. 11 shows TCR induced CD69 upregulation: IRES-GFP vector
control.
[0047] FIG. 12 shows that dn-syk inhibits both TCR and BCR
signaling.
[0048] FIG. 13 shows known TCR regulators identified from a CD69
cDNA screen.
[0049] FIG. 14 shows primary, novel TCR regulators identified from
a CD69 cDNA screen.
[0050] FIG. 15 shows a schematic of EDG-1 and C-terminally
truncated variants.
[0051] FIG. 16 shows a dendogram of EDG family members.
[0052] FIG. 17 shows that wild-type EDG-1 and the c-terminally
truncated variants inhibit anti-BCR-induced CD69 upregulation in
BJAB cells.
[0053] FIG. 18 shows that wild-type EDG-1 and the c-terminally
truncated variants inhibit anti-TCR-induced CD69 upregulation in
Jurkat cells.
[0054] FIG. 19 shows that wild-type EDG-1 and the c-terminally
truncated variants inhibit anti-BCR-induced CD69 upregulation in
BJAB cells in TICD8.
[0055] FIG. 20 shows that wild-type EDG-1 and the c-terminally
truncated variants have no effect on anti-BCR-induced NFAT
upregulation in BJAB/NFAT cells
[0056] FIG. 21 shows that wild-type EDG-1 and the c-terminally
truncated variants have no effect on anti-BCR-induced IgH promoter
activation in BJAB/IgH cells.
[0057] FIG. 22 shows the relative level of EDG-1 message in
selected human tissues.
[0058] FIG. 23 shows the relative level of EDG-1 message in
selected, purified human cells.
[0059] FIG. 24 shows that in regular serum, SPP has no effect on
anti-BCR/TCR-induced CD69 upregulation in naive BJAB and Jurkat
cells.
[0060] FIG. 25 shows that SPP has no effect on anti-BCR/TCR-induced
CD69 upregulation in naive BJAB and Jurkat cells in the presence of
CS-FBS.
[0061] FIG. 26 shows a schematic of SPP induction in BJAB cells
expressing TIG-EDG-1 constructs.
[0062] FIG. 27 shows that SPP has no effect on anti-BCR induced
CD69 upregulation in BJAB cells expressing TIG vector.
[0063] FIG. 28 shows that SPP potentiates wild-type EDG-1
inhibition of anti-BCR induced CD69 upregulation in BJAB cells.
[0064] FIGS. 29 and 30 show that SPP has no effect on the
inhibition of EDG-1 mediated anti-BCR induced CD69 upregulation in
BJAB cells.
[0065] FIG. 31 shows a summary of SPP effect on wild-type EDG-1 and
the c-truncated variants in BJAB cells.
[0066] FIG. 32 shows that wild-type EDG-1 inhibits anti-BCR induced
CD69 upregulation in an SPP dose-dependent manner.
[0067] FIG. 33A shows a migration assay in Jurkat cells. FIG. 33B
shows SPP-induced migration of Jurkat cells.
[0068] FIG. 34A demonstrates that FTY720 has no effect on
SPP-induced migration in Jurkat cells. FIG. 34B demonstrates that
EDG-1 wild-type and EDG-1.DELTA.L potentiates SPP induced migration
in Jurkat cells.
[0069] FIG. 35 demonstrates that FTY720 inhibits SPP-induced
primary T cell migration.
[0070] FIG. 36A shows that FTY720 inhibits EDG-1 wild-type enhanced
SPP-induced migration in Jurkat cells. FIG. 36B shows that FTY720
has no effect on EDG-1 wild-type induced CD69 inhibition in Jurkat
cells.
[0071] FIG. 37 shows a summary of EDG-1 effects.
[0072] FIG. 38 shows the effect of EDG-1, 2, 4, 5, 7, and 8 on SPP
induced migration.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
[0073] For the first time, a protein from the EDG G-protein coupled
receptor (GPCR) family has been functionally identified as a
protein involved in regulating lymphocyte activation and migration.
EDG-1 was identified in a functional genetic screen using CD 69 as
a readout of lymphocyte activation. Truncated forms of nucleic
acids encoding C-terminally truncated variants of EDG-1 (SEQ ID
NOS:2-4) were recovered as inhibitors of lymphocyte
activation-induced CD69 expression. Wild type EDG-1 was also found
to inhibit T cell activation-induced CD69 expression. EDG-1
expression also inhibited IL-2 production in human primary
lymphocytes. In the absence of SPP, a ligand for wild-type EDG-1,
truncated forms of EDG-1 inhibited B cell activation-induced CD69
upregulation, while wild-type EDG-1 had a similar but weaker
effect. However, when SPP was added to the overexpressing wild-type
EDG-1, the wild type EDG-1 inhibited B cell activation-induced CD69
expression in a dose dependent manner. These results indicate that
EDG-1, other EDG family members such as EDG 8, and EDG family
modulators, e.g., agonists or agonists, can be used for inhibition
or activation of TCR and BCR signaling and lymphocyte activation.
In one embodiment, modulators of EDG family proteins are used to
inhibit lymphocyte activation. In one embodiment, agonists of EDG-1
are used for inhibition of lymphocyte activation.
[0074] In addition, EDG-1 mediates SPP-induced lymphocyte
migration, which is blocked by
2-amino-2(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride
(FTY720). The mutant EDG-1 protein lacking amino acids 315-382
(only one remaining amino acid of the C-terminal cytoplasmic tail)
does not mediate ligand-induced migration, demonstrating that the
C-terminal tail of EDG-1 is required for SPP-induced migration.
EDG-5 blocks SPP mediated migration. These results indicate that
EDG-1, other EDG family members such as EDG-5, and EDG modulators,
e.g., antagonists or agonists, can be used for inhibition or
activation of lymphocyte migration. In one embodiment, modulators
of EDG family proteins are used to inhibit lymphocyte migration. In
one embodiment, antagonists of EDG-1 are used for inhibition of
lymphocyte migration.
[0075] Previously, EDG family proteins were known to be G-protein
coupled receptors (GPCR, see, e.g., WO 94/05695 and U.S. Pat. No.
5,508,384) that are expressed in a wide variety of cells (see,
e.g., Goetzl et al., J. Immunol. 164:4669-4999 (2000)). However,
the function of EDG proteins was unknown. EDG-1 was identified as
expressed in endothelial cells as well as in many other cells, and
a role in angiogenesis has been proposed for this protein (see,
e.g., WO 91/15583; Bornfeldt et al., J. Cell Biol. 130:193-206
(1995); and Wang et al., J. Biol. Chem. 274:35343-35350 (1999)). It
has also been speculated that EDG-1 is involved in numerous diverse
disease states (see, e.g., WO 99/46277). EDG-1 is ubiquitously
expressed. EDG-4 has been identified as expressed in T lymphocytes,
among other cells (see, e.g., Goetzl et al., J. Immunol.
164:4669-4999 (2000)). A role for EDG-2 and other EDG family
members in apoptosis, e.g., in lymphocytes, has also been proposed
(see, e.g., WO 99/19513).
[0076] EDG-1 and other EDG family members EDG-2 to -8 were known to
bind sphingolipid ligands, e.g., sphingosine-1-phosphate (SPP,
EDG-1, 3, 5, 6, and 8) or lysophosphatidic acid (LPA), EDG-2, 4,
and 7) (see, e.g., Okamoto et al., J. Biol. Chem. 273:27104-27110
(1998); Lee et al., Science 279:1552-1555 (1998); Lee et al., J.
Biol. Chem. 273:22105-22112 (1998); Pyne & Pyne, Biochem. J.
349:385-402 (2000); and Windh et al., J. Biol. Chem.
274:27351-27358 (1999); and Prieschl & Baumruker, Immunology
Today 21:555-560 (2000)). Recent screening for immunosuppressants
has re-identified myriocin, a sphingosine-like natural fungal
product (Chen et al., Chemistry & Biology 6:221-235 (1999)).
FTY720 is a synthetic analog of myriocin and has immunosuppressant
activity, e.g., for organ transplant and graft vs. host disease
(2-amino-2(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride).
Its primary molecular target, however, is unknown (see, e.g.,
Brinkmann et al., TIPS 21:49-52 (2000); Pinschewer et al., J.
Immunol 164:5761-5770 (2000)). Although extracellular ligands SPP
and LPA were known to bind to EDG proteins, the function of the EDG
proteins remained unknown.
[0077] The present invention, therefore, has functionally
identified EDG-1, and other EDG family members such as EDG 3, 5, 6,
and 8 and EDG 2, 4, and 7 as drug targets for compounds that
suppress or activate lymphocyte activation and migration, e.g., for
the treatment of diseases in which modulation of the immune
response is desired, e.g., for treating diseases related to
lymphocyte activation and migration, such as delayed type
hypersensitivity reactions; asthma; allergies; autoimmune diseases
such as scleroderma, pernicious anemia, multiple sclerosis,
myasthenia gravis, IDDM, rheumatoid arthritis, systemic lupus
erythematosus, and Crohn's disease; and conditions related to organ
and tissue transplant, such as graft vs. host disease; and acute
and chronic inflammation; as well as in diseases in which
activation of the immune response and stimulation of lymphocyte
migration is desired, e.g., in immunocompromised subjects, e.g.,
due to HIV infection or cancer; and in infectious disease caused by
viral, fungal, protozoal, and bacterial infections. Preferably,
EDG-1, 3, 5, 6, 7, and 8 are used as drug targets for compounds
that activate or inhibit T cell activation and migration.
DEFINITIONS
[0078] By "disorder associated with lymphocyte activation or
migration" or "disease associated with lymphocyte activation or
migration" herein is meant a disease state which is marked by
either an excess or a deficit of B or T cell activation or
migration. For example, lymphocyte activation disorders associated
with increased activation or migration include, but are not limited
to, acute and chronic inflammation, asthma, allergies, autoimmune
disease and transplant rejection. Pathological states for which it
may be desirable to increase lymphocyte activation or migration
include HIV infection that results in immunocompromise, cancer, and
infectious disease such as viral, fungal, protozoal, and bacterial
infections. Different compounds may be used to modulate lymphocyte
activation and migration, or the same compound may be used to
modulate lymphocyte activation and migration.
[0079] "Lymphocyte migration" refers to migration of B and T
lymphocytes to and from primary and secondary lymphoid organs
(e.g., bone marrow, thymus, lymph nodes, spleen, Peyer's patch, and
tonsils), the periphery, and non-lymphoid tissues via the blood
stream, lymphatic vessels, and by penetration of capillary walls
(see, e.g., Paul, Immunology (3.sup.rd ed., 1993) (Chapters 4 and
6)). Without being bound to a particular theory, the present
invention demonstrates that EDG proteins, e.g., EDG-1 and EDG-5,
participate in the process of lymphocyte migration via ligand
binding to and or activation of the EDG protein (e.g., using SPP or
LPA or analogs thereof, and/or cytokines). SPP and LPA are present
in serum and are produced by a number of cells, including platelets
and fibroblasts. Ligand-induced lymphocyte migration can be
measured using the assay described in FIG. 33A, in which
lymphocytes migrate toward the ligand from an upper to a lower
chamber. The sphingolipid analog compound
2-amino-2(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride and
analogs thereof inhibit such migration. The C-terminus of EDG-1
appears to be involved in migration. Such domains (e.g., the
cytoplasmic tail of EDG-1) can be used in high throughput binding
assays for compounds that modulate lymphocyte migration.
[0080] "Lymphocyte activation" refers to the process of stimulating
quiescent (G.sub.0 phase of cell cycle), mature B and T cells by
encounter with antigen, either directly or indirectly (e.g., via a
helper cell and antigen presenting cells as well as via direct
antigen contact with a cell surface molecule of the lymphocyte).
Characteristics of activation can include, e.g., increase in cell
surface markers such as CD69, entry into the G.sub.1 phase of the
cell cycle, cytokine production, and proliferation (see, e.g.,
Paul, Immunology (3.sup.rd ed., 1993) (Chapters 13 and 14)).
Without being bound to a particular theory, the present invention
demonstrates that EDG proteins, e.g., EDG-1, participate in
modulation of lymphocyte activation, e.g., EDG-1 and EDG-8 inhibit
lymphocyte activation.
[0081] The terms "EDG" protein or fragment thereof, or a nucleic
acid encoding "EDG" or a fragment thereof refer to nucleic acids
and polypeptide polymorphic variants, alleles, mutants, and
interspecies homologs that: (1) have an amino acid sequence that
has greater than about 60% amino acid sequence identity, 65%, 70%,
75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98% or 99% or greater amino acid sequence identity, preferably over
a region of at least about 25, 50, 100, 200, 500, 1000, or more
amino acids, to an amino acid sequence encoded by an EDG nucleic
acid or amino acid sequence of an EDG protein, e.g., EDG-1, 3, 5,
6, 8, or EDG-2, 4, and 7; (2) specifically bind to antibodies,
e.g., polyclonal antibodies, raised against an immunogen comprising
an amino acid sequence of an EDG protein, e.g., EDG-1, 3, 5, 6, 8,
or EDG-2, 4, and 7, immunogenic fragments thereof, and
conservatively modified variants thereof, (3) specifically
hybridize under stringent hybridization conditions to an anti-sense
strand corresponding to a nucleic acid sequence encoding an EDG
protein, e.g., EDG-1, 3, 5, 6, 8, or EDG-2, 4, and 7, and
conservatively modified variants thereof, (4) have a nucleic acid
sequence that has greater than about 60% sequence identity, 65%,
70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99%, or higher nucleotide sequence identity, preferably
over a region of at least about 25, 50, 100, 200, 500, 1000, or
more nucleotides, to an EDG nucleic acid, e.g., EDG-1, 3, 5, 6, 8,
or EDG-2, 4, and 7.
[0082] An EDG polynucleotide or polypeptide sequence is typically
from a mammal including, but not limited to, primate, e.g., human;
rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any
mammal. The nucleic acids and proteins of the invention include
both naturally occurring or recombinant molecules. The EDG
polypeptide further has the ability to bind its naturally occurring
ligand, e.g., SPP or LPA, as well as other naturally occurring and
synthetic EDG family ligands and their analogs, including
sphingolipid-like compounds. The ability to bind an EDG family
protein identifies a compound as a sphingolipid analog, e.g., a
sphingolipid-like compound. EDG proteins often have GPCR activity,
e.g., the ability to transduce a signal via a G protein in response
to extracellular ligand binding. For example, EDG-1 is coupled to
G.sub.1, a pertussis toxin-sensitive G protein. Binding of SPP to
EDG-1 results in inhibition of adenylate cyclase and activation of
MAPK (both G.sub.1-mediated) as well as upregulation of P- and
E-cadherin expression and Rho-dependent morphogenesis.
[0083] The terms "EDG-1" protein or a fragment thereof, or a
nucleic acid encoding "EDG-1" protein or a fragment thereof refer
to nucleic acid and polypeptide polymorphic variants, alleles,
mutants, and interspecies homologs that: (1) have an amino acid
sequence that has greater than about 60% amino acid sequence
identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence
identity, preferably over a region of over a region of at least
about 25, 50, 100, 200, 500, 1000, or more amino acids, to an amino
acid sequence encoded by SEQ ID NO:1, 2, 3, or 4; (2) specifically
bind to antibodies, e.g., polyclonal antibodies, raised against an
immunogen comprising an amino acid sequence encoded by SEQ ID NO:1,
2, 3, or 4, immunogenic fragments thereof, and conservatively
modified variants thereof, (3) specifically hybridize under
stringent hybridization conditions to an anti-sense strand
corresponding to a nucleic acid sequence encoding an EDG protein,
e.g., SEQ ID NO:1, 2, 3, or 4, or their complements, and
conservatively modified variants thereof; (4) have a nucleic acid
sequence that has greater than about 60% sequence identity, 65%,
70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98% or 99%, or higher nucleotide sequence identity, preferably
over a region of at least about 25, 50, 100, 200, 500, 1000, or
more nucleotides, to SEQ ID NO:1, 2, 3, or 4 or their complements.
The EDG-1 polypeptide further has the ability to bind its naturally
occurring ligand, e.g., SPP, as well as other naturally occurring
and synthetic EDG-1 ligands and their analogs, including
sphingolipid-like compounds.
[0084] The Unigene number for EDG-1 is Hs.154210, and GenBank
accession numbers for exemplary nucleotide and amino acids
sequences are NM.sub.--001400, XM.sub.--001499, NP.sub.--001391,
XP.sub.--00149, AAC51905, AAF43420, and AAA52336. The chromosomal
location is Chr 1p21. The OMIM reference number for EDG-1 is
601974. EDG-1 is expressed in, e.g., in endothelial cells, vascular
smooth muscle cells, fibroblasts, melanocytes and cells of
epithelioid origin (see, e.g., Hla & Maciag, J. Biol. Chem.
265:9308-9313 (1990); Hobson et al., Science 291:1800-1803 (2001);
and Lee et al., Science 279:1552-1555 (1998)).
[0085] Exemplary wild type nucleic acid and protein sequences for
additional members of the EDG family are provided by the following
OMIM reference numbers (see also FIG. 2 for exemplary amino acid
sequences of EDG family members):
[0086] For EDG-2, OMIM reference number 602282. The GenBank
accession numbers for exemplary nucleotide and amino acids
sequences are NM.sub.--001401, XM.sub.--005557, XM.sub.--036690,
XM.sub.--036691, NP.sub.--001392, XP)--5557, XP.sub.--036690,
XP.sub.--036691, AAC00530, AAC51139, CAA70686, and CAA70687. (see,
e.g., An et al., Molec. Pharm. 54:881-888 (1998); An et al.,
Biochem. Biophys. Res. Commun. 231:619-622 (1997); Contos et al.,
Genomics 51:364-378 (1998); Hecht et al., J. Cell. Biol.
135:1071-1083 (1996); and Moolenaar et al., Curr. Opin. Cell Biol.
9:168-173 (1997)).
[0087] For EDG-3, OMIM reference number 601965. The GenBank
accession numbers for exemplary nucleotide and amino acids
sequences are NM.sub.--005226, NP.sub.--005217, CAA58744 and
AAC51906. (see, e.g., An et al., FEBS Lett. 417:279-282 (1997); and
Yamaguchi et al., Biochem. Biophys. Res. Commun. 227:608-614
(1996)).
[0088] For EDG-4, OMIM reference number 605110. The GenBank
accession numbers for exemplary nucleotide and amino acids
sequences are NM.sub.--004720, XM.sub.--012893, XM.sub.--048494,
XM.sub.--048495, NP.sub.--004711, XP.sub.--012893, XP.sub.--048494,
XP.sub.--048495, AAB61528, AAC27728 and AAF43409. (see, e.g., An et
al., J. Biol. Chem. 273:7906-7910 (1998); An et al., Molec. Pharm.
54:881-888 (1998); Contos et al., Genomics 64:155-169 (2000); and
Goetzl et al., J. Immunol. 164:4996-4999 (2000)).
[0089] For EDG-5, OMIM reference number 605111. The GenBank
accession numbers for exemplary nucleotide and amino acids
sequences are NM.sub.--004230, XM.sub.--008898, NP.sub.--004221,
XP.sub.--008898, and AAC98919. (see, e.g., An et al., J. Biol.
Chem. 275:288-296 (2000); Kupperman et al., Nature 406:192-195
(2000); and MacLennan et al., Molec. Cell. Neurosci. 5:201-209
(1994)).
[0090] For EDG-6, OMIM reference number 603751. The GenBank
accession numbers for exemplary nucleotide and amino acids
sequences are NM.sub.--003775, XM.sub.--009219, NP.sub.--003766,
XP.sub.--009219, and CAA04118. (see, e.g., Graler et al., Genomics
53:164-169 (1998); and Jedlicka et al., Cytogenet. Cell. Genet.
65:140 (1994)).
[0091] For EDG-7, OMIM reference number 605106. The GenBank
accession numbers for exemplary nucleotide and amino acids
sequences are NM.sub.--012152, XM.sub.--002057, XM.sub.--035234,
NP.sub.--036284, XP.sub.--002057, XP.sub.--035234, AAD56311,
AAF00530, and AAF91291. (see, e.g., Bandoh et al., J. Biol. Chem.
274:27776-27785 (1999)).
[0092] For EDG-8, OMIM reference number 605146. The GenBank
accession numbers for exemplary nucleotide and amino acids
sequences are NM.sub.--030760, XM.sub.--049584, NP.sub.--110387,
XP.sub.--049584, and AAG3813. (see, e.g., Im et al., J. Biol. Chem.
275:14281-14286 (2000)).
[0093] As described above, EDG proteins have "G-protein coupled
receptor activity," e.g., they bind to G-proteins in response to
extracellular stimuli, such as ligand binding, and promote
production of second messengers such as IP3, cAMP, and Ca.sup.2+
via stimulation of enzymes such as phospholipase C and adenylate
cyclase. Such activity can be measured in a heterologous cell, by
coupling a GPCR (or a chimeric GPCR) to a G-protein, e.g., a
promiscuous G-protein such as G.alpha.15, and an enzyme such as
PLC, and measuring increases in intracellular calcium using
(Offermans & Simon, J. Biol. Chem. 270:15175-15180 (1995)).
Receptor activity can be effectively measured, e.g., by recording
ligand-induced changes in [Ca.sup.2+].sub.i and calcium influx
using fluorescent Ca.sup.2+-indicator dyes and fluorometric
imaging.
[0094] G protein coupled receptors are glycoproteins that share
certain structural similarities (see, e.g., Gilman, Ann. Rev.
Biochem. 56:615-649 (1987), Strader et al., The FASEB J.
3:1825-1832 (1989), Kobilka et al., Nature 329:75-79 (1985), and
Young et al., Cell 45:711-719 (1986)). For example, G protein
coupled receptors have an extracellular domain, seven hydrophobic
stretches of about 20-25 amino acids in length interspersed with
eight hydrophilic regions (collectively known as the transmembrane
domain), and a cytoplasmic tail. Each of the seven hydrophobic
regions forms a transmembrane alpha helix, with the intervening
hydrophilic regions forming alternatively intracellular and
extracellular loops. The third cytosolic loop between transmembrane
domains five and six is involved in G-protein interaction. These
transmembrane hydrophobic domains, hydrophilic loop domains,
extracellular domains, and cytoplasmic tail domains can be
structurally identified using methods known to those of skill in
the art, such as sequence analysis programs that identify
hydrophobic and hydrophilic domains (see, e.g., Kyte &
Doolittle, J. Mol. Biol. 157:105-132 (1982)). Such domains are
useful for making chimeric proteins and for in vitro assays of the
invention (see, e.g., WO 94/05695 and U.S. Pat. No. 5,508,384).
Such domains are also considered "fragments" of EDG proteins, and
as such are useful in the assays of the invention, e.g., for ligand
binding studies, or for signal transduction studies using chimeric
proteins.
[0095] The phrase "functional effects" in the context of assays for
testing compounds that modulate activity of an EDG protein includes
the determination of a parameter that is indirectly or directly
under the influence of an EDG GPCR, e.g., an indirect, chemical or
phenotypic effect such as inhibition of lymphocyte activation or
migration represented by a change in expression of a cell surface
marker or cytokine production upon TCR stimulation, or changes in
cellular proliferation or apoptosis, or signal transduction leading
to increases in intracellular calcium; or, e.g., a direct, physical
effect such as ligand binding or inhibition of ligand binding or
movement from one chamber to another in response to ligand. A
functional effect therefore includes ligand binding activity, the
ability of cells to proliferate, the ability of cells to migrate,
apoptosis, gene expression in cells undergoing activation,
expression of cell surface molecules such as CD69, signal
transduction, production of cytokines, calcium influx, and other
characteristics of activated and/or migrating lymphocytes.
"Functional effects" include in vitro, in vivo, and ex vivo
activities.
[0096] By "determining the functional effect" is meant assaying for
a compound that increases or decreases a parameter that is
indirectly or directly under the influence of an EDG GPCR protein,
e.g., measuring physical and chemical or phenotypic effects. Such
functional effects can be measured by any means known to those
skilled in the art, e.g., changes in spectroscopic (e.g.,
fluorescence, absorbance, refractive index), hydrodynamic (e.g.,
shape), chromatographic, or solubility properties for the protein;
measuring inducible markers or transcriptional activation of the
protein; measuring binding activity or binding assays, e.g. binding
to antibodies; measuring changes in ligand binding affinity, e.g.,
SPP or LPA or analogs thereof or sphingolipid-like compounds,
either naturally occurring or synthetic; measuring cellular
proliferation; measuring cellular movement towards a ligand;
measuring apoptosis; measuring cell surface marker expression,
e.g., CD69; measuring cytokine, e.g., IL-2, production; measurement
of calcium influx; measurement of changes in protein levels for
EDG-associated sequences; measurement of RNA stability; G-protein
binding; GPCR phosphorylation or dephosphorylation; signal
transduction, e.g., receptor-ligand interactions, second messenger
concentrations (e.g., cAMP, IP3, or intracellular Ca.sup.2+);
identification of downstream or reporter gene expression (CAT,
luciferase, .beta.-gal, GFP and the like), e.g., via
chemiluminescence, fluorescence, colorimetric reactions, antibody
binding, inducible markers, and ligand binding assays.
[0097] "Inhibitors", "activators", and "modulators" of EDG
polynucleotide and polypeptide sequences are used to refer to
activating, inhibitory, or modulating molecules identified using in
vitro and in vivo assays of EDG polynucleotide and polypeptide
sequences. Inhibitors are compounds that, e.g., bind to, partially
or totally block activity, decrease, prevent, delay activation,
inactivate, desensitize, or down regulate the activity or
expression of EDG proteins, e.g., antagonists. "Activators" are
compounds that increase, open, activate, facilitate, enhance
activation, sensitize, agonize, or up regulate EDG protein
activity. Inhibitors, activators, or modulators also include
genetically modified versions of EDG proteins, e.g., versions with
altered activity, as well as naturally occurring and synthetic
ligands, antagonists, agonists, peptides, cyclic peptides, nucleic
acids, antibodies, antisense molecules, ribozymes, small organic
molecules and the like. Such assays for inhibitors and activators
include, e.g., expressing EDG protein in vitro, in cells, cell
extracts, or cell membranes, applying putative modulator compounds,
and then determining the functional effects on activity, as
described above.
[0098] Samples or assays comprising EDG proteins that are treated
with a potential activator, inhibitor, or modulator are compared to
control samples without the inhibitor, activator, or modulator to
examine the extent of activation or migration modulation. Control
samples (untreated with inhibitors) are assigned a relative protein
activity value of 100%. Inhibition of EDG is achieved when the
activity value relative to the control is about 80%, preferably
50%, more preferably 25-0%. Activation of EDG is achieved when the
activity value relative to the control (untreated with activators)
is 110%, more preferably 150%, more preferably 200-500% (i.e., two
to five fold higher relative to the control), more preferably
1000-3000% higher.
[0099] The term "test compound" or "drug candidate" or "modulator"
or grammatical equivalents as used herein describes any molecule,
either naturally occurring or synthetic, e.g., protein,
oligopeptide (e.g., from about 5 to about 25 amino acids in length,
preferably from about 10 to 20 or 12 to 18 amino acids in length,
preferably 12, 15, or 18 amino acids in length), small organic
molecule, polysaccharide, lipid (e.g., a sphingolipid), fatty acid,
polynucleotide, oligonucleotide, etc., to be tested for the
capacity to directly or indirectly modulation lymphocyte activation
or migration. The test compound can be in the form of a library of
test compounds, such as a combinatorial or randomized library that
provides a sufficient range of diversity. Test compounds are
optionally linked to a fusion partner, e.g., targeting compounds,
rescue compounds, dimerization compounds, stabilizing compounds,
addressable compounds, and other functional moieties.
Conventionally, new chemical entities with useful properties are
generated by identifying a test compound (called a "lead compound")
with some desirable property or activity, e.g., inhibiting
activity, creating variants of the lead compound, and evaluating
the property and activity of those variant compounds. Often, high
throughput screening (HTS) methods are employed for such an
analysis.
[0100] "FTY720" refers to a chemical molecule of the formula
2-amino-2(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride.
FTY720 is a sphingolipid analog. FTY720 and analogs thereof are
useful for inhibiting EDG-1 and EDG family mediated lymphocyte
migration. FTY720 and analogs thereof are designed and made
according to methods known to those of skill in the art (see, e.g.,
U.S. Pat. No. 6,004,565, U.S. Pat. No. 5,604,229, and PCT
application PCT/JP95/01654, and Fujita et al., J. Antibiotics
47:216-224 (1994)).
[0101] A "small organic molecule" refers to an organic molecule,
either naturally occurring or synthetic, that has a molecular
weight of more than about 50 daltons and less than about 2500
daltons, preferably less than about 2000 daltons, preferably
between about 100 to about 1000 daltons, more preferably between
about 200 to about 500 daltons.
[0102] "Biological sample" include sections of tissues such as
biopsy and autopsy samples, and frozen sections taken for
histologic purposes. Such samples include blood, sputum, tissue,
cultured cells, e.g., primary cultures, explants, and transformed
cells, stool, urine, etc. A biological sample is typically obtained
from a eukaryotic organism, most preferably a mammal such as a
primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g.,
guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
[0103] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region (e.g., nucleotide sequences SEQ ID
NO:1, 2, 3, or 4), when compared and aligned for maximum
correspondence over a comparison window or designated region) as
measured using a BLAST or BLAST 2.0 sequence comparison algorithms
with default parameters described below, or by manual alignment and
visual inspection (see, e.g., NCBI web site or the like). Such
sequences are then said to be "substantially identical." This
definition also refers to, or may be applied to, the compliment of
a test sequence. The definition also includes sequences that have
deletions and/or additions, as well as those that have
substitutions. As described below, the preferred algorithms can
account for gaps and the like. Preferably, identity exists over a
region that is at least about 25 amino acids or nucleotides in
length, or more preferably over a region that is 50-100 amino acids
or nucleotides in length.
[0104] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer; subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0105] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0106] A preferred example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J.
Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0
are used, with the parameters described herein, to determine
percent sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology
Information. This algorithm involves first identifying high scoring
sequence pairs (HSPs) by identifying short words of length W in the
query sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands.
[0107] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0108] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0109] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0110] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence with respect to the expression product, but not with
respect to actual probe sequences.
[0111] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0112] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0113] Macromolecular structures such as polypeptide structures can
be described in terms of various levels of organization. For a
general discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell (3.sup.rd ed., 1994) and Cantor and
Schimmel, Biophysical Chemistry Part I: The Conformation of
Biological Macromolecules (1980). "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains, e.g.,
transmembrane domains, pore domains, and cytoplasmic tail domains.
Domains are portions of a polypeptide that form a compact unit of
the polypeptide and are typically 15 to 350 amino acids long.
Exemplary domains include extracellular domains, transmembrane
domains, and cytoplasmic domains. Typical domains are made up of
sections of lesser organization such as stretches of .beta.-sheet
and .alpha.-helices. "Tertiary structure" refers to the complete
three dimensional structure of a polypeptide monomer. "Quaternary
structure" refers to the three dimensional structure formed by the
noncovalent association of independent tertiary units. Anisotropic
terms are also known as energy terms.
[0114] A particular nucleic acid sequence also implicitly
encompasses "splice variants." Similarly, a particular protein
encoded by a nucleic acid implicitly encompasses any protein
encoded by a splice variant of that nucleic acid. "Splice
variants," as the name suggests, are products of alternative
splicing of a gene. After transcription, an initial nucleic acid
transcript may be spliced such that different (alternate) nucleic
acid splice products encode different polypeptides. Mechanisms for
the production of splice variants vary, but include alternate
splicing of exons. Alternate polypeptides derived from the same
nucleic acid by read-through transcription are also encompassed by
this definition. Any products of a splicing reaction, including
recombinant forms of the splice products, are included in this
definition.
[0115] A "label" or a "detectable moiety" is a composition
detectable by spectroscopic, photochemical, biochemical,
immunochemical, chemical, or other physical means. For example,
useful labels include .sup.32P, fluorescent dyes, electron-dense
reagents, enzymes (e.g., as commonly used in an ELISA), biotin,
digoxigenin, or haptens and proteins which can be made detectable,
e.g., by incorporating a radiolabel into the peptide or used to
detect antibodies specifically reactive with the peptide.
[0116] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all.
[0117] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0118] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization. Exemplary stringent
hybridization conditions can be as following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C.
[0119] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., and Current Protocols in Molecular Biology, ed.
Ausubel, et al.
[0120] For PCR, a temperature of about 36.degree. C. is typical for
low stringency amplification, although annealing temperatures may
vary between about 32.degree. C. and 48.degree. C. depending on
primer length. For high stringency PCR amplification, a temperature
of about 62.degree. C. is typical, although high stringency
annealing temperatures can range from about 50.degree. C. to about
65.degree. C., depending on the primer length and specificity.
Typical cycle conditions for both high and low stringency
amplifications include a denaturation phase of 90.degree.
C.-95.degree. C. for 30 sec-2 min., an annealing phase lasting 30
sec.-2 min., and an extension phase of about 72.degree. C. for 1-2
min. Protocols and guidelines for low and high stringency
amplification reactions are provided, e.g., in Innis et al. (1990)
PCR Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y.).
[0121] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody will be most
critical in specificity and affinity of binding.
[0122] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0123] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well-characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially Fab with part of the
hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
While various antibody fragments are defined in terms of the
digestion of an intact antibody, one of skill will appreciate that
such fragments may be synthesized de novo either chemically or by
using recombinant DNA methodology. Thus, the term antibody, as used
herein, also includes antibody fragments either produced by the
modification of whole antibodies, or those synthesized de novo
using recombinant DNA methodologies (e.g., single chain Fv) or
those identified using phage display libraries (see, e.g.,
McCafferty et al., Nature 348:552-554 (1990))
[0124] For preparation of antibodies, e.g., recombinant,
monoclonal, or polyclonal antibodies, many technique known in the
art can be used (see, e.g., Kohler & Milstein, Nature
256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology
(1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988);
and Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
1986)). The genes encoding the heavy and light chains of an
antibody of interest can be cloned from a cell, e.g., the genes
encoding a monoclonal antibody can be cloned from a hybridoma and
used to produce a recombinant monoclonal antibody. Gene libraries
encoding heavy and light chains of monoclonal antibodies can also
be made from hybridoma or plasma cells. Random combinations of the
heavy and light chain gene products generate a large pool of
antibodies with different antigenic specificity (see, e.g., Kuby,
Immunology (3.sup.rd ed. 1997)). Techniques for the production of
single chain antibodies or recombinant antibodies (U.S. Pat. No.
4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce
antibodies to polypeptides of this invention. Also, transgenic
mice, or other organisms such as other mammals, may be used to
express humanized or human antibodies (see, e.g., U.S. Pat. Nos.
5,545,807; 5,545,806, 5,569,825; 5,625,126; 5,633,425; 5,661,016,
Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al.,
Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994);
Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger,
Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,
Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage
display technology can be used to identify antibodies and
heteromeric Fab fragments that specifically bind to selected
antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990);
Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also
be made bispecific, i.e., able to recognize two different antigens
(see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659
(1991); and Suresh et al., Methods in Enzymology 121:210 (1986)).
Antibodies can also be heteroconjugates, e.g., two covalently
joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No.
4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
[0125] Methods for humanizing or primatizing non-human antibodies
are well known in the art. Generally, a humanized antibody has one
or more amino acid residues introduced into it from a source which
is non-human. These non-human amino acid residues are often
referred to as import residues, which are typically taken from an
import variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (see, e.g., Jones et
al., Nature 321:522-525 (1986); Riechmann et al., Nature
332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such humanized
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0126] A "chimeric antibody" is an antibody molecule in which (a)
the constant region, or a portion thereof, is altered, replaced or
exchanged so that the antigen binding site (variable region) is
linked to a constant region of a different or altered class,
effector function and/or species, or an entirely different molecule
which confers new properties to the chimeric antibody, e.g., an
enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the
variable region, or a portion thereof, is altered, replaced or
exchanged with a variable region having a different or altered
antigen specificity.
[0127] In one embodiment, the antibody is conjugated to an
"effector" moiety. The effector moiety can be any number of
molecules, including labeling moieties such as radioactive labels
or fluorescent labels, or can be a therapeutic moiety. In one
aspect the antibody modulates the activity of the protein.
[0128] The phrase "specifically (or selectively) binds" to an
antibody or "specifically (or selectively) immunoreactive with,"
when referring to a protein or peptide, refers to a binding
reaction that is determinative of the presence of the protein,
often in a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein at least two
times the background and more typically more than 10 to 100 times
background. Specific binding to an antibody under such conditions
requires an antibody that is selected for its specificity for a
particular protein. For example, polyclonal antibodies raised to
EDG protein as encoded by SEQ ID NO:1, 2, 3, or 4, polymorphic
variants, alleles, orthologs, and conservatively modified variants,
or splice variants, or portions thereof, can be selected to obtain
only those polyclonal antibodies that are specifically
immunoreactive with EDG proteins and not with other proteins. This
selection may be achieved by subtracting out antibodies that
cross-react with other molecules. A variety of immunoassay formats
may be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select antibodies specifically immunoreactive
with a protein (see, e.g., Harlow & Lane, Antibodies, A
Laboratory Manual (1988) for a description of immunoassay formats
and conditions that can be used to determine specific
immunoreactivity).
[0129] By "therapeutically effective dose" herein is meant a dose
that produces effects for which it is administered. The exact dose
will depend on the purpose of the treatment, and will be
ascertainable by one skilled in the art using known techniques
(see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3,
1992); Lloyd, The Art, Science and Technology of Pharmaceutical
Compounding (1999); and Pickar, Dosage Calculations (1999)).
Assays for Proteins that Modulation Lymphocyte Activation
[0130] High throughput functional genomics assays can be used to
identify modulators of lymphocyte activation. Such assays can
monitor changes in cell surface marker expression, cytokine
production, antibody production, proliferation and differentiation,
and apoptosis, using either cell lines or primary cells. Typically,
the lymphocytes are contacted with a cDNA or a random peptide
library (encoded by nucleic acids). The cDNA library can comprise
sense, antisense, full length, and truncated cDNAs. The peptide
library is encoded by nucleic acids. The lymphocytes are then
activated, e.g., by activating either the T cell receptor (TCR,
also known as CD3) or the B cell receptor (BCR, also known as
surface or mIg), as appropriate, e.g., using antibodies to the
receptor. The effect of the cDNA or peptide library on the
phenotype of lymphocyte activation is then monitored, using an
assay as described above. The effect of the cDNA or peptide can be
validated and distinguished from somatic mutations, using, e.g.,
regulatable expression of the nucleic acid such as expression from
a tetracycline promoter. cDNAs and nucleic acids encoding peptides
can be rescued using techniques known to those of skill in the art,
e.g., using a sequence tag.
[0131] Proteins interacting with the peptide or with the protein
encoded by the cDNA (e.g., EDG) can be isolated using a yeast
two-hybrid system, mammalian two hybrid system, or phage display
screen, etc. Targets so identified can be further used as bait in
these assays to identify additional members of the lymphocyte
activation pathway, which members are also targets for drug
development (see, e.g., Fields et al., Nature 340:245 (1989);
Vasavada et al., Proc. Nat'l Acad. Sci. USA 88:10686 (1991); Fearon
et al., Proc. Nat'l Acad. Sci. USA 89:7958 (1992); Dang et al.,
Mol. Cell. Biol. 11:954 (1991); Chien et al., Proc. Nat'l Acad.
Sci. USA 9578 (1991); and U.S. Pat. Nos. 5,283,173, 5,667,973,
5,468,614, 5,525,490, and 5,637,463).
[0132] Suitable B cell lines include surface Ig(+) lines such as
CL-01, LA350, and CA46, as well as other mature and immature B cell
lines and primary B cells known to those of skill in the art.
Suitable T cell lines include Jurkat, HPB-ALL, HSB-2, and PEER, as
well as other mature and immature T cell lines and primary T cells
known to those of skill in the art. Suitable B cell surface
markers, for assaying B cell activation, include MHC class I, MHC
class II, CD23, CD40, CD58, CD69, CD72, CD80, CD86, LFA-1, LFA-3,
and ICAM-1, as well as other cell surface markers known to those of
skill in the art. Suitable T cell surface markers include MHC class
II, CD2, CD3, CD4, CD5, CD8, CD25, CD28, CD69, CD40L, LFA-1, and
ICAM-1 as well as other cell surface markers known to those of
skill in the art (see, e.g., Yablonski et al., Science 281:413-416
(1998)). Suitable cytokines, for measuring either production or
response, include IL-2, IL-4, IL-5, IL-6, IL-10, INF-.gamma., and
TGF-.beta., as well as their corresponding receptors.
[0133] Cell surface markers can be assayed using fluorescently
labeled antibodies and FACS. Cell proliferation can be measured
using .sup.3H-thymidine or dye inclusion. Apoptosis can be measured
using dye inclusion, or by assaying for DNA laddering or increases
in intracellular calcium. Cytokine production can be measured using
an immunoassay such as ELISA.
[0134] cDNA libraries are made from any suitable source, preferably
from primary human lymphoid organs such as thymus, spleen, lymph
node, and bone marrow. Libraries encoding random peptides are made
according to techniques well known to those of skill in the art
(see, e.g., U.S. Pat. Nos. 6,153,380, 6,114,111, and 6,180,343).
Any suitable vector can be used for the cDNA and peptide libraries,
including, e.g., retroviral vectors.
[0135] In a preferred embodiment, target proteins that modulate
lymphocyte activation, preferably T cell activation, are identified
using a high throughput cell based assay (using a microtiter plate
format) and FACS screening for CD69 cell surface expression (see
FIGS. 3-10 and Example I). cDNA libraries are made from primary
lymphocyte organs. These cDNA libraries include, e.g., sense,
antisense, full length, and truncated cDNAs. The cDNAs are cloned
into a retroviral vector with a tet-regulatable promoter. Jurkat
cells are infected with the library, the cells are stimulated with
anti-TCR antibodies, and then the cells are sorted using
fluorescent antibodies and FACS for CD69 low/CD3+ cells. Cells with
the desired phenotype are recovered, expanded, and cloned. A
Tet-regulatable phenotype is established to distinguish somatic
mutations. The cDNA is rescued. Optionally, the phenotype is
validated by assaying for IL-2 production using primary
lymphocytes. Optionally, a marker such as GFP can be used to select
for retrovirally infected cells. Using this system, cDNAs encoding
EDG-1 were identified as inhibitors of T cell activation.
Isolation of Nucleic Acids Encoding EDG Family Members
[0136] This invention relies on routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of
use in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994)).
[0137] EDG nucleic acids, polymorphic variants, orthologs, and
alleles that are substantially identical to an amino acid sequence
encoded by SEQ ID NO:1-4, as well as other EDG family members, can
be isolated using EDG nucleic acid probes and oligonucleotides
under stringent hybridization conditions, by screening libraries.
Alternatively, expression libraries can be used to clone EDG
protein, polymorphic variants, orthologs, and alleles by detecting
expressed homologs immunologically with antisera or purified
antibodies made against human EDG or portions thereof.
[0138] To make a cDNA library, one should choose a source that is
rich in EDG RNA. The mRNA is then made into cDNA using reverse
transcriptase, ligated into a recombinant vector, and transfected
into a recombinant host for propagation, screening and cloning.
Methods for making and screening cDNA libraries are well known
(see, e.g., Gubler & Hoffman, Gene 25:263-269 (1983); Sambrook
et al., supra; Ausubel et al., supra).
[0139] For a genomic library, the DNA is extracted from the tissue
and either mechanically sheared or enzymatically digested to yield
fragments of about 12-20 kb. The fragments are then separated by
gradient centrifugation from undesired sizes and are constructed in
bacteriophage lambda vectors. These vectors and phage are packaged
in vitro. Recombinant phage are analyzed by plaque hybridization as
described in Benton & Davis, Science 196:180-182 (1977). Colony
hybridization is carried out as generally described in Grunstein et
al., Proc. Natl. Acad. Sci. USA., 72:3961-3965 (1975).
[0140] An alternative method of isolating EDG nucleic acid and its
orthologs, alleles, mutants, polymorphic variants, and
conservatively modified variants combines the use of synthetic
oligonucleotide primers and amplification of an RNA or DNA template
(see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide
to Methods and Applications (Innis et al., eds, 1990)). Methods
such as polymerase chain reaction (PCR) and ligase chain reaction
(LCR) can be used to amplify nucleic acid sequences of human EDG
directly from mRNA, from cDNA, from genomic libraries or cDNA
libraries. Degenerate oligonucleotides can be designed to amplify
EDG homologs using the sequences provided herein. Restriction
endonuclease sites can be incorporated into the primers. Polymerase
chain reaction or other in vitro amplification methods may also be
useful, for example, to clone nucleic acid sequences that code for
proteins to be expressed, to make nucleic acids to use as probes
for detecting the presence of EDG encoding mRNA in physiological
samples, for nucleic acid sequencing, or for other purposes. Genes
amplified by the PCR reaction can be purified from agarose gels and
cloned into an appropriate vector.
[0141] Gene expression of EDG can also be analyzed by techniques
known in the art, e.g., reverse transcription and amplification of
mRNA, isolation of total RNA or poly A.sup.+ RNA, northern
blotting, dot blotting, in situ hybridization, RNase protection,
high density polynucleotide array technology, e.g., and the
like.
[0142] Nucleic acids encoding EDG protein can be used with high
density oligonucleotide array technology (e.g., GeneChip.TM.) to
identify EDG protein, orthologs, alleles, conservatively modified
variants, and polymorphic variants in this invention. In the case
where the homologs being identified are linked to modulation of T
cell activation and migration, they can be used with GeneChip as a
diagnostic tool in detecting the disease in a biological sample,
see, e.g., Gunthand et al, AIDS Res. Hum. Retroviruses 14: 869-876
(1998); Kozal et al., Nat. Med. 2:753-759 (1996); Matson et al.,
Anal. Biochem. 224:110-106 (1995); Lockhart et al., Nat.
Biotechnol. 14:1675-1680 (1996); Gingeras et al., Genome Res.
8:435-448 (1998); Hacia et al., Nucleic Acids Res. 26:3865-3866
(1998).
[0143] The gene for EDG is typically cloned into intermediate
vectors before transformation into prokaryotic or eukaryotic cells
for replication and/or expression. These intermediate vectors are
typically prokaryote vectors, e.g., plasmids, or shuttle
vectors.
Expression in Prokaryotes and Eukaryotes
[0144] To obtain high level expression of a cloned gene, such as
those cDNAs encoding EDG, one typically subclones EDG into an
expression vector that contains a strong promoter to direct
transcription, a transcription/translation terminator, and if for a
nucleic acid encoding a protein, a ribosome binding site for
translational initiation. Suitable bacterial promoters are well
known in the art and described, e.g., in Sambrook et al., and
Ausubel et al, supra. Bacterial expression systems for expressing
the EDG protein are available in, e.g., E. coli, Bacillus sp., and
Salmonella (Palva et al., Gene 22:229-235 (1983); Mosbach et al.,
Nature 302:543-545 (1983). Kits for such expression systems are
commercially available. Eukaryotic expression systems for mammalian
cells, yeast, and insect cells are well known in the art and are
also commercially available. In one preferred embodiment,
retroviral expression systems are used in the present
invention.
[0145] Selection of the promoter used to direct expression of a
heterologous nucleic acid depends on the particular application.
The promoter is preferably positioned about the same distance from
the heterologous transcription start site as it is from the
transcription start site in its natural setting. As is known in the
art, however, some variation in this distance can be accommodated
without loss of promoter function.
[0146] In addition to the promoter, the expression vector typically
contains a transcription unit or expression cassette that contains
all the additional elements required for the expression of the EDG
encoding nucleic acid in host cells. A typical expression cassette
thus contains a promoter operably linked to the nucleic acid
sequence encoding EDG and signals required for efficient
polyadenylation of the transcript, ribosome binding sites, and
translation termination. Additional elements of the cassette may
include enhancers and, if genomic DNA is used as the structural
gene, introns with functional splice donor and acceptor sites.
[0147] In addition to a promoter sequence, the expression cassette
should also contain a transcription termination region downstream
of the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes.
[0148] The particular expression vector used to transport the
genetic information into the cell is not particularly critical. Any
of the conventional vectors used for expression in eukaryotic or
prokaryotic cells may be used. Standard bacterial expression
vectors include plasmids such as pBR322 based plasmids, pSKF,
pET23D, and fusion expression systems such as MBP, GST, and LacZ.
Epitope tags can also be added to recombinant proteins to provide
convenient methods of isolation, e.g., c-myc. Sequence tags may be
included in an expression cassette for nucleic acid rescue. Markers
such as fluorescent proteins, green or red fluorescent protein,
.beta.-gal, CAT, and the like can be included in the vectors as
markers for vector transduction.
[0149] Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 vectors, papilloma virus vectors, retroviral
vectors, and vectors derived from Epstein-Barr virus. Other
exemplary eukaryotic vectors include pMSG, pAV009/A.sup.+,
pMTO10/A.sup.+, pMAMneo-5, baculovirus pDSVE, and any other vector
allowing expression of proteins under the direction of the CMV
promoter, SV40 early promoter, SV40 later promoter, metallothionein
promoter, murine mammary tumor virus promoter, Rous sarcoma virus
promoter, polyhedrin promoter, or other promoters shown effective
for expression in eukaryotic cells.
[0150] Expression of proteins from eukaryotic vectors can be also
be regulated using inducible promoters. With inducible promoters,
expression levels are tied to the concentration of inducing agents,
such as tetracycline or ecdysone, by the incorporation of response
elements for these agents into the promoter. Generally, high level
expression is obtained from inducible promoters only in the
presence of the inducing agent; basal expression levels are
minimal.
[0151] In one embodiment, the vectors of the invention have a
regulatable promoter, e.g., tet-regulated systems and the RU-486
system (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992);
Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene
Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996);
and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)). These
impart small molecule control on the expression of the candidate
target nucleic acids. This beneficial feature can be used to
determine that a desired phenotype is caused by a transfected cDNA
rather than a somatic mutation.
[0152] Some expression systems have markers that provide gene
amplification such as thymidine kinase and dihydrofolate reductase.
Alternatively, high yield expression systems not involving gene
amplification are also suitable, such as using a baculovirus vector
in insect cells, with a EDG encoding sequence under the direction
of the polyhedrin promoter or other strong baculovirus
promoters.
[0153] The elements that are typically included in expression
vectors also include a replicon that functions in E. coli, a gene
encoding antibiotic resistance to permit selection of bacteria that
harbor recombinant plasmids, and unique restriction sites in
nonessential regions of the plasmid to allow insertion of
eukaryotic sequences. The particular antibiotic resistance gene
chosen is not critical, any of the many resistance genes known in
the art are suitable. The prokaryotic sequences are preferably
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells, if necessary.
[0154] Standard transfection methods are used to produce bacterial,
mammalian, yeast or insect cell lines that express large quantities
of EDG protein, which are then purified using standard techniques
(see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989);
Guide to Protein Purification, in Methods in Enzymology, vol. 182
(Deutscher, ed., 1990)). Transformation of eukaryotic and
prokaryotic cells are performed according to standard techniques
(see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss
& Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds,
1983).
[0155] Any of the well-known procedures for introducing foreign
nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, polybrene, protoplast
fusion, electroporation, biolistics, liposomes, microinjection,
plasma vectors, viral vectors and any of the other well known
methods for introducing cloned genomic DNA, cDNA, synthetic DNA or
other foreign genetic material into a host cell (see, e.g.,
Sambrook et al., supra). It is only necessary that the particular
genetic engineering procedure used be capable of successfully
introducing at least one gene into the host cell capable of
expressing EDG.
[0156] After the expression vector is introduced into the cells,
the transfected cells are cultured under conditions favoring
expression of EDG, which is recovered from the culture using
standard techniques identified below.
Purification of EDG Polypeptides
[0157] Either naturally occurring or recombinant EDG can be
purified for use in functional assays. Naturally occurring EDG can
be purified, e.g., from human tissue. Recombinant EDG can be
purified from any suitable expression system.
[0158] The EDG protein may be purified to substantial purity by
standard techniques, including selective precipitation with such
substances as ammonium sulfate; column chromatography,
immunopurification methods, and others (see, e.g., Scopes, Protein
Purification: Principles and Practice (1982); U.S. Pat. No.
4,673,641; Ausubel et al., supra; and Sambrook et al., supra).
[0159] A number of procedures can be employed when recombinant EDG
protein is being purified. For example, proteins having established
molecular adhesion properties can be reversible fused to the EDG
protein. With the appropriate ligand, EDG protein can be
selectively adsorbed to a purification column and then freed from
the column in a relatively pure form. The fused protein is then
removed by enzymatic activity. Finally, EDG protein could be
purified using immunoaffinity columns.
[0160] A. Purification of EDG from Recombinant Bacteria
[0161] Recombinant proteins are expressed by transformed bacteria
in large amounts, typically after promoter induction; but
expression can be constitutive. Promoter induction with IPTG is one
example of an inducible promoter system. Bacteria are grown
according to standard procedures in the art. Fresh or frozen
bacteria cells are used for isolation of protein.
[0162] Proteins expressed in bacteria may form insoluble aggregates
("inclusion bodies"). Several protocols are suitable for
purification of EDG protein inclusion bodies. For example,
purification of inclusion bodies typically involves the extraction,
separation and/or purification of inclusion bodies by disruption of
bacterial cells, e.g., by incubation in a buffer of 50 mM TRIS/HCL
pH 7.5, 50 mM NaCl, 5 mM MgCl.sub.2, 1 mM DTT, 0.1 mM ATP, and 1 mM
PMSF. The cell suspension can be lysed using 2-3 passages through a
French Press, homogenized using a Polytron (Brinkman Instruments)
or sonicated on ice. Alternate methods of lysing bacteria are
apparent to those of skill in the art (see, e.g., Sambrook et al.,
supra; Ausubel et al., supra).
[0163] If necessary, the inclusion bodies are solubilized, and the
lysed cell suspension is typically centrifuged to remove unwanted
insoluble matter. Proteins that formed the inclusion bodies may be
renatured by dilution or dialysis with a compatible buffer.
Suitable solvents include, but are not limited to urea (from about
4 M to about 8 M), formamide (at least about 80%, volume/volume
basis), and guanidine hydrochloride (from about 4 M to about 8 M).
Some solvents which are capable of solubilizing aggregate-forming
proteins, for example SDS (sodium dodecyl sulfate), 70% formic
acid, are inappropriate for use in this procedure due to the
possibility of irreversible denaturation of the proteins,
accompanied by a lack of immunogenicity and/or activity. Although
guanidine hydrochloride and similar agents are denaturants, this
denaturation is not irreversible and renaturation may occur upon
removal (by dialysis, for example) or dilution of the denaturant,
allowing re-formation of immunologically and/or biologically active
protein. Other suitable buffers are known to those skilled in the
art. Human EDG proteins are separated from other bacterial proteins
by standard separation techniques, e.g., with Ni-NTA agarose
resin.
[0164] Alternatively, it is possible to purify EDG protein from
bacteria periplasm. After lysis of the bacteria, when the EDG
protein exported into the periplasm of the bacteria, the
periplasmic fraction of the bacteria can be isolated by cold
osmotic shock in addition to other methods known to skill in the
art. To isolate recombinant proteins from the periplasm, the
bacterial cells are centrifuged to form a pellet. The pellet is
resuspended in a buffer containing 20% sucrose. To lyse the cells,
the bacteria are centrifuged and the pellet is resuspended in
ice-cold 5 mM MgSO.sub.4 and kept in an ice bath for approximately
10 minutes. The cell suspension is centrifuged and the supernatant
decanted and saved. The recombinant proteins present in the
supernatant can be separated from the host proteins by standard
separation techniques sell known to those of skill in the art.
[0165] B. Standard Protein Separation Techniques for Purifying EDG
Proteins
[0166] Solubility Fractionation
[0167] Often as an initial step, particularly if the protein
mixture is complex, an initial salt fractionation can separate many
of the unwanted host cell proteins (or proteins derived from the
cell culture media) from the recombinant protein of interest. The
preferred salt is ammonium sulfate. Ammonium sulfate precipitates
proteins by effectively reducing the amount of water in the protein
mixture. Proteins then precipitate on the basis of their
solubility. The more hydrophobic a protein is, the more likely it
is to precipitate at lower ammonium sulfate concentrations. A
typical protocol includes adding saturated ammonium sulfate to a
protein solution so that the resultant ammonium sulfate
concentration is between 20-30%. This concentration will
precipitate the most hydrophobic of proteins. The precipitate is
then discarded (unless the protein of interest is hydrophobic) and
ammonium sulfate is added to the supernatant to a concentration
known to precipitate the protein of interest. The precipitate is
then solubilized in buffer and the excess salt removed if
necessary, either through dialysis or diafiltration. Other methods
that rely on solubility of proteins, such as cold ethanol
precipitation, are well known to those of skill in the art and can
be used to fractionate complex protein mixtures.
[0168] Size Differential Filtration
[0169] The molecular weight of the EDG proteins can be used to
isolate it from proteins of greater and lesser size using
ultrafiltration through membranes of different pore size (for
example, Amicon or Millipore membranes). As a first step, the
protein mixture is ultrafiltered through a membrane with a pore
size that has a lower molecular weight cut-off than the molecular
weight of the protein of interest. The retentate of the
ultrafiltration is then ultrafiltered against a membrane with a
molecular cut off greater than the molecular weight of the protein
of interest. The recombinant protein will pass through the membrane
into the filtrate. The filtrate can then be chromatographed as
described below.
[0170] Column Chromatography
[0171] The EDG proteins can also be separated from other proteins
on the basis of its size, net surface charge, hydrophobicity, and
affinity for ligands. In addition, antibodies raised against
proteins can be conjugated to column matrices and the proteins
immunopurified. All of these methods are well known in the art. It
will be apparent to one of skill that chromatographic techniques
can be performed at any scale and using equipment from many
different manufacturers (e.g., Pharmacia Biotech).
Assays for Modulators of EDG Protein
[0172] A. Assays
[0173] Modulation of an EDG protein, and corresponding modulation
of lymphocyte activation and/or migration, can be assessed using a
variety of in vitro and in vivo assays, including cell-based models
as described above. Such assays can be used to test for inhibitors
and activators of EDG protein or fragments thereof, and,
consequently, inhibitors and activators of lymphocyte activation
and migration. Such modulators of EDG protein, which is involved in
lymphocyte activation and migration, are useful for treating
disorders related to T and B cell activation and migration.
Modulators of EDG protein are tested using either recombinant or
naturally occurring EDG, preferably human EDG.
[0174] Preferably, the EDG protein will have the sequence as
encoded by SEQ ID NO:1-4, or an exemplary Genbank Accession number
as provided herein, or a conservatively modified variant thereof.
Alternatively, the EDG protein of the assay will be derived from a
eukaryote and include an amino acid subsequence having substantial
amino acid sequence identity to SEQ ID NO:1-4. Generally, the amino
acid sequence identity will be at least 60%, preferably at least
65%, 70%, 75%, 80%, 85%, or 90%, most preferably at least 95%.
[0175] Measurement of lymphocyte activation, migration, or
loss-of-lymphocyte activation or migration phenotype on EDG protein
or cell expressing EDG protein, either recombinant or naturally
occurring, can be performed using a variety of assays, in vitro, in
vivo, and ex vivo, as described herein. A suitable physical,
chemical or phenotypic change that affects activity or binding can
be used to assess the influence of a test compound on the
polypeptide of this invention. When the functional effects are
determined using intact cells or animals, one can also measure a
variety of effects such as, in the case of signal transduction,
e.g., ligand binding (SPP, LPA, GTP), hormone release,
transcriptional changes to both known and uncharacterized genetic
markers (e.g., northern blots), cellular movement towards a ligand,
movement of labeled cells, changes in cell metabolism such as pH
changes, and changes in intracellular second messengers such as
Ca.sup.2+, IP3, cGMP, or cAMP; as well as changes related to
lymphocyte activation and migration, e.g., cellular proliferation,
cell surface marker expression, e.g., CD69, cytokine production,
and apoptosis.
[0176] In one preferred embodiment, described herein in Example I,
measurement of CD69 activation and FACS sorting is used to identify
modulators of lymphocyte, e.g., T cell, activation. In another
preferred embodiment, shown in FIG. 33A, measurement of cellular
migration toward a ligand is used to identify modulators of
lymphocyte, e.g., T cell, migration.
[0177] In Vitro Assays
[0178] Assays to identify compounds with EDG modulating activity
can be performed in vitro. Such assays can used full length EDG
protein or a variant thereof (see, e.g., SEQ ID NOS: 1-4), or a
fragment of an EDG protein, such as an extracellular domain or a
cytoplasmic domain, optionally fused to a heterologous protein to
form a chimera. In one embodiment, different domains can be used to
assay for activation and migration. In another embodiment, the same
domain can be used to assay for activation and migration. In one
embodiment, the C-terminal cytoplasmic tail (e.g., amino acids
315-382) can be used in high throughput binding assays to identify
compounds that modulate lymphocyte migration. Purified recombinant
or naturally occurring EDG protein can be used in the in vitro
methods of the invention. In addition to purified EDG protein or
fragment thereof, the recombinant or naturally occurring EDG
protein can be part of a cellular lysate or a cell membrane. As
described below, the binding assay can be either solid state or
soluble. Preferably, the protein, fragment thereof or membrane is
bound to a solid support, either covalently or non-covalently.
Often, the in vitro assays of the invention are ligand binding or
ligand affinity assays, either non-competitive or competitive (with
known extracellular ligands SPP or LPA, or with a known
intracellular ligand GTP). Other in vitro assays include measuring
changes in spectroscopic (e.g., fluorescence, absorbance,
refractive index), hydrodynamic (e.g., shape), chromatographic, or
solubility properties for the protein.
[0179] In one embodiment, a high throughput binding assay is
performed in which the EDG protein or fragment thereof is contacted
with a potential modulator and incubated for a suitable amount of
time. In one embodiment, the potential modulator is bound to a
solid support, and the EDG protein is added. In another embodiment,
the EDG protein is bound to a solid support. A wide variety of
modulators can be used, as described below, including small organic
molecules, peptides, antibodies, and EDG ligand analogs. A wide
variety of assays can be used to identify EDG-modulator binding,
including labeled protein-protein binding assays, electrophoretic
mobility shifts, immunoassays, enzymatic assays such as
phosphorylation assays, and the like. In some cases, the binding of
the candidate modulator is determined through the use of
competitive binding assays, where interference with binding of a
known ligand is measured in the presence of a potential modulator.
Ligands for the EDG family are known (SPP, LPA, and GTP). Either
the modulator or the known ligand is bound first, and then the
competitor is added. After the EDG protein is washed, interference
with binding, either of the potential modulator or of the known
ligand, is determined. Often, either the potential modulator or the
known ligand is labeled.
[0180] Cell-Based In Vivo Assays
[0181] In another embodiment, EDG protein is expressed in a cell,
and functional, e.g., physical and chemical or phenotypic, changes
are assayed to identify EDG and lymphocyte activation and migration
modulators. Cells expressing EDG proteins can also be used in
binding assays. Any suitable functional effect can be measured, as
described herein. For example, ligand binding, cell surface marker
expression, cellular proliferation, apoptosis, cytokine production,
and GPCR signal transduction, e.g., changes in intracellular
Ca.sup.2+ levels, are all suitable assays to identify potential
modulators using a cell based system. Suitable cells for such cell
based assays include both primary lymphocytes and cell lines, as
described herein. The EDG protein can be naturally occurring or
recombinant. Also, as described above, fragments of EDG proteins or
chimeras with GPCR activity can be used in cell based assays. For
example, the extracellular domain of an EDG protein can be fused to
the transmembrane and/or cytoplasmic domain of a heterologous
protein, preferably a heterologous GPCR. Such a chimeric GPCR would
have GPCR activity and could be used in cell based assays of the
invention. In another embodiment, a domain of the EDG protein, such
as the extracellular or cytoplasmic domain, is used in the
cell-based assays of the invention.
[0182] As described above, in one embodiment, lymphocyte activation
is measured by contacting T cells comprising an EDG target with a
potential modulator and activating the cells with an anti-TCR
antibody. Modulation of T cell activation is identified by
screening for cell surface marker expression, e.g., CD69 expression
levels, using fluorescent antibodies and FACS sorting. In another
embodiment, lymphocyte migration is measured by observing T cell
migration from an upper to a lower chamber containing an EDG ligand
such as SPP.
[0183] In another embodiment, cellular proliferation, migration, or
apoptosis can be measured using .sup.3H-thymidine incorporation or
dye inclusion. Cytokine production can be measured using an
immunoassay such as an ELISA.
[0184] In another embodiment, cellular EDG polypeptide levels are
determined by measuring the level of protein or mRNA. The level of
EDG protein or proteins related to EDG signal transduction are
measured using immunoassays such as western blotting, ELISA and the
like with an antibody that selectively binds to the EDG polypeptide
or a fragment thereof. For measurement of mRNA, amplification,
e.g., using PCR, LCR, or hybridization assays, e.g., northern
hybridization, RNAse protection, dot blotting, are preferred. The
level of protein or mRNA is detected using directly or indirectly
labeled detection agents, e.g., fluorescently or radioactively
labeled nucleic acids, radioactively or enzymatically labeled
antibodies, and the like, as described herein.
[0185] Alternatively, EDG expression can be measured using a
reporter gene system. Such a system can be devised using an EDG
protein promoter operably linked to a reporter gene such as
chloramphenicol acetyltransferase, firefly luciferase, bacterial
luciferase, .beta.-galactosidase and alkaline phosphatase.
Furthermore, the protein of interest can be used as an indirect
reporter via attachment to a second reporter such as red or green
fluorescent protein (see, e.g., Mistili & Spector, Nature
Biotechnology 15:961-964 (1997)). The reporter construct is
typically transfected into a cell. After treatment with a potential
modulator, the amount of reporter gene transcription, translation,
or activity is measured according to standard techniques known to
those of skill in the art.
[0186] In another embodiment, a functional effect related to GPCR
signal transduction can be measured. An activated or inhibited EDG
G-coupled protein receptor will alter the properties of target
enzymes, second messengers, channels, and other effector proteins.
The examples include the activation of cGMP phosphodiesterase,
adenylate cyclase, phospholipase C, IP3, and modulation of diverse
channels by G proteins. Downstream consequences can also be
examined such as generation of diacyl glycerol and IP3 by
phospholipase C, and in turn, for calcium mobilization by IP3.
Activated GPCR receptors become substrates for kinases that
phosphorylate the C-terminal tail of the receptor (and possibly
other sites as well). Thus, activators will promote the transfer of
.sup.32P from gamma-labeled GTP to the receptor, which can be
assayed with a scintillation counter. The phosphorylation of the
C-terminal tail will promote the binding of arrestin-like proteins
and will interfere with the binding of G-proteins. For a general
review of GPCR signal transduction and methods of assaying signal
transduction, see, e.g., Methods in Enzymology, vols. 237 and 238
(1994) and volume 96 (1983); Bourne et al., Nature 10:349:117-27
(1991); Bourne et al., Nature 348:125-32 (1990); Pitcher et al.,
Annu. Rev. Biochem. 67:653-92 (1998).
[0187] As described above, activation of some G-protein coupled
receptors stimulates the formation of inositol triphosphate (IP3)
through phospholipase C-mediated hydrolysis of phosphatidylinositol
(Berridge & Irvine, Nature 312:315-21 (1984)). IP3 in turn
stimulates the release of intracellular calcium ion stores. Thus, a
change in cytoplasmic calcium ion levels, or a change in second
messenger levels such as IP3 can be used to assess G-protein
coupled receptor function. Cells expressing such G-protein coupled
receptors may exhibit increased cytoplasmic calcium levels as a
result of contribution from both intracellular stores and via
activation of ion channels, in which case it may be desirable
although not necessary to conduct such assays in calcium-free
buffer, optionally supplemented with a chelating agent such as
EGTA, to distinguish fluorescence response resulting from calcium
release from internal stores.
[0188] In one example, EDG GPCR activity is measured by expressing
an EDG GPCR in a heterologous cell with a promiscuous G-protein
that links the receptor to a phospholipase C signal transduction
pathway (see Offermanns & Simon, J. Biol. Chem. 270:15175-15180
(1995)). Modulation of signal transduction is assayed by measuring
changes in intracellular Ca.sup.2+ levels, which change in response
to modulation of the GPCR signal transduction pathway via
administration of a molecule that associates with an EDG GPCR.
Changes in Ca.sup.2+ levels are optionally measured using
fluorescent Ca.sup.2+ indicator dyes and fluorometric imaging.
[0189] In another example, phosphatidyl inositol (PI) hydrolysis
can be analyzed according to U.S. Pat. No. 5,436,128, herein
incorporated by reference. Briefly, the assay involves labeling of
cells with .sup.3H-myoinositol for 48 or more hrs. The labeled
cells are treated with a test compound for one hour. The treated
cells are lysed and extracted in chloroform-methanol-water after
which the inositol phosphates were separated by ion exchange
chromatography and quantified by scintillation counting. Fold
stimulation is determined by calculating the ratio of cpm in the
presence of agonist to cpm in the presence of buffer control.
Likewise, fold inhibition is determined by calculating the ratio of
cpm in the presence of antagonist to cpm in the presence of buffer
control (which may or may not contain an agonist).
[0190] Other assays can involve determining the activity of
receptors which, when activated, result in a change in the level of
intracellular cyclic nucleotides, e.g., cAMP or cGMP, by activating
or inhibiting enzymes such as adenylate cyclase. In cases where
activation of the receptor results in a decrease in cyclic
nucleotide levels, it may be preferable to expose the cells to
agents that increase intracellular cyclic nucleotide levels, e.g.,
forskolin, prior to adding a receptor-activating compound to the
cells in the assay.
[0191] In one example, the changes in intracellular cAMP or cGMP
can be measured using immunoassays. The method described in
Offermanns & Simon, J. Biol. Chem. 270:15175-15180 (1995) may
be used to determine the level of cAMP. Also, the method described
in Felley-Bosco et al., Am. J. Resp. Cell and Mol. Biol. 11:159-164
(1994) may be used to determine the level of cGMP. Further, an
assay kit for measuring cAMP and/or cGMP is described in U.S. Pat.
No. 4,115,538, herein incorporated by reference.
[0192] In one example, assays for G-protein coupled receptor
activity include cells that are loaded with ion or voltage
sensitive dyes to report receptor activity, e.g., by observing
calcium influx or intracellular calcium release. Assays for
determining activity of such receptors can also use known agonists
and antagonists for other G-protein coupled receptors as negative
or positive controls to assess activity of tested compounds. In
assays for identifying modulatory compounds (e.g., agonists,
antagonists), changes in the level of ions in the cytoplasm or
membrane voltage will be monitored using an ion sensitive or
membrane voltage fluorescent indicator, respectively. Among the
ion-sensitive indicators and voltage probes that may be employed
are those disclosed in the Molecular Probes 1997 Catalog. For
G-protein coupled receptors, promiscuous G-proteins such as
G.alpha.15 and G.alpha.16 can be used in the assay of choice
(Wilkie et al., Proc. Nat'l Acad. Sci. USA 88:10049-10053 (1991)).
Such promiscuous G-proteins allow coupling of a wide range of
receptors.
[0193] Animal Models
[0194] Animal models of lymphocyte activation and migration also
find use in screening for modulators of lymphocyte activation or
migration. Similarly, transgenic animal technology including gene
knockout technology, for example as a result of homologous
recombination with an appropriate gene targeting vector, or gene
overexpression, will result in the absence or increased expression
of the EDG protein. The same technology can also be applied to make
knock-out cells. When desired, tissue-specific expression or
knockout of the EDG protein may be necessary. Transgenic animals
generated by such methods find use as animal models of lymphocyte
activation and migration and are additionally useful in screening
for modulators of lymphocyte activation and migration.
[0195] Knock-out cells and transgenic mice can be made by insertion
of a marker gene or other heterologous gene into an endogenous EDG
gene site in the mouse genome via homologous recombination. Such
mice can also be made by substituting an endogenous EDG with a
mutated version of the EDG gene, or by mutating an endogenous EDG,
e.g., by exposure to carcinogens.
[0196] A DNA construct is introduced into the nuclei of embryonic
stem cells. Cells containing the newly engineered genetic lesion
are injected into a host mouse embryo, which is re-implanted into a
recipient female. Some of these embryos develop into chimeric mice
that possess germ cells partially derived from the mutant cell
line. Therefore, by breeding the chimeric mice it is possible to
obtain a new line of mice containing the introduced genetic lesion
(see, e.g., Capecchi et al., Science 244:1288 (1989)). Chimeric
targeted mice can be derived according to Hogan et al.,
Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring
Harbor Laboratory (1988) and Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach, Robertson, ed., IRL Press, Washington,
D.C., (1987).
[0197] B. Modulators
[0198] The compounds tested as modulators of EDG protein can be any
small organic molecule, or a biological entity, such as a protein,
e.g., an antibody or peptide, a sugar, a nucleic acid, e.g., an
antisense oligonucleotide or a ribozyme, or a lipid. Alternatively,
modulators can be genetically altered versions of an EDG protein.
Typically, test compounds will be small organic molecules,
peptides, lipids, and lipid analogs. In one embodiment, the
compound is a sphingolipid analog, either naturally occurring or
synthetic. In another embodiment, the compound is
2-amino-2(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride
(also known as FTY720) or an analog thereof.
[0199] Essentially any chemical compound can be used as a potential
modulator or ligand in the assays of the invention, although most
often compounds can be dissolved in aqueous or organic (especially
DMSO-based) solutions are used. The assays are designed to screen
large chemical libraries by automating the assay steps and
providing compounds from any convenient source to assays, which are
typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays). It will be appreciated that
there are many suppliers of chemical compounds, including Sigma
(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St.
Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs Switzerland)
and the like.
[0200] In one preferred embodiment, high throughput screening
methods involve providing a combinatorial small organic molecule or
peptide library containing a large number of potential therapeutic
compounds (potential modulator or ligand compounds). Such
"combinatorial chemical libraries" or "ligand libraries" are then
screened in one or more assays, as described herein, to identify
those library members (particular chemical species or subclasses)
that display a desired characteristic activity. The compounds thus
identified can serve as conventional "lead compounds" or can
themselves be used as potential or actual therapeutics.
[0201] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0202] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication No. WO
93/20242), random bio-oligomers (e.g., PCT Publication No. WO
92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514),
diversomers such as hydantoins, benzodiazepines and dipeptides
(Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)),
vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.
114:6568 (1992)), nonpeptidal peptidomimetics with glucose
scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218
(1992)), analogous organic syntheses of small compound libraries
(Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates
(Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates
(Campbell et al., J. Org. Chem. 59:658 (1994)), nucleic acid
libraries (see Ausubel, Berger and Sambrook, all supra), peptide
nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083),
antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology,
14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries
(see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S.
Pat. No. 5,593,853), small organic molecule libraries (see, e.g.,
benzodiazepines, Baum C&EN, January 18, page 33 (1993);
isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and
metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat.
Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. Nos.
5,506,337; benzodiazepines, 5,288,514, and the like).
[0203] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd, Moscow, Ru, 3D Pharmaceuticals, Exton, Pa., Martek
Biosciences, Columbia, Md., etc.).
[0204] C. Solid State and Soluble High Throughput Assays
[0205] In one embodiment the invention provides soluble assays
using a EDG protein, or a cell or tissue expressing an EDG protein,
either naturally occurring or recombinant. In another embodiment,
the invention provides solid phase based in vitro assays in a high
throughput format, where the EDG protein or fragment thereof, such
as the cytoplasmic domain, is attached to a solid phase substrate.
Any one of the assays described herein can be adapted for high
throughput screening, e.g., ligand binding, cellular proliferation,
cell surface marker flux, e.g., CD-69, screening, radiolabeled GTP
binding, second messenger flux, e.g., Ca.sup.2+, IP3, cGMP, or
cAMP, cytokine production, etc. In one preferred embodiment, the
cell-based system using CD-69 modulation and FACS assays is used in
a high throughput format for identifying modulators of EDG
proteins, and therefore modulators of T cell activation.
[0206] In the high throughput assays of the invention, either
soluble or solid state, it is possible to screen up to several
thousand different modulators or ligands in a single day. This
methodology can be used for EDG proteins in vitro, or for
cell-based or membrane-based assays comprising an EDG protein. In
particular, each well of a microtiter plate can be used to run a
separate assay against a selected potential modulator, or, if
concentration or incubation time effects are to be observed, every
5-10 wells can test a single modulator. Thus, a single standard
microtiter plate can assay about 100 (e.g., 96) modulators. If 1536
well plates are used, then a single plate can easily assay from
about 100-about 1500 different compounds. It is possible to assay
many plates per day; assay screens for up to about 6,000, 20,000,
50,000, or more than 100,000 different compounds are possible using
the integrated systems of the invention.
[0207] For a solid state reaction, the protein of interest or a
fragment thereof, e.g., an extracellular domain, or a cell or
membrane comprising the protein of interest or a fragment thereof
as part of a fusion protein can be bound to the solid state
component, directly or indirectly, via covalent or non covalent
linkage e.g., via a tag. The tag can be any of a variety of
components. In general, a molecule which binds the tag (a tag
binder) is fixed to a solid support, and the tagged molecule of
interest is attached to the solid support by interaction of the tag
and the tag binder.
[0208] A number of tags and tag binders can be used, based upon
known molecular interactions well described in the literature. For
example, where a tag has a natural binder, for example, biotin,
protein A, or protein G, it can be used in conjunction with
appropriate tag binders (avidin, streptavidin, neutravidin, the Fc
region of an immunoglobulin, etc.) Antibodies to molecules with
natural binders such as biotin are also widely available and
appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue
SIGMA, St. Louis Mo.).
[0209] Similarly, any haptenic or antigenic compound can be used in
combination with an appropriate antibody to form a tag/tag binder
pair. Thousands of specific antibodies are commercially available
and many additional antibodies are described in the literature. For
example, in one common configuration, the tag is a first antibody
and the tag binder is a second antibody which recognizes the first
antibody. In addition to antibody-antigen interactions,
receptor-ligand interactions are also appropriate as tag and
tag-binder pairs. For example, agonists and antagonists of cell
membrane receptors (e.g., cell receptor-ligand interactions such as
transferrin, c-kit, viral receptor ligands, cytokine receptors,
chemokine receptors, interleukin receptors, immunoglobulin
receptors and antibodies, the cadherein family, the integrin
family, the selectin family, and the like; see, e.g., Pigott &
Power, The Adhesion Molecule Facts Book I (1993). Similarly, toxins
and venoms, viral epitopes, hormones (e.g., opiates, steroids,
etc.), intracellular receptors (e.g. which mediate the effects of
various small ligands, including steroids, thyroid hormone,
retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic
acids (both linear and cyclic polymer configurations),
oligosaccharides, proteins, phospholipids and antibodies can all
interact with various cell receptors.
[0210] Synthetic polymers, such as polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, and polyacetates
can also form an appropriate tag or tag binder. Many other tag/tag
binder pairs are also useful in assay systems described herein, as
would be apparent to one of skill upon review of this
disclosure.
[0211] Common linkers such as peptides, polyethers, and the like
can also serve as tags, and include polypeptide sequences, such as
poly gly sequences of between about and 200 amino acids. Such
flexible linkers are known to persons of skill in the art. For
example, poly(ethelyne glycol) linkers are available from
Shearwater Polymers, Inc. Huntsville, Ala. These linkers optionally
have amide linkages, sulfhydryl linkages, or heterofunctional
linkages.
[0212] Tag binders are fixed to solid substrates using any of a
variety of methods currently available. Solid substrates are
commonly derivatized or functionalized by exposing all or a portion
of the substrate to a chemical reagent which fixes a chemical group
to the surface which is reactive with a portion of the tag binder.
For example, groups which are suitable for attachment to a longer
chain portion would include amines, hydroxyl, thiol, and carboxyl
groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to
functionalize a variety of surfaces, such as glass surfaces. The
construction of such solid phase biopolymer arrays is well
described in the literature. See, e.g., Merrifield, J. Am. Chem.
Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,
e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)
(describing synthesis of solid phase components on pins); Frank
& Doring, Tetrahedron 44:60316040 (1988) (describing synthesis
of various peptide sequences on cellulose disks); Fodor et al.,
Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry
39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759
(1996) (all describing arrays of biopolymers fixed to solid
substrates). Non-chemical approaches for fixing tag binders to
substrates include other common methods, such as heat,
cross-linking by UV radiation, and the like.
Immunological Detection of EDG Polypeptides
[0213] In addition to the detection of EDG gene and gene expression
using nucleic acid hybridization technology, one can also use
immunoassays to detect EDG proteins of the invention. Such assays
are useful for screening for modulators of EDG and lymphocyte
activation and migration, as well as for therapeutic and diagnostic
applications. Immunoassays can be used to qualitatively or
quantitatively analyze EDG protein. A general overview of the
applicable technology can be found in Harlow & Lane,
Antibodies: A Laboratory Manual (1988).
[0214] A. Production of Antibodies
[0215] Methods of producing polyclonal and monoclonal antibodies
that react specifically with the EDG proteins are known to those of
skill in the art (see, e.g., Coligan, Current Protocols in
Immunology (1991); Harlow & Lane, supra; Goding, Monoclonal
Antibodies: Principles and Practice (2d ed. 1986); and Kohler &
Milstein, Nature 256:495-497 (1975). Such techniques include
antibody preparation by selection of antibodies from libraries of
recombinant antibodies in phage or similar vectors, as well as
preparation of polyclonal and monoclonal antibodies by immunizing
rabbits or mice (see, e.g., Huse et al., Science 246:1275-1281
(1989); Ward et al., Nature 341:544-546 (1989)).
[0216] A number of immunogens comprising portions of EDG protein
may be used to produce antibodies specifically reactive with EDG
protein. For example, recombinant EDG protein or an antigenic
fragment thereof, can be isolated as described herein. Recombinant
protein can be expressed in eukaryotic or prokaryotic cells as
described above, and purified as generally described above.
Recombinant protein is the preferred immunogen for the production
of monoclonal or polyclonal antibodies. Alternatively, a synthetic
peptide derived from the sequences disclosed herein and conjugated
to a carrier protein can be used an immunogen. Naturally occurring
protein may also be used either in pure or impure form. The product
is then injected into an animal capable of producing antibodies.
Either monoclonal or polyclonal antibodies may be generated, for
subsequent use in immunoassays to measure the protein.
[0217] Methods of production of polyclonal antibodies are known to
those of skill in the art. An inbred strain of mice (e.g., BALB/C
mice) or rabbits is immunized with the protein using a standard
adjuvant, such as Freund's adjuvant, and a standard immunization
protocol. The animal's immune response to the immunogen preparation
is monitored by taking test bleeds and determining the titer of
reactivity to the beta subunits. When appropriately high titers of
antibody to the immunogen are obtained, blood is collected from the
animal and antisera are prepared. Further fractionation of the
antisera to enrich for antibodies reactive to the protein can be
done if desired (see, Harlow & Lane, supra).
[0218] Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Briefly, spleen cells from an
animal immunized with a desired antigen are immortalized, commonly
by fusion with a myeloma cell (see, Kohler & Milstein, Eur. J.
Immunol. 6:511-519 (1976)). Alternative methods of immortalization
include transformation with Epstein Barr Virus, oncogenes, or
retroviruses, or other methods well known in the art. Colonies
arising from single immortalized cells are screened for production
of antibodies of the desired specificity and affinity for the
antigen, and yield of the monoclonal antibodies produced by such
cells may be enhanced by various techniques, including injection
into the peritoneal cavity of a vertebrate host. Alternatively, one
may isolate DNA sequences which encode a monoclonal antibody or a
binding fragment thereof by screening a DNA library from human B
cells according to the general protocol outlined by Huse, et al.,
Science 246:1275-1281 (1989).
[0219] Monoclonal antibodies and polyclonal sera are collected and
titered against the immunogen protein in an immunoassay, for
example, a solid phase immunoassay with the immunogen immobilized
on a solid support. Typically, polyclonal antisera with a titer of
10.sup.4 or greater are selected and tested for their cross
reactivity against non-EDG proteins, using a competitive binding
immunoassay. Specific polyclonal antisera and monoclonal antibodies
will usually bind with a K.sub.d of at least about 0.1 mM, more
usually at least about 1 .mu.M, preferably at least about 0.1 .mu.M
or better, and most preferably, 0.01 .mu.M or better. Antibodies
specific only for a particular EDG family member, such as EDG 1, or
a particular EDG-1 ortholog, such as human EDG1, can also be made,
by subtracting out other cross-reacting EDG family members or
orthologs from a species such as a non-human mammal. In this
manner, antibodies that bind only to a particular EDG protein or
ortholog may be obtained.
[0220] Once the specific antibodies against EDG protein are
available, the protein can be detected by a variety of immunoassay
methods. In addition: the antibody can be used therapeutically as a
EDG modulators. For a review of immunological and immunoassay
procedures, see Basic and Clinical Immunology (Stites & Terr
eds., 7.sup.th ed. 1991). Moreover, the immunoassays of the present
invention can be performed in any of several configurations, which
are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980);
and Harlow & Lane, supra.
[0221] B. Immunological Binding Assays
[0222] EDG protein can be detected and/or quantified using any of a
number of well recognized immunological binding assays (see, e.g.,
U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For
a review of the general immunoassays, see also Methods in Cell
Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993);
Basic and Clinical Immunology (Stites & Terr, eds., 7th ed.
1991). Immunological binding assays (or immunoassays) typically use
an antibody that specifically binds to a protein or antigen of
choice (in this case the EDG protein or antigenic subsequence
thereof). The antibody (e.g., anti-EDG) may be produced by any of a
number of means well known to those of skill in the art and as
described above.
[0223] Immunoassays also often use a labeling agent to specifically
bind to and label the complex formed by the antibody and antigen.
The labeling agent may itself be one of the moieties comprising the
antibody/antigen complex. Thus, the labeling agent may be a labeled
EDG or a labeled anti-EDG antibody. Alternatively, the labeling
agent may be a third moiety, such a secondary antibody, that
specifically binds to the antibody/EDG complex (a secondary
antibody is typically specific to antibodies of the species from
which the first antibody is derived). Other proteins capable of
specifically binding immunoglobulin constant regions, such as
protein A or protein G may also be used as the label agent. These
proteins exhibit a strong non-immunogenic reactivity with
immunoglobulin constant regions from a variety of species (see,
e.g., Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom
et al., J. Immunol. 135:2589-2542 (1985)). The labeling agent can
be modified with a detectable moiety, such as biotin, to which
another molecule can specifically bind, such as streptavidin. A
variety of detectable moieties are well known to those skilled in
the art.
[0224] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, optionally from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, antigen, volume of solution,
concentrations, and the like. Usually, the assays will be carried
out at ambient temperature, although they can be conducted over a
range of temperatures, such as 110.degree. C. to 40.degree. C.
[0225] Non-Competitive Assay Formats
[0226] Immunoassays for detecting EDG in samples may be either
competitive or noncompetitive. Noncompetitive immunoassays are
assays in which the amount of antigen is directly measured. In one
preferred "sandwich" assay, for example, the anti-EDG antibodies
can be bound directly to a solid substrate on which they are
immobilized. These immobilized antibodies then capture EDG present
in the test sample. EDG proteins thus immobilized are then bound by
a labeling agent, such as a second EDG antibody bearing a label.
Alternatively, the second antibody may lack a label, but it may, in
turn, be bound by a labeled third antibody specific to antibodies
of the species from which the second antibody is derived. The
second or third antibody is typically modified with a detectable
moiety, such as biotin, to which another molecule specifically
binds, e.g., streptavidin, to provide a detectable moiety.
[0227] Competitive Assay Formats
[0228] In competitive assays, the amount of EDG protein present in
the sample is measured indirectly by measuring the amount of a
known, added (exogenous) EDG protein displaced (competed away) from
an anti-EDG antibody by the unknown EDG protein present in a
sample. In one competitive assay, a known amount of EDG protein is
added to a sample and the sample is then contacted with an antibody
that specifically binds to EDG protein. The amount of exogenous EDG
protein bound to the antibody is inversely proportional to the
concentration of EDG protein present in the sample. In a
particularly preferred embodiment, the antibody is immobilized on a
solid substrate. The amount of EDG protein bound to the antibody
may be determined either by measuring the amount of EDG present in
EDG protein/antibody complex, or alternatively by measuring the
amount of remaining uncomplexed protein. The amount of EDG protein
may be detected by providing a labeled EDG molecule.
[0229] A hapten inhibition assay is another preferred competitive
assay. In this assay the known EDG protein is immobilized on a
solid substrate. A known amount of anti-EDG antibody is added to
the sample, and the sample is then contacted with the immobilized
EDG. The amount of anti-EDG antibody bound to the known immobilized
EDG is inversely proportional to the amount of EDG protein present
in the sample. Again, the amount of immobilized antibody may be
detected by detecting either the immobilized fraction of antibody
or the fraction of the antibody that remains in solution. Detection
may be direct where the antibody is labeled or indirect by the
subsequent addition of a labeled moiety that specifically binds to
the antibody as described above.
[0230] Cross-Reactivity Determinations
[0231] Immunoassays in the competitive binding format can also be
used for crossreactivity determinations. For example, an EDG
protein can be immobilized to a solid support. Proteins (e.g., EDG
and homologs) are added to the assay that compete for binding of
the antisera to the immobilized antigen. The ability of the added
proteins to compete for binding of the antisera to the immobilized
protein is compared to the ability of the EDG protein to compete
with itself. The percent crossreactivity for the above proteins is
calculated, using standard calculations. Those antisera with less
than 10% crossreactivity with each of the added proteins listed
above are selected and pooled. The cross-reacting antibodies are
optionally removed from the pooled antisera by immunoabsorption
with the added considered proteins, e.g., distantly related
homologs.
[0232] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein, thought to be perhaps an allele or polymorphic
variant of an EDG protein, to the immunogen protein. In order to
make this comparison, the two proteins are each assayed at a wide
range of concentrations and the amount of each protein required to
inhibit 50% of the binding of the antisera to the immobilized
protein is determined. If the amount of the second protein required
to inhibit 50% of binding is less than 10 times the amount of the
EDG protein that is required to inhibit 50% of binding, then the
second protein is said to specifically bind to the polyclonal
antibodies generated to EDG immunogen.
[0233] Other Assay Formats
[0234] Western blot (immunoblot) analysis is used to detect and
quantify the presence of EDG in the sample. The technique generally
comprises separating sample proteins by gel electrophoresis on the
basis of molecular weight, transferring the separated proteins to a
suitable solid support, (such as a nitrocellulose filter, a nylon
filter, or derivatized nylon filter), and incubating the sample
with the antibodies that specifically bind EDG. The anti-EDG
antibodies specifically bind to the EDG on the solid support. These
antibodies may be directly labeled or alternatively may be
subsequently detected using labeled antibodies (e.g., labeled sheep
anti-mouse antibodies) that specifically bind to the anti-EDG
antibodies.
[0235] Other assay formats include liposome immunoassays (LIA),
which use liposomes designed to bind specific molecules (e.g.,
antibodies) and release encapsulated reagents or markers. The
released chemicals are then detected according to standard
techniques (see Monroe et al., Amer. Clin. Prod. Rev. 5:34-41
(1986)).
[0236] Reduction of Non-Specific Binding
[0237] One of skill in the art will appreciate that it is often
desirable to minimize non-specific binding in immunoassays.
Particularly, where the assay involves an antigen or antibody
immobilized on a solid substrate it is desirable to minimize the
amount of non-specific binding to the substrate. Means of reducing
such non-specific binding are well known to those of skill in the
art. Typically, this technique involves coating the substrate with
a proteinaceous composition. In particular, protein compositions
such as bovine serum albumin (BSA), nonfat powdered milk, and
gelatin are widely used with powdered milk being most
preferred.
[0238] Labels
[0239] The particular label or detectable group used in the assay
is not a critical aspect of the invention, as long as it does not
significantly interfere with the specific binding of the antibody
used in the assay. The detectable group can be any material having
a detectable physical or chemical property. Such detectable labels
have been well-developed in the field of immunoassays and, in
general, most any label useful in such methods can be applied to
the present invention. Thus, a label is any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g., DYNABEADS.TM.), fluorescent
dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and
the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S,
.sup.14C, or .sup.32P), enzymes (e.g., horse radish peroxidase,
alkaline phosphatase and others commonly used in an ELISA), and
colorimetric labels such as colloidal gold or colored glass or
plastic beads (e.g., polystyrene, polypropylene, latex, etc.).
[0240] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. As indicated above, a wide variety of labels may be used,
with the choice of label depending on sensitivity required, ease of
conjugation with the compound, stability requirements, available
instrumentation, and disposal provisions.
[0241] Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to
the molecule. The ligand then binds to another molecules (e.g.,
streptavidin) molecule, which is either inherently detectable or
covalently bound to a signal system, such as a detectable enzyme, a
fluorescent compound, or a chemiluminescent compound. The ligands
and their targets can be used in any suitable combination with
antibodies that recognize EDG protein, or secondary antibodies that
recognize anti-EDG.
[0242] The molecules can also be conjugated directly to signal
generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes of interest as labels will primarily be
hydrolases, particularly phosphatases, esterases and glycosidases,
or oxidotases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds
include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
For a review of various labeling or signal producing systems that
may be used, see U.S. Pat. No. 4,391,904.
[0243] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is a
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence. The fluorescence may be detected visually,
by means of photographic film, by the use of electronic detectors
such as charge coupled devices (CCDs) or photomultipliers and the
like. Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the resulting
reaction product. Finally simple colorimetric labels may be
detected simply by observing the color associated with the label.
Thus, in various dipstick assays, conjugated gold often appears
pink, while various conjugated beads appear the color of the
bead.
[0244] Some assay formats do not require the use of labeled
components. For instance, agglutination assays can be used to
detect the presence of the target antibodies. In this case,
antigen-coated particles are agglutinated by samples comprising the
target antibodies. In this format, none of the components need be
labeled and the presence of the target antibody is detected by
simple visual inspection.
Cellular Transfection and Gene Therapy
[0245] The present invention provides the nucleic acids of EDG
protein for the transfection of cells in vitro and in vivo. These
nucleic acids can be inserted into any of a number of well-known
vectors for the transfection of target cells and organisms as
described below. The nucleic acids are transfected into cells, ex
vivo or in vivo, through the interaction of the vector and the
target cell. The nucleic acid, under the control of a promoter,
then expresses a EDG protein of the present invention, thereby
mitigating the effects of absent, partial inactivation, or abnormal
expression of an EDG gene, particularly as it relates to T cell
activation and migration. The compositions are administered to a
patient in an amount sufficient to elicit a therapeutic response in
the patient. An amount adequate to accomplish this is defined as
"therapeutically effective dose or amount."
[0246] Such gene therapy procedures have been used to correct
acquired and inherited genetic defects, cancer, and other diseases
in a number of contexts. The ability to express artificial genes in
humans facilitates the prevention and/or cure of many important
human diseases, including many diseases which are not amenable to
treatment by other therapies (for a review of gene therapy
procedures, see Anderson, Science 256:808-813 (1992); Nabel &
Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH
11: 162-166 (1993); Mulligan, Science 926-932 (1993); Dillon,
TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van
Brunt, Biotechnology 6(10):1149-1154 (1998); Vigne, Restorative
Neurology and Neuroscience 8:35-36 (1995); Kremer &
Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada
et al., in Current Topics in Microbiology and Immunology (Doerfler
& Bohm eds., 1995); and Yu et al., Gene Therapy 1: 13-26
(1994)).
Pharmaceutical Compositions and Administration
[0247] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered (e.g., nucleic
acid, protein, modulatory compounds or transduced cell), as well as
by the particular method used to administer the composition.
Accordingly, there are a wide variety of suitable formulations of
pharmaceutical compositions of the present invention (see, e.g.,
Remington's Pharmaceutical Sciences, 17.sup.th ed., 1989).
Administration can be in any convenient manner, e.g., by injection,
oral administration, inhalation, transdermal application, or rectal
administration.
[0248] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the packaged
nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Tablet forms can include one or more of
lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, microcrystalline cellulose, gelatin,
colloidal silicon dioxide, talc, magnesium stearate, stearic acid,
and other excipients, colorants, fillers, binders, diluents,
buffering agents, moistening agents, preservatives, flavoring
agents, dyes, disintegrating agents, and pharmaceutically
compatible carriers. Lozenge forms can comprise the active
ingredient in a flavor, e.g., sucrose, as well as pastilles
comprising the active ingredient in an inert base, such as gelatin
and glycerin or sucrose and acacia emulsions, gels, and the like
containing, in addition to the active ingredient, carriers known in
the art.
[0249] The compound of choice, alone or in combination with other
suitable components, can be made into aerosol formulations (i.e.,
they can be "nebulized") to be administered via inhalation. Aerosol
formulations can be placed into pressurized acceptable propellants,
such as dichlorodifluoromethane, propane, nitrogen, and the
like.
[0250] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. In the practice
of this invention, compositions can be administered, for example,
by intravenous infusion, orally, topically, intraperitoneally,
intravesically or intrathecally. Parenteral administration and
intravenous administration are the preferred methods of
administration. The formulations of commends can be presented in
unit-dose or multi-dose sealed containers, such as ampules and
vials.
[0251] Injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described. Cells transduced by nucleic acids for ex vivo therapy
can also be administered intravenously or parenterally as described
above.
[0252] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
therapeutic response in the patient over time. The dose will be
determined by the efficacy of the particular vector employed and
the condition of the patient, as well as the body weight or surface
area of the patient to be treated. The size of the dose also will
be determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
vector, or transduced cell type in a particular patient.
[0253] In determining the effective amount of the vector to be
administered in the treatment or prophylaxis of conditions owing to
diminished or aberrant expression of the EDG protein, the physician
evaluates circulating plasma levels of the vector, vector
toxicities, progression of the disease, and the production of
anti-vector antibodies. In general, the dose equivalent of a naked
nucleic acid from a vector is from about 1 .mu.g to 100 .mu.g for a
typical 70 kilogram patient, and doses of vectors which include a
retroviral particle are calculated to yield an equivalent amount of
therapeutic nucleic acid.
[0254] For administration, compounds and transduced cells of the
present invention can be administered at a rate determined by the
LD-50 of the inhibitor, vector, or transduced cell type, and the
side-effects of the inhibitor, vector or cell type at various
concentrations, as applied to the mass and overall health of the
patient. Administration can be accomplished via single or divided
doses.
EXAMPLES
[0255] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
Identification of EDG1 and Other Genes Involved in Modulation of T
Cell Activation and Migration
[0256] A. Introduction
[0257] In this study, an approach to identify new targets for
immune suppressive drugs is provided. It is known that following T
cell activation, expression of numerous cell surface markers such
as CD25, CD69, and CD40L are upregulated. CD69 has been shown to be
an early activation marker in T, B, and NK cells. CD69 is a
disulfide-linked dimer. It is not expressed in resting lymphocytes
but appears on T, B and NK cells after activation in vitro. Its
relevance as a TCR signaling outcome has been validated using T
cell deficient in certain key signaling molecules such as LAT and
SLP76 (Yablonski, supra). Furthermore, re-introducing SLP76 to the
deficient cells results in restoration of CD69 expression. CD69
upregulation was therefore to be used to monitor TCR signal
transduction. The rationale of the functional genomics screen was
then to identify cell clones whose CD69 upregulation was repressed
following introduction of a retroviral cDNA library. The library
members conferring such repression would then represent immune
modulators that function to block TCR signal transduction.
[0258] b. Results
[0259] Several T cell lines, including Jurkat, HPB-ALL, HSB-2 and
PEER were tested for the presence of surface CD3, CD25, CD28,
CD40L, CD69, CD95, and CD95L. Those that express CD3 were cultured
with anti-CD3 or anti-TCR to crosslink the TCR and examined for the
upregulation of CD69. Jurkat T cell line was selected for its
ability to upregulate CD69 in response to crosslinking of their TCR
with a kinetics mimicking that of primary T lymphocytes (data not
shown). The population of Jurkat cells was sorted for low basal and
highly inducible CD69 expression following anti-TCR stimulation.
Clone 4D9 was selected because CD69 in this clone was uniformly and
strongly induced following TCR stimulation in 24 hours.
[0260] In order to regulate the expression of the retroviral
library, the Tet-Off system was used. Basically, cDNA inserts in
the retroviral library were cloned behind the tetracycline
regulatory element (TRE) and the minimal promoter of TK.
Transcription of the cDNA inserts were then dependent on the
presence of tetracycline-controlled trans-activator (tTA), a fusion
of Tet repression protein and the VP16 activation domain, and the
absence of tetracyaline or its derivatives such as doxycycline
(Dox). To shut off the cDNA expression, one can simply add
doxycycline in the medium. To obtain a Jurkat clone stably
expresses tTA, retroviral LTR-driven tTA was introduced in
conjunction with a TRE-dependent reporter construct, namely
TRA-Lyt2. Through sorting of Lyt2 positive cells in the absence of
Dox and Lyt2 negative cells in the presence of Dox, coupled with
clonal evaluation, a derivative of Jurkat clone 4D9 was obtained,
called 4D9#32, that showed the best Dox regulation of Lyt2
expression.
[0261] Positive controls: ZAP70 is a positive regulator of T cell
activation. A kinase-inactivated (KI) ZAP70 and a truncated ZAP70
(SH2 N+C) were subcloned into the retroviral vector under TRE
control. ZAP70 SH2 (N+C) and ZAP70 KI both inhibited TCR-induced
CD69 expression. Consistent with the published report on dominant
negative forms of ZAP70 on NFAT activity, the truncated protein is
also a more potent inhibitor of CD69 induction. In addition, the
higher protein expression, as shown by adjusting GFP-gating, the
stronger the inhibition was. When one puts the marker M1 at bottom
1% of the uninfected cells, one has a 40% likelihood of obtaining
cells whose phenotype resembled that of ZAP70 SH2 (N+C). This
translates into a 40:1 enrichment of the desired phenotype.
[0262] The CD69 inhibitory phenotype is dependent on expression of
dominant negative forms of ZAP70. When Dox was added for 7 days
before TCR was stimulated, there was no inhibition of CD69
expression. Analysis of cellular phenotype by FACS of GFP, which
was produced from the bi-cistronic mRNA ZAP70 SH2 (N+C)-IRES-GFP,
revealed a lack of GFP+ cells. The lack of ZAP70 SH2 (N+C)
expression in the presence of Dox was confirmed by Western.
[0263] Screening for cells lacking CD69 upregulation: Jurkat 4D9#32
cells were infected with cDNA libraries made form primary human
lymphoid organs such as thymus, spleen, lymph node and bone marrow.
The library complexity was 5.times.10.sup.7 and was built on the
TRE vector. A total of 7.1.times.10.sup.8 cells were screened with
an infection rate of 52%, as judged by parallel infection of the
same cells with TRA-dsGFP (data not shown). After infection, the
cells will be stimulated with the anti-TCR antibody C305 for
overnight and sorted for CD69 low and CD3+ phenotype by FACS. If
the sorting gate was set to include the bottom 3% cells based on
the single parameter of CD69 level, 2/3 cells in the sorting gate
lacked TCR/CD3 complex, which explained their refractory to
stimulation. The second parameter of CD3 expression was then
incorporated. Even though there was a significant reduction of
CD3/TCR complex on the surface following receptor-mediated
internalization, the CD3- population was still distinguishable from
the CD3+ population. The resulting sort gate contained 1% of the
total cells, which translated into a 100-fold enrichment based on
cell numbers. The recovered cells with CD69 low CD3+ phenotype were
allowed to rest in complete medium for 5 days before being
stimulated again for a new round of sorting. In subsequent round of
sortings, the sort gate was always maintained to contain the
equivalent of 1% of the unsorted control population. Obvious
enrichment was achieved after 3 rounds of reiterative sorting.
Cells with the desired phenotype increased from 1% to 22.3%. In
addition, the overall population's geometric mean for CD69 was also
reduced.
[0264] In order to ascertain that the phenotype was due to
expression of the cDNA library rather than entirely due to
spontaneous or retroviral insertion-mediated somatic mutation, the
cells recovered after the third round of sorting were split into
two halves. One half of the cells were grown in the absence of Dox
while the other half in the presence of Dox. A week later, CD69
expression was compared following anti-TCR stimulation. There was a
significant numbers of cells (11%) whose CD69 repression was lost
in the presence of Dox, suggesting that the CD69 inhibition
phenotype was indeed caused by the expression of library members.
Single cell clones in conjunction with the fourth round of CD69 low
CD3+ sorting (LLLL) were deposited.
[0265] In order to reduce the number of cells whose phenotype was
not Dox-regulatable, the half of the cells grown in the presence of
Dox were subjected to a fourth round of sorting for enrichment of
CD69 high phenotype (LLLH). The cells recovered from LLLH sort were
cultured in the absence of Dox for subsequence sorting and single
cell cloning of CD69 low CD3+ phenotypes.
[0266] Dox regulation of CD69 expression was expressed as the ratio
of geometric mean fluorescent intensity (GMFI) in the presence of
Dox over that in the absence of Dox. In uninfected cells, Dox had
limited effect on the induction of CD69 expression so that the
ratio of GMFI (+Dox)/GMFI (-Dox) remained to be 1.00+/-0.25. The
2.times. standard deviation was therefore used as a cut-off
criterion and clones with a ratio above 1.5 were regarded as
Dox-regulated clones.
[0267] RNA samples were prepared from clones with Dox-regulatable
phenotypes. Using primers specific for the vector sequence flanking
the cDNA library insert, the cDNA insert of selected clones were
captured by RT-PCR. Most clones generated only on DNA band, whereas
a few clones generated two or more bands. Sequencing analysis
revealed that the additional bands were caused by double or
multiple insertions.
[0268] Characterization of proteins involved in T cell activation:
Known TCR regulators such as Lck, ZAP70, PLC.gamma.1 and Raf were
obtained. In addition, the BCR regulator SYK was also uncovered.
EDG1, a GPCR not previously known to be involved in B and T cell
activation, was also identified using this assay (see FIGS.
14-32).
[0269] Lck is a non-receptor protein tyrosine kinase. Its role in T
cell development and activation has been widely documented. So far,
dominant negative form of Lck has no been reported. Our discovery
that over expression of the kinase-truncated form of Lck caused
inhibition of CD69, similar to the phenotype of Jurkat somatic
mutant lacking Lck, suggests that kinase deletion of Lck could also
work as a dominant negative form of Lck.
[0270] The two ZAP70 hits ended at aa 262 and 269, respectively.
They both missed the catalytic domain. The deletions are very close
to the positive control for the screen, ZAP70 SH2 (N+C), which
ended at aa 276. Since ZAP70 SH2 (N+C) was shown to be a dominant
negative protein, it appears that the two ZAP70 hits also behaved
as dominant negative proteins of ZAP70.
[0271] SYK is a non-receptor tyrosine kinase belonging to the
SYK/ZAP70 family of kinases. Since it has also been shown that the
lack of SYK expression in Jurkat cells did not appear to
significantly alter the TCR-mediated responses compared with Jurkat
clones expressing SYK, it appears that the SYK hit obtained from
our screen worked mainly to block ZAP70-function. SYK's similarity
to ZAP70 and its ability to associate with phosphorylated TCR zeta
chains also support this notion.
[0272] PLC.gamma.1 plays a crucial role in coupling T cell receptor
ligation to IL-2 gene expression in activated T lymphocytes. TCR
engagement leads to rapid tyrosine phosphorylation and activation
of PLC.gamma.1. The activated enzyme converts
phosphatidylinositol-4,5-bisphosphate (PIP2) to
inositol-1,3,5-trisphosphate ((IP3) and diacylglycerol (DAG). IP3
triggers intracellular Ca2+ increase and DAG is a potent activator
of protein kinase C(PKC). PLC.gamma.1 has a split catalytic domain
comprised of conserved X and Y subdomains. Single point mutation in
the catalytic X box completely abolished the enzyme activity and
also blocked IL-2 reporter gene expression when introduced into
PLC.gamma.-deficient Jurkat cells. Our hit contained the PH domain
and the N and C terminal SH2 domains of PLC.gamma.1. Significantly
this hit also deleted the crucial tyrosine Y783 between the SH2 and
SH3 domains. It was reported that Y783 was essential for coupling
of TCR stimulation to IL-2 promoter activation and that mutation of
Y783 to F (phenylalanine) generated a very potent dominant negative
form of PLC.gamma.1. Indeed, the original clone encoding the
PLC.gamma.1 hit had the highest Dox +/-ratio for CD69 expression
among all clones from the cDNA screen, indicating the strong
repression of CD69 induction by the hit as well as the total
de-repression in the absence of the hit. When introduced to naive
Jurkat cells, this fragment caused severe block of TCR-induced CD69
expression.
[0273] Raf is a MAP kinase kinase kinase. It interacts with Ras and
leads to activation of the MAP kinase pathway. The Raf hit obtained
also had a truncation of the kinase domain, creating a dominant
negative form of the kinase. Other signaling molecules known to
involve in TCR pathway were also discovered in our screen. They
included PAG, CSK, SHP-1 and nucleolin.
[0274] Function in primary T lymphocytes: The relevance of the CD69
screen hits to physiological function of T cells was investigated
in primary T lymphocytes. The hit was subcloned into a retroviral
vector under a constitutively active promoter, followed by
IRES-GFP. A protocol was also developed to couple successful
retroviral infection to subsequence T cell activation. Primary T
lymphocytes are at the quiescent stage when isolated from healthy
donors. In order to be infected by retrovirus, primary lymphocytes
need to be activated to progress in cell cycle. Fresh peripheral
blood lymphocytes (PBL) contained typically T cells and B cells.
The combined CD4+ and CD8+ cells represented total T cell
percentage, which was 81% in this particular donor. The remaining
19% CD4-CD8- cells were B cells as stained by CD19 (data not
shown). Upon culturing on anti-CD3 and anti-CD28 coated dishes,
primary T lymphocytes were expanded and primary B cells and other
cell types gradually died off in the culture. After infection, the
culture contained virtually all T cells. Furthermore, primary T
lymphocytes were successfully infected by retroviruses.
[0275] As seen with Jurkat cells (data not shown), GFP translated
by way of IRES was not as abundant as GFP translated using the
conventional Kozak sequence (comparing GFP geometric mean from
CRU5-IRES-GFP and CRU5-GFP). Nevertheless the percentage infection
remained similar. Insertion of a gene in front of IRES-GFP further
reduced the expression level of GFP, which was observed with cell
lines (data not shown) and here primary T lymphocytes. After
allowing cells to rest following infection, FACS sorted cells were
divided into two populations: GFP- and GFP+. The sorted cells were
immediately put into culture. Anti-CD3 alone did not induce IL-2
production. This observation was consistent with previous report on
freshly isolated primary T lymphocytes and confirmed the notion
that prior culture and retroviral infection did not damage the
physiological properties of these primary T lymphocytes. Addition
of anti-CD28 in conjunction with anti-CD3 led to robust IL-2
production with vector-infected cells and the GFP- population of
LckDN and PLC.gamma.1DN-infected cells. The GFP+ cell population
from LckDN and PLC.gamma.1DN-infected cells, however, were severely
impaired in IL-2 production. As expect, the defect caused by LckDN
and PLC.gamma.1DN can be completely rescued by stimulation using
PMA and ionomycin. Taken together, these results showed that Lck
and PLC.gamma.1 plays a role in IL-2 production from primary T
lymphocytes, consistently with their involvement membrane proximal
signaling events of T cell activation. These results also
demonstrated a successful system to quickly validate hits from our
functional genetic screens in primary cells.
[0276] Use of CD69 upregulation in drug screening: The discovery of
important immune regulatory molecules from the B and T cell
activation-induced CD69 upregulation validated the relevance of
this cell-based assay. Essentially such a cell-based assay offers
the opportunity to discover inhibitors of multiple targets such as
Lck, ZAP70, PLC.gamma.1, and EDG family proteins such as EDG1. It
is the equivalent of multiplexing enzymatic assays with the
additional advantage of cell permeability of compounds. It may even
be possible to identify novel compounds that block adaptor protein
functions. Towards this end, the FACS assay of cell surface CD69
expression was converted to a micro-titer plate based assay, for
both T and B cell regulation assays.
[0277] In conclusion, the strategy presented in this study
demonstrates a successful approach to discover and validate
important immune regulators on a genome-wide scale. This approach,
which requires no prior sequence information, provides a tool for
functional cloning of regulators in numerous signal transduction
pathways. For example, B cell activation-induced CD69 expression,
IL-4-induced IgE class switch and TNF-induced NF-kB reporter gene
expression are all amendable to the genetic perturbation following
introduction of retroviral cDNA libraries. The outlined strategy is
less biased compared to forced introduction of a handful of
signaling molecules discovered in other context such as growth
factor signal transduction. It also opens the door for discovering
peptide inhibitors of immune modulatory proteins by screening
random peptide libraries, including cyclic peptides, expressed from
the retroviral vector.
[0278] C. Methods
[0279] Cell culture: Human Jurkat T cells (clone N) were routinely
cultured in RPMI 1640 medium supplemented with 10% fetal calf serum
(Hyclone), penicillin and streptamycin. Phoenix A cells were grown
in DMEM supplemented with 10% fetal calf serum, penicillin and
streptamycin. To produce the tTA-Jurkat cell line, Jurkat cells
were infected with a retroviral construct which constitutively
expresses the tetracycline transactivator protein and a reporter
construct which expresses LyT2 driven by a tetracycline responsive
element (TRE). The tTA-Jurkat cell population was optimized by
sorting multiple sounds for high TRE-dependent expression of LyT2
in the absence of Dox and strong repression of LyT2 expression in
the presence Dox. The cells were also sorted for maximal anti-TCR
induced expression of CD69. Doxycycline was used at a final
concentration of 10 ng/ml for at least 6 days to downregulate
expression of cDNAs from the TRE promoter.
[0280] Transfection and infection: Phoenix A packaging cells were
transfected with retroviral vectors using calcium phosphate for 6
hours as standard protocols. After 24 hours, supernatant was
replaced with complete RPMI medium and virus was allowed to
accumulate for an additional 24 hours. Viral supernatant was
collected, filtered through a 0.2 .mu.M filter and mixed with
Jurkat cells at a density of 2.5.times.10.sup.5 cells/ml. Cells
were spun at room temperature for 3 hours at 3000 rpm, followed by
overnight incubation at 37.degree. C. Transfection and infection
efficiencies were monitored by GFP expression and functional
analysis was carried out 2-4 days after infection.
[0281] Libraries: RNA extracted from human lymph node, thymus,
spleen and bone marrow was used to produce two cDNA libraries; one
random primed and directionally cloned and the second
non-directionally cloned and provided with 3 exogenous ATG in 3
frames. cDNAs were cloned into the pTRA-exs vector giving robust
doxycycline-regulable transcription of cDNAs from the TRE promoter.
The total combined library complexity was 5.times.10.sup.7
independent clones.
[0282] Stimulation: For CD69 upregulation experiments, tTA-Jurkat
cells were split to 2.5.times.10.sup.5 cells/ml 24 hours prior to
stimulation. Cells were spun and resuspended at 5.times.10.sup.5
cells/ml in fresh complete RPMI medium in the presence of 100 ng/ml
C305 (anti-Jurkat clonotypic TCR) or 5 ng/ml PMA hybridoma
supernatant for 20-26 hours at 37.degree. C., and then assayed for
surface CD69 expression.
[0283] Cell surface marker analysis: Jurkat-N cells were stained
with an APC-conjugated mouse monoclonal anti-human CD69 antibody
(Caltag) at 4.degree. C. for 20 minutes and analyzed using a
Facscalibur instrument (Becton Dickinson) with Cellquest software.
Cell sorts were performed on a MoFlo (Cytomation).
[0284] cDNA screen: Phoenix A packaging cells were transfected with
a mixture of the two tTA regulated retroviral pTRA-exs cDNA
libraries. Supernatant containing packaged viral particles was used
to infect tTA-Jurkat cells with an efficiency of .about.85%. After
4 days of cDNA expression, library infected cells were stimulated
with 0.3 .mu.g/ml C305 for 20-26 hours, stained with APC-conjugated
anti-CD69, and lowest CD69-expressing cells still expressing CD3
(CD69.sup.lowCD3.sup.+) were isolated using a fluorescence
activated cell sorter. Sorting was repeated over multiple rounds
with a 6-day rest period between stimulations until the population
was significantly enriched for non-responders. Single cells were
deposited from 4 separate rounds of sorting. Cell clones were
expanded in the presence and absence of Dox, stimulated and
analyzed for CD69 upregulation.
[0285] Isolation of cDNA inserts: PCR primers were designed to
amplify cDNA inserts from both libraries and did not amplify Lyt2
that was also under TRE regulation. The primers used contained
flanking BstXI sites for subsequent cloning to pTRA-IRES-GFP
vector. RT-PCR cloning was achieved with kits from Clontech or Life
Technologies. The gel-purified RT-PCR products were submitted for
sequencing directly and simultaneously digested for subcloning.
Dominant negative ZAP70 (KI) and ZAP70SH2 (N+C) as well as selected
hits from cDNA screens were subcloned to the retroviral
pTRA-IRES-GFP vector. Selected hits form cDNA screens were also
subcloned to CRU5-IRES-GFP for infection of human primary T
lymphocytes and examination of IL-2 production.
Example 2
Identification of Additional EDG Family Genes Involved in
Modulation of T Cell Activation
[0286] Using the CD69 lymphocyte activation assays and other
methods described in Example 1, nucleic acids encoding EDG 2-8 are
tested, and EDG2-8 are identified as proteins involved in
modulation of lymphocyte activation. Wild-type EDG 2-8 nucleotide
sequences are described herein. EDG 2-8 are therefore useful in
assays to identify compounds that modulate lymphocyte
activation.
[0287] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
1511149DNAHomo sapienswild-type human endothelial differentiation
G-protein coupled receptor (GPCR) 1 (EDG1) 1atggggccca ccagcgtccc
gctggtcaag gcccaccgca gctcggtctc tgactacgtc 60aactatgata tcatcgtccg
gcattacaac tacacgggaa agctgaatat cagcgcggac 120aaggagaaca
gcattaaact gacctcggtg gtgttcattc tcatctgctg ctttatcatc
180ctggagaaca tctttgtctt gctgaccatt tggaaaacca agaaattcca
ccgacccatg 240tactatttta ttggcaatct ggccctctca gacctgttgg
caggagtagc ctacacagct 300aacctgctct tgtctggggc caccacctac
aagctcactc ccgcccagtg gtttctgcgg 360gaagggagta tgtttgtggc
cctgtcagcc tccgtgttca gtctcctcgc catcgccatt 420gagcgctata
tcacaatgct gaaaatgaaa ctccacaacg ggagcaataa cttccgcctc
480ttcctgctaa tcagcgcctg ctgggtcatc tccctcatcc tgggtggcct
gcctatcatg 540ggctggaact gcatcagtgc gctgtccagc tgctccaccg
tgctgccgct ctaccacaag 600cactatatcc tcttctgcac cacggtcttc
actctgcttc tgctctccat cgtcattctg 660tactgcagaa tctactcctt
ggtcaggact cggagccgcc gcctgacgtt ccgcaagaac 720atttccaagg
ccagccgcag ctctgagaag tcgctggcgc tgctcaagac cgtaattatc
780gtcctgagcg tcttcatcgc ctgctgggca ccgctcttca tcctgctcct
gctggatgtg 840ggctgcaagg tgaagacctg tgacatcctc ttcagagcgg
agtacttcct ggtgttagct 900gtgctcaact ccggcaccaa ccccatcatt
tacactctga ccaacaagga gatgcgtcgg 960gccttcatcc ggatcatgtc
ctgctgcaag tgcccgagcg gagactctgc tggcaaattc 1020aagcgaccca
tcatcgccgg catggaattc agccgcagca aatcggacaa ttcctcccac
1080ccccagaaag acgaagggga caacccagag accattatgt cttctggaaa
cgtcaactct 1140tcttcctag 114921032DNAHomo sapiensmutant #1,
C-terminally truncated variant of human endothelial differentiation
G-protein coupled receptor (GPCR) 1 (EDG1) 2ttggcaccat ggggcccacc
agcgtcccgc tggtcaaggc ccaccgcagc tcggtctctg 60actacgtcaa ctatgatatc
atcgtccggc attacaacta cacgggaaag ctgaatatca 120gcgcggacaa
ggagaacagc attaaactga cctcggtggt gttcattctc atctgctgct
180ttatcatcct ggagaacatc tttgtcttgc tgaccatttg gaaaaccaag
aaattccacc 240gacccatgta ctattttatt ggcaatctgg ccctctcaga
cctgttggca ggagtagcct 300acacagctaa cctgctcttg tctggggcca
ccacctacaa gctcactccc gcccagtggt 360ttctgcggga agggagtatg
tttgtggccc tgtcagcctc cgtgttcagt ctcctcgcca 420tcgccattga
gcgctatatc acaatgctga aaatgaaact ccacaacggg agcaataact
480tccgcctctt cctgctaatc agcgcctgct gggtcatctc cctcatcctg
ggtggcctgc 540ctatcatggg ctggaactgc atcagtgcgc tgtccagctg
ctccaccgtg ctgccgctct 600accacaagca ctatatcctc ttctgcacca
cggtcttcac tctgcttctg ctctccatcg 660tcattctgta ctgcagaatc
tactccttgg tcaggactcg gagccgccgc ctgacgttcc 720gcaagaacat
ttccaaggcc agccgcagct ctgagaagtc gctggcgctg ctcaagaccg
780taattatcgt cctgagcgtc ttcatcgcct gctgggcacc gctcttcatc
ctgctcctgc 840tggatgtggg ctgcaaggtg aagacctgtg acatcctctt
cagagcggag tacttcctgg 900tgttagctgt gctcaactcc ggcaccaacc
ccatcattta cactctgacc aacaaggaga 960tgcgtcgggc cttcatccgg
atcatgtcct gctgcaagtg cccgagcgga gactctgctg 1020gcaaattcaa gc
103231437DNAHomo sapiensmutant #2, C-terminally truncated variant
of human endothelial differentiation G-protein coupled receptor
(GPCR) 1 (EDG1) 3gcggccgcgt cgacgtgcgt ctcagcagtt cagatccggg
ggcccccagc tgacagaggg 60cgtggggggt taaggcatta acccctccca gcctcttcct
gaagaaacca cccagccttg 120gcgcggcgct gggtgacttc gcgtagcagg
cagggaactg gccgcggcga gcgggactgg 180ccattggagt gctccgctgc
ggagggaggg gaccccgact cgagtaagtt tgcgagagca 240ctacgcagtc
agtcgggggc agcagcaaga tgcgaagcga gccgtacaga tcccgggctc
300tccgaacgca acttcgccct gcttgagcga ggctgcggtt tccgaggccc
tctccagcca 360aggaaaagct acacaaaaag cctggatcac tcatcgaacc
acccctgaag ccagtgaagg 420ctctctcgcc tcgccctcta gcgttcgtct
ggagtagcgc caccccggct tcctggggac 480acagggttgg caccatgggg
cccaccagcg tcccgctggt caaggcccac cgcagctcgg 540tctctgacta
cgtcaactat gatatcatcg tccggcatta caactacacg ggaaagctga
600atatcagcgc ggacaaggag aacagcatta aactgacctc ggtggtgttc
attctcatct 660gctgctttat catcctggag aacatctttg tcttgctgac
catttggaaa accaagaaat 720tccaccgacc catgtactat tttattggca
atctggccct ctcagacctg ttggcaggag 780tagcctacac agctaacctg
ctcttgtctg gggccaccac ctacaagctc actcccgccc 840agtggtttct
gcgggaaggg agtatgtttg tggccctgtc agcctccgtg ttcagtctcc
900tcgccatcgc cattgagcgc tatatcacaa tgctgaaaat gaaactccac
aacgggagca 960ataacttccg cctcttcctg ctaatcagcg cctgctgggt
catctccctc atcctgggtg 1020gcctgcctat catgggctgg aactgcatca
gtgcgctgtc cagctgctcc accgtgctgc 1080cgctctacca caagcactat
atcctcttct gcaccacggt cttcactctg cttctgctct 1140ccatcgtcat
tctgtactgc agaatctact ccttggtcag gactcggagc cgccgcctga
1200cgttccgcaa gaacatttcc aaggccagcc gcagctctga gaagtcgctg
gcgctgctca 1260agaccgtaat tatcgtcctg agcgtcttca tcgcctgctg
ggcaccgctc ttcatcctgc 1320tcctgctgga tgtgggctgc aaggtgaaga
cctgtgacat cctcttcaga gcggagtact 1380tcctggtgtt agctgtgctc
aactccggca ccaaccccat catttacact ctgacca 143741376DNAHomo
sapiensmutant #3, C-terminally truncated variant of human
endothelial differentiation G-protein coupled receptor (GPCR) 1
(EDG1) 4ggcacgaggc gagcgggact ggccattgga gtgctccgct gcggagggag
gggaccccgt 60actcgagtaa gtttgcgaga gcactacgca gtcagtcggg ggcagcagca
agatgcgaag 120cgagccgtac agatcccggg ctctccgaac gcaacttcgc
cctgcttgag cgaggccgcg 180gtttccgagg ccctctccag ccaaggaaaa
gctacacaaa aagcctggat cactcatcga 240accacccctg aagccagtga
aggctctctc gcctcgccct ctagcgttcg tctggagtag 300cgccaccccg
gcttcctggg gacacagggt tggcaccatg gggcccacca gcgtcccgct
360ggtcaaggcc caccgcagct cggtctctga ctacgtcaac tatgatatca
tcgtccggca 420ttacaactac acgggaaagc cgaatatcag cgcggacaag
gagaacagca ttaaactgac 480ctcggtggtg ttcattctca tctgctgctt
tatcatcctg gagaacatct ttgtcttgct 540gaccatttgg aaaaccaaga
aattccaccg acccatgtac tattttattg gcaatctggc 600cctctcagac
ctgttggcag gagtagccta cacagctaac ctgctcttgt ctggggccac
660cacctacaag ctcactcccg cccagtggtt tctgcgggaa gggagtatgt
ttgtggccct 720gtcagcctcc gtgttcagtc tcctcgccat cgccattgag
cgctatatca caatgctgaa 780aatgaaactc cacaacggga gcaataactt
ccgcctcttc ctgctaatca gcgcctgctg 840ggtcatctcc ctcatcctgg
gtggcctgcc tatcatgggc tggaactgca tcagtgcgct 900gtccagctgc
tccaccgtgc tgccgctcta ccacaagcac tatatcctct tctgcaccac
960ggtcttcact ctgcttctgc tctccatcgt cattctgtac tgcagaatct
actccttggt 1020caggactcgg agccgccgcc tgacgttccg caagaacatt
tccaaggcca gccgcagctc 1080tgagaagtcg ctggcgctgc tcaggaccgt
aattatcgtc ctgagcgtct tcatcgcctg 1140ctgggcaccg ctcttcatcc
tgctcctgct ggatgtgggc tgcaaggtga agacctgtga 1200catcctcttc
agagcggagt acttcctggt gttagctgtg ctcaactccg gcaccaaccc
1260catcatttac actctgacca acaaggagat gcgtcgggcc ttcatccgga
tcatgtcctg 1320ctgcaagtgc ccgagcggag actctgctgg caaattcaag
cgacccatca tcgccg 13765382PRTHomo sapienswild-type human
endothelial differentiation G-protein coupled receptor (GPCR) 1
(EDG1) 5Met Gly Pro Thr Ser Val Pro Leu Val Lys Ala His Arg Ser Ser
Val 1 5 10 15Ser Asp Tyr Val Asn Tyr Asp Ile Ile Val Arg His Tyr
Asn Tyr Thr 20 25 30Gly Lys Leu Asn Ile Ser Ala Asp Lys Glu Asn Ser
Ile Lys Leu Thr 35 40 45Ser Val Val Phe Ile Leu Ile Cys Cys Phe Ile
Ile Leu Glu Asn Ile 50 55 60Phe Val Leu Leu Thr Ile Trp Lys Thr Lys
Lys Phe His Arg Pro Met 65 70 75 80Tyr Tyr Phe Ile Gly Asn Leu Ala
Leu Ser Asp Leu Leu Ala Gly Val 85 90 95Ala Tyr Thr Ala Asn Leu Leu
Leu Ser Gly Ala Thr Thr Tyr Lys Leu 100 105 110Thr Pro Ala Gln Trp
Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu 115 120 125Ser Ala Ser
Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg Tyr Ile 130 135 140Thr
Met Leu Lys Met Lys Leu His Asn Gly Ser Asn Asn Phe Arg Leu145 150
155 160Phe Leu Leu Ile Ser Ala Cys Trp Val Ile Ser Leu Ile Leu Gly
Gly 165 170 175Leu Pro Ile Met Gly Trp Asn Cys Ile Ser Ala Leu Ser
Ser Cys Ser 180 185 190Thr Val Leu Pro Leu Tyr His Lys His Tyr Ile
Leu Phe Cys Thr Thr 195 200 205Val Phe Thr Leu Leu Leu Leu Ser Ile
Val Ile Leu Tyr Cys Arg Ile 210 215 220Tyr Ser Leu Val Arg Thr Arg
Ser Arg Arg Leu Thr Phe Arg Lys Asn225 230 235 240Ile Ser Lys Ala
Ser Arg Ser Ser Glu Lys Ser Leu Ala Leu Leu Lys 245 250 255Thr Val
Ile Ile Val Leu Ser Val Phe Ile Ala Cys Trp Ala Pro Leu 260 265
270Phe Ile Leu Leu Leu Leu Asp Val Gly Cys Lys Val Lys Thr Cys Asp
275 280 285Ile Leu Phe Arg Ala Glu Tyr Phe Leu Val Leu Ala Val Leu
Asn Ser 290 295 300Gly Thr Asn Pro Ile Ile Tyr Thr Leu Thr Asn Lys
Glu Met Arg Arg305 310 315 320Ala Phe Ile Arg Ile Met Ser Cys Cys
Lys Cys Pro Ser Gly Asp Ser 325 330 335Ala Gly Lys Phe Lys Arg Pro
Ile Ile Ala Gly Met Glu Phe Ser Arg 340 345 350Ser Lys Ser Asp Asn
Ser Ser His Pro Gln Lys Asp Glu Gly Asp Asn 355 360 365Pro Glu Thr
Ile Met Ser Ser Gly Asn Val Asn Ser Ser Ser 370 375 3806364PRTHomo
sapienshuman endothelial differentiation G-protein coupled receptor
(GPCR) 2 (EDG2) 6Met Ala Ala Ile Ser Thr Ser Ile Pro Val Ile Ser
Gln Pro Gln Phe 1 5 10 15Thr Ala Met Asn Glu Pro Gln Cys Phe Tyr
Asn Glu Ser Ile Ala Phe 20 25 30Phe Tyr Asn Arg Ser Gly Lys His Leu
Ala Thr Glu Trp Asn Thr Val 35 40 45Ser Lys Leu Val Met Gly Leu Gly
Ile Thr Val Cys Ile Phe Ile Met 50 55 60Leu Ala Asn Leu Leu Val Met
Val Ala Ile Tyr Val Asn Arg Arg Phe 65 70 75 80His Phe Pro Ile Tyr
Tyr Leu Met Ala Asn Leu Ala Ala Ala Asp Phe 85 90 95Phe Ala Gly Leu
Ala Tyr Phe Tyr Leu Met Phe Asn Thr Gly Pro Asn 100 105 110Thr Arg
Arg Leu Thr Val Ser Thr Trp Leu Leu Arg Gln Gly Leu Ile 115 120
125Asp Thr Ser Leu Thr Ala Ser Val Ala Asn Leu Leu Ala Ile Ala Ile
130 135 140Glu Arg His Ile Thr Val Phe Arg Met Gln Leu His Thr Arg
Met Ser145 150 155 160Asn Arg Arg Val Val Val Val Ile Val Val Ile
Trp Thr Met Ala Ile 165 170 175Val Met Gly Ala Ile Pro Ser Val Gly
Trp Asn Cys Ile Cys Asp Ile 180 185 190Glu Asn Cys Ser Asn Met Ala
Pro Leu Tyr Ser Asp Ser Tyr Leu Val 195 200 205Phe Trp Ala Ile Phe
Asn Leu Val Thr Phe Val Val Met Val Val Leu 210 215 220Tyr Ala His
Ile Phe Gly Tyr Val Arg Gln Arg Thr Met Arg Met Ser225 230 235
240Arg His Ser Ser Gly Pro Arg Arg Asn Arg Asp Thr Met Met Ser Leu
245 250 255Leu Lys Thr Val Val Ile Val Leu Gly Ala Phe Ile Ile Cys
Trp Thr 260 265 270Pro Gly Leu Val Leu Leu Leu Leu Asp Val Cys Cys
Pro Gln Cys Asp 275 280 285Val Leu Ala Tyr Glu Lys Phe Phe Leu Leu
Leu Ala Glu Phe Asn Ser 290 295 300Ala Met Asn Pro Ile Ile Tyr Ser
Tyr Arg Asp Lys Glu Met Ser Ala305 310 315 320Thr Phe Arg Gln Ile
Leu Cys Cys Gln Arg Ser Glu Asn Pro Thr Gly 325 330 335Pro Thr Glu
Ser Ser Asp Arg Ser Ala Ser Ser Leu Asn His Thr Ile 340 345 350Leu
Ala Gly Val His Ser Asn Asp His Ser Val Val 355 3607378PRTHomo
sapienshuman endothelial differentiation G-protein coupled receptor
(GPCR) 3 (EDG3) 7Met Ala Thr Ala Leu Pro Pro Arg Leu Gln Pro Val
Arg Gly Asn Glu 1 5 10 15Thr Leu Arg Glu His Tyr Gln Tyr Val Gly
Lys Leu Ala Gly Arg Leu 20 25 30Lys Glu Ala Ser Glu Gly Ser Thr Leu
Thr Thr Val Leu Phe Leu Val 35 40 45Ile Cys Ser Phe Ile Val Leu Glu
Asn Leu Met Val Leu Ile Ala Ile 50 55 60Trp Lys Asn Asn Lys Phe His
Asn Arg Met Tyr Phe Phe Ile Gly Asn 65 70 75 80Leu Ala Leu Cys Asp
Leu Leu Ala Gly Ile Ala Tyr Lys Val Asn Ile 85 90 95Leu Met Ser Gly
Lys Lys Thr Phe Ser Leu Ser Pro Thr Val Trp Phe 100 105 110Leu Arg
Glu Gly Ser Met Phe Val Ala Leu Gly Ala Ser Thr Cys Ser 115 120
125Leu Leu Ala Ile Ala Ile Glu Arg His Leu Thr Met Ile Lys Met Arg
130 135 140Pro Tyr Asp Ala Asn Lys Arg His Arg Val Phe Leu Leu Ile
Gly Met145 150 155 160Cys Trp Leu Ile Ala Phe Thr Leu Gly Ala Leu
Pro Ile Leu Gly Trp 165 170 175Asn Cys Leu His Asn Leu Pro Asp Cys
Ser Thr Ile Leu Pro Leu Tyr 180 185 190Ser Lys Lys Tyr Ile Ala Phe
Cys Ile Ser Ile Phe Thr Ala Ile Leu 195 200 205Val Thr Ile Val Ile
Leu Tyr Ala Arg Ile Tyr Phe Leu Val Lys Ser 210 215 220Ser Ser Arg
Lys Val Ala Asn His Asn Asn Ser Glu Arg Ser Met Ala225 230 235
240Leu Leu Arg Thr Val Val Ile Val Val Ser Val Phe Ile Ala Cys Trp
245 250 255Ser Pro Leu Phe Ile Leu Phe Leu Ile Asp Val Ala Cys Arg
Val Gln 260 265 270Ala Cys Pro Ile Leu Phe Lys Ala Gln Trp Phe Ile
Val Leu Ala Val 275 280 285Leu Asn Ser Ala Met Asn Pro Val Ile Tyr
Thr Leu Ala Ser Lys Glu 290 295 300Met Arg Arg Ala Phe Phe Arg Leu
Val Cys Asn Cys Leu Val Arg Gly305 310 315 320Arg Gly Ala Arg Ala
Ser Pro Ile Gln Pro Ala Leu Asp Pro Ser Arg 325 330 335Ser Lys Ser
Ser Ser Ser Asn Asn Ser Ser His Ser Pro Lys Val Lys 340 345 350Glu
Asp Leu Pro His Thr Asp Pro Ser Ser Cys Ile Met Asp Lys Asn 355 360
365Ala Ala Leu Gln Asn Gly Ile Phe Cys Asn 370 3758351PRTHomo
sapienshuman endothelial differentiation G-protein coupled receptor
(GPCR) 4 (EDG4) 8Met Val Ile Met Gly Gln Cys Tyr Tyr Asn Glu Thr
Ile Gly Phe Phe 1 5 10 15Tyr Asn Asn Ser Gly Lys Glu Leu Ser Ser
His Trp Arg Pro Lys Asp 20 25 30Val Val Val Val Ala Leu Gly Leu Thr
Val Ser Val Leu Val Leu Leu 35 40 45Thr Asn Leu Leu Val Ile Ala Ala
Ile Ala Ser Asn Arg Arg Phe His 50 55 60Gln Pro Ile Tyr Tyr Leu Leu
Gly Asn Leu Ala Ala Ala Asp Leu Phe 65 70 75 80Ala Gly Val Ala Tyr
Leu Phe Leu Met Phe His Thr Gly Pro Arg Thr 85 90 95Ala Arg Leu Ser
Leu Glu Gly Trp Phe Leu Arg Gln Gly Leu Leu Asp 100 105 110Thr Ser
Leu Thr Ala Ser Val Ala Thr Leu Leu Ala Ile Ala Val Glu 115 120
125Arg His Arg Ser Val Met Ala Val Gln Leu His Ser Arg Leu Pro Arg
130 135 140Gly Arg Val Val Met Leu Ile Val Gly Val Trp Val Ala Ala
Leu Gly145 150 155 160Leu Gly Leu Leu Pro Ala His Ser Trp His Cys
Leu Cys Ala Leu Asp 165 170 175Arg Cys Ser Arg Met Ala Pro Leu Leu
Ser Arg Ser Tyr Leu Ala Val 180 185 190Trp Ala Leu Ser Ser Leu Leu
Val Phe Leu Leu Met Val Ala Val Tyr 195 200 205Thr Arg Ile Phe Phe
Tyr Val Arg Arg Arg Val Gln Arg Met Ala Glu 210 215 220His Val Ser
Cys His Pro Arg Tyr Arg Glu Thr Thr Leu Ser Leu Val225 230 235
240Lys Thr Val Val Ile Ile Leu Gly Ala Phe Val Val Cys Trp Thr Pro
245 250 255Gly Gln Val Val Leu Leu Leu Asp Gly Leu Gly Cys Glu Ser
Cys Asn 260 265 270Val Leu Ala Val Glu Lys Tyr Phe Leu Leu Leu Ala
Glu Ala Asn Ser 275 280 285Leu Val Asn Ala Ala Val Tyr Ser Cys Arg
Asp Ala Glu Met Arg Arg 290 295 300Thr Phe Arg Arg Leu Leu Cys Cys
Ala Cys Leu Arg Gln Ser Thr Arg305 310 315 320Glu Ser Val His Tyr
Thr Ser Ser Ala Gln Gly Gly Ala Ser Thr Arg 325 330 335Ile Met Leu
Pro Glu Asn Gly His Pro Leu Met Asp Ser Thr Leu 340 345
3509353PRTHomo sapienshuman endothelial differentiation G-protein
coupled receptor (GPCR) 5 (EDG5) 9Met Gly Ser Leu Tyr Ser
Glu Tyr Leu Asn Pro Asn Lys Val Gln Glu 1 5 10 15His Tyr Asn Tyr
Thr Lys Glu Thr Leu Glu Thr Gln Glu Thr Thr Ser 20 25 30Arg Gln Val
Ala Ser Ala Phe Ile Val Ile Leu Cys Cys Ala Ile Val 35 40 45Val Glu
Asn Leu Leu Val Leu Ile Ala Val Ala Arg Asn Ser Lys Phe 50 55 60His
Ser Ala Met Tyr Leu Phe Leu Gly Asn Leu Ala Ala Ser Asp Leu 65 70
75 80Leu Ala Gly Val Ala Phe Val Ala Asn Thr Leu Leu Ser Gly Ser
Val 85 90 95Thr Leu Arg Leu Thr Pro Val Gln Trp Phe Ala Arg Glu Gly
Ser Ala 100 105 110Ser Ile Thr Leu Ser Ala Ser Val Phe Ser Leu Leu
Ala Ile Ala Ile 115 120 125Glu Arg His Val Ala Ile Ala Lys Val Lys
Leu Tyr Gly Ser Asp Lys 130 135 140Ser Cys Arg Met Leu Leu Leu Ile
Gly Ala Ser Trp Leu Ile Ser Leu145 150 155 160Val Leu Gly Gly Leu
Pro Ile Leu Gly Trp Asn Cys Leu Gly His Leu 165 170 175Glu Ala Cys
Ser Thr Val Leu Pro Leu Tyr Ala Lys His Tyr Val Leu 180 185 190Cys
Val Val Thr Ile Phe Ser Ile Ile Leu Leu Ala Ile Val Ala Leu 195 200
205Tyr Val Arg Ile Tyr Cys Val Val Arg Ser Ser His Ala Asp Met Ala
210 215 220Ala Pro Gln Thr Leu Ala Leu Leu Lys Thr Val Thr Ile Val
Leu Gly225 230 235 240Val Phe Ile Val Cys Trp Leu Pro Ala Phe Ser
Ile Leu Leu Leu Asp 245 250 255Tyr Ala Cys Pro Val His Ser Cys Pro
Ile Leu Tyr Lys Ala His Tyr 260 265 270Phe Phe Ala Val Ser Thr Leu
Asn Ser Leu Leu Asn Pro Val Ile Tyr 275 280 285Thr Trp Arg Ser Arg
Asp Leu Arg Arg Glu Val Leu Arg Pro Leu Gln 290 295 300Cys Trp Arg
Pro Gly Val Gly Val Gln Gly Arg Arg Arg Val Gly Thr305 310 315
320Pro Gly His His Leu Leu Pro Leu Arg Ser Ser Ser Ser Leu Glu Arg
325 330 335Gly Met His Met Pro Thr Ser Pro Thr Phe Leu Glu Gly Asn
Thr Val 340 345 350Val10384PRTHomo sapienshuman endothelial
differentiation G-protein coupled receptor (GPCR) 6 (EDG6) 10Met
Asn Ala Thr Gly Thr Pro Val Ala Pro Glu Ser Cys Gln Gln Leu 1 5 10
15Ala Ala Gly Gly His Ser Arg Leu Ile Val Leu His Tyr Asn His Ser
20 25 30Gly Arg Leu Ala Gly Arg Gly Gly Pro Glu Asp Gly Gly Leu Gly
Ala 35 40 45Leu Arg Gly Leu Ser Val Ala Ala Ser Cys Leu Val Val Leu
Glu Asn 50 55 60Leu Leu Val Leu Ala Ala Ile Thr Ser His Met Arg Ser
Arg Arg Trp 65 70 75 80Val Tyr Tyr Cys Leu Val Asn Ile Thr Leu Ser
Asp Leu Leu Thr Gly 85 90 95Ala Ala Tyr Leu Ala Asn Val Leu Leu Ser
Gly Ala Arg Thr Phe Arg 100 105 110Leu Ala Pro Ala Gln Trp Phe Leu
Arg Glu Gly Leu Leu Phe Thr Ala 115 120 125Leu Ala Ala Ser Thr Phe
Ser Leu Leu Phe Thr Ala Gly Glu Arg Phe 130 135 140Ala Thr Met Val
Arg Pro Val Ala Glu Ser Gly Ala Thr Lys Thr Ser145 150 155 160Arg
Val Tyr Gly Phe Ile Gly Leu Cys Trp Leu Leu Ala Ala Leu Leu 165 170
175Gly Met Leu Pro Leu Leu Gly Trp Asn Cys Leu Cys Ala Phe Asp Arg
180 185 190Cys Ser Ser Leu Leu Pro Leu Tyr Ser Lys Arg Tyr Ile Leu
Phe Cys 195 200 205Leu Val Ile Phe Ala Gly Val Leu Ala Thr Ile Met
Gly Leu Tyr Gly 210 215 220Ala Ile Phe Arg Leu Val Gln Ala Ser Gly
Gln Lys Ala Pro Arg Pro225 230 235 240Ala Ala Arg Arg Lys Ala Arg
Arg Leu Leu Lys Thr Val Leu Met Ile 245 250 255Leu Leu Ala Phe Leu
Val Cys Trp Gly Pro Leu Phe Gly Leu Leu Leu 260 265 270Ala Asp Val
Phe Gly Ser Asn Leu Trp Ala Gln Glu Tyr Leu Arg Gly 275 280 285Met
Asp Trp Ile Leu Ala Leu Ala Val Leu Asn Ser Ala Val Asn Pro 290 295
300Ile Ile Tyr Ser Phe Arg Ser Arg Glu Val Cys Arg Ala Val Leu
Ser305 310 315 320Phe Leu Cys Cys Gly Cys Leu Arg Leu Gly Met Arg
Gly Pro Gly Asp 325 330 335Cys Leu Ala Arg Ala Val Glu Ala His Ser
Gly Ala Ser Thr Thr Asp 340 345 350Ser Ser Leu Arg Pro Arg Asp Ser
Phe Arg Gly Ser Arg Ser Leu Ser 355 360 365Phe Arg Met Arg Glu Pro
Leu Ser Ser Ile Ser Ser Val Arg Ser Ile 370 375 38011353PRTHomo
sapienshuman endothelial differentiation G-protein coupled receptor
(GPCR) 7 (EDG7) 11Met Asn Glu Cys His Tyr Asp Lys His Met Asp Phe
Phe Tyr Asn Arg 1 5 10 15Ser Asn Thr Asp Thr Val Asp Asp Trp Thr
Gly Thr Lys Leu Val Ile 20 25 30Val Leu Cys Val Gly Thr Phe Phe Cys
Leu Phe Ile Phe Phe Ser Asn 35 40 45Ser Leu Val Ile Ala Ala Val Ile
Lys Asn Arg Lys Phe His Phe Pro 50 55 60Phe Tyr Tyr Leu Leu Ala Asn
Leu Ala Ala Ala Asp Phe Phe Ala Gly 65 70 75 80Ile Ala Tyr Val Phe
Leu Met Phe Asn Thr Gly Pro Val Ser Lys Thr 85 90 95Leu Thr Val Asn
Arg Trp Phe Leu Arg Gln Gly Leu Leu Asp Ser Ser 100 105 110Leu Thr
Ala Ser Leu Thr Asn Leu Leu Val Ile Ala Val Glu Arg His 115 120
125Met Ser Ile Met Arg Met Arg Val His Ser Asn Leu Thr Lys Lys Arg
130 135 140Val Thr Leu Leu Ile Leu Leu Val Trp Ala Ile Ala Ile Phe
Met Gly145 150 155 160Ala Val Pro Thr Leu Gly Trp Asn Cys Leu Cys
Asn Ile Ser Ala Cys 165 170 175Ser Ser Leu Ala Pro Ile Tyr Ser Arg
Ser Tyr Leu Val Phe Trp Thr 180 185 190Val Ser Asn Leu Met Ala Phe
Leu Ile Met Val Val Val Tyr Leu Arg 195 200 205Ile Tyr Val Tyr Val
Lys Arg Lys Thr Asn Val Leu Ser Pro His Thr 210 215 220Ser Gly Ser
Ile Ser Arg Arg Arg Thr Pro Met Lys Leu Met Lys Thr225 230 235
240Val Met Thr Val Leu Gly Ala Phe Val Val Cys Trp Thr Pro Gly Leu
245 250 255Val Val Leu Leu Leu Asp Gly Leu Asn Cys Arg Gln Cys Gly
Val Gln 260 265 270His Val Lys Arg Trp Phe Leu Leu Leu Ala Leu Leu
Asn Ser Val Val 275 280 285Asn Pro Ile Ile Tyr Ser Tyr Lys Asp Glu
Asp Met Tyr Gly Thr Met 290 295 300Lys Lys Met Ile Cys Cys Phe Ser
Gln Glu Asn Pro Glu Arg Arg Pro305 310 315 320Ser Arg Ile Pro Ser
Thr Val Leu Ser Arg Ser Asp Thr Gly Ser Gln 325 330 335Tyr Ile Glu
Asp Ser Ile Ser Gln Gly Ala Val Cys Asn Lys Ser Thr 340 345
350Ser12400PRTRattus norvegicusrat endothelial differentiation
G-protein coupled receptor (GPCR) 8 (EDG8) 12Met Glu Ser Gly Leu
Leu Arg Pro Ala Pro Val Ser Glu Val Ile Val 1 5 10 15Leu His Tyr
Asn Tyr Thr Gly Lys Leu Arg Gly Ala Arg Tyr Gln Pro 20 25 30Gly Ala
Gly Leu Arg Ala Asp Ala Ala Val Cys Leu Ala Val Cys Ala 35 40 45Phe
Ile Val Leu Glu Asn Leu Ala Val Leu Leu Val Leu Gly Arg His 50 55
60Pro Arg Phe His Ala Pro Met Phe Leu Leu Leu Gly Ser Leu Thr Leu
65 70 75 80Ser Asp Leu Leu Ala Gly Ala Ala Tyr Ala Thr Asn Ile Leu
Leu Ser 85 90 95Gly Pro Leu Thr Leu Arg Leu Ser Pro Ala Leu Trp Phe
Ala Arg Glu 100 105 110Gly Gly Val Phe Val Ala Leu Ala Ala Ser Val
Leu Ser Leu Leu Ala 115 120 125Ile Ala Leu Glu Arg His Leu Thr Met
Ala Arg Arg Gly Pro Ala Pro 130 135 140Ala Ala Ser Arg Ala Arg Thr
Leu Ala Met Ala Val Ala Ala Trp Gly145 150 155 160Leu Ser Leu Leu
Leu Gly Leu Leu Pro Ala Leu Gly Trp Asn Cys Leu 165 170 175Gly Arg
Leu Glu Ala Cys Ser Thr Val Leu Pro Leu Tyr Ala Lys Ala 180 185
190Tyr Val Leu Phe Cys Val Leu Ala Phe Leu Gly Ile Leu Ala Ala Ile
195 200 205Cys Ala Leu Tyr Ala Arg Ile Tyr Cys Gln Val Arg Ala Asn
Ala Arg 210 215 220Arg Leu Arg Ala Gly Pro Gly Ser Arg Arg Ala Thr
Ser Ser Ser Arg225 230 235 240Ser Arg His Thr Pro Arg Ser Leu Ala
Leu Leu Arg Thr Leu Ser Val 245 250 255Val Leu Leu Ala Phe Val Ala
Cys Trp Gly Pro Leu Phe Leu Leu Leu 260 265 270Leu Leu Asp Val Ala
Cys Pro Ala Arg Ala Cys Pro Val Leu Leu Gln 275 280 285Ala Asp Pro
Phe Leu Gly Leu Ala Met Ala Asn Ser Leu Leu Asn Pro 290 295 300Ile
Ile Tyr Thr Phe Thr Asn Arg Asp Leu Arg His Ala Leu Leu Arg305 310
315 320Leu Leu Cys Cys Gly Arg Gly Pro Cys Asn Gln Asp Ser Ser Asn
Ser 325 330 335Leu Gln Arg Ser Pro Ser Ala Val Gly Pro Ser Gly Gly
Gly Leu Arg 340 345 350Arg Cys Leu Pro Pro Thr Leu Asp Arg Ser Ser
Ser Pro Ser Glu His 355 360 365Ser Cys Pro Gln Arg Asp Gly Met Asp
Thr Ser Cys Ser Thr Gly Ser 370 375 380Pro Gly Ala Ala Thr Ala Asn
Arg Thr Leu Val Pro Asp Ala Thr Asp385 390 395 40013341PRTHomo
sapiensmutant #1, C-terminally truncated variant of human
endothelial differentiation G-protein coupled receptor (GPCR) 1
(EDG1) 13Met Gly Pro Thr Ser Val Pro Leu Val Lys Ala His Arg Ser
Ser Val 1 5 10 15Ser Asp Tyr Val Asn Tyr Asp Ile Ile Val Arg His
Tyr Asn Tyr Thr 20 25 30Gly Lys Leu Asn Ile Ser Ala Asp Lys Glu Asn
Ser Ile Lys Leu Thr 35 40 45Ser Val Val Phe Ile Leu Ile Cys Cys Phe
Ile Ile Leu Glu Asn Ile 50 55 60Phe Val Leu Leu Thr Ile Trp Lys Thr
Lys Lys Phe His Arg Pro Met 65 70 75 80Tyr Tyr Phe Ile Gly Asn Leu
Ala Leu Ser Asp Leu Leu Ala Gly Val 85 90 95Ala Tyr Thr Ala Asn Leu
Leu Leu Ser Gly Ala Thr Thr Tyr Lys Leu 100 105 110Thr Pro Ala Gln
Trp Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu 115 120 125Ser Ala
Ser Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg Tyr Ile 130 135
140Thr Met Leu Lys Met Lys Leu His Asn Gly Ser Asn Asn Phe Arg
Leu145 150 155 160Phe Leu Leu Ile Ser Ala Cys Trp Val Ile Ser Leu
Ile Leu Gly Gly 165 170 175Leu Pro Ile Met Gly Trp Asn Cys Ile Ser
Ala Leu Ser Ser Cys Ser 180 185 190Thr Val Leu Pro Leu Tyr His Lys
His Tyr Ile Leu Phe Cys Thr Thr 195 200 205Val Phe Thr Leu Leu Leu
Leu Ser Ile Val Ile Leu Tyr Cys Arg Ile 210 215 220Tyr Ser Leu Val
Arg Thr Arg Ser Arg Arg Leu Thr Phe Arg Lys Asn225 230 235 240Ile
Ser Lys Ala Ser Arg Ser Ser Glu Lys Ser Leu Ala Leu Leu Lys 245 250
255Thr Val Ile Ile Val Leu Ser Val Phe Ile Ala Cys Trp Ala Pro Leu
260 265 270Phe Ile Leu Leu Leu Leu Asp Val Gly Cys Lys Val Lys Thr
Cys Asp 275 280 285Ile Leu Phe Arg Ala Glu Tyr Phe Leu Val Leu Ala
Val Leu Asn Ser 290 295 300Gly Thr Asn Pro Ile Ile Tyr Thr Leu Thr
Asn Lys Glu Met Arg Arg305 310 315 320Ala Phe Ile Arg Ile Met Ser
Cys Cys Lys Cys Pro Ser Gly Asp Ser 325 330 335Ala Gly Lys Phe Lys
34014314PRTHomo sapiensmutant #2, C-terminally truncated variant of
human endothelial differentiation G-protein coupled receptor (GPCR)
1 (EDG1) 14Met Gly Pro Thr Ser Val Pro Leu Val Lys Ala His Arg Ser
Ser Val 1 5 10 15Ser Asp Tyr Val Asn Tyr Asp Ile Ile Val Arg His
Tyr Asn Tyr Thr 20 25 30Gly Lys Leu Asn Ile Ser Ala Asp Lys Glu Asn
Ser Ile Lys Leu Thr 35 40 45Ser Val Val Phe Ile Leu Ile Cys Cys Phe
Ile Ile Leu Glu Asn Ile 50 55 60Phe Val Leu Leu Thr Ile Trp Lys Thr
Lys Lys Phe His Arg Pro Met 65 70 75 80Tyr Tyr Phe Ile Gly Asn Leu
Ala Leu Ser Asp Leu Leu Ala Gly Val 85 90 95Ala Tyr Thr Ala Asn Leu
Leu Leu Ser Gly Ala Thr Thr Tyr Lys Leu 100 105 110Thr Pro Ala Gln
Trp Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu 115 120 125Ser Ala
Ser Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg Tyr Ile 130 135
140Thr Met Leu Lys Met Lys Leu His Asn Gly Ser Asn Asn Phe Arg
Leu145 150 155 160Phe Leu Leu Ile Ser Ala Cys Trp Val Ile Ser Leu
Ile Leu Gly Gly 165 170 175Leu Pro Ile Met Gly Trp Asn Cys Ile Ser
Ala Leu Ser Ser Cys Ser 180 185 190Thr Val Leu Pro Leu Tyr His Lys
His Tyr Ile Leu Phe Cys Thr Thr 195 200 205Val Phe Thr Leu Leu Leu
Leu Ser Ile Val Ile Leu Tyr Cys Arg Ile 210 215 220Tyr Ser Leu Val
Arg Thr Arg Ser Arg Arg Leu Thr Phe Arg Lys Asn225 230 235 240Ile
Ser Lys Ala Ser Arg Ser Ser Glu Lys Ser Leu Ala Leu Leu Lys 245 250
255Thr Val Ile Ile Val Leu Ser Val Phe Ile Ala Cys Trp Ala Pro Leu
260 265 270Phe Ile Leu Leu Leu Leu Asp Val Gly Cys Lys Val Lys Thr
Cys Asp 275 280 285Ile Leu Phe Arg Ala Glu Tyr Phe Leu Val Leu Ala
Val Leu Asn Ser 290 295 300Gly Thr Asn Pro Ile Ile Tyr Thr Leu
Thr305 31015346PRTHomo sapiensmutant #3, C-terminally truncated
variant of human endothelial differentiation G-protein coupled
receptor (GPCR) 1 (EDG1) 15Met Gly Pro Thr Ser Val Pro Leu Val Lys
Ala His Arg Ser Ser Val 1 5 10 15Ser Asp Tyr Val Asn Tyr Asp Ile
Ile Val Arg His Tyr Asn Tyr Thr 20 25 30Gly Lys Pro Asn Ile Ser Ala
Asp Lys Glu Asn Ser Ile Lys Leu Thr 35 40 45Ser Val Val Phe Ile Leu
Ile Cys Cys Phe Ile Ile Leu Glu Asn Ile 50 55 60Phe Val Leu Leu Thr
Ile Trp Lys Thr Lys Lys Phe His Arg Pro Met 65 70 75 80Tyr Tyr Phe
Ile Gly Asn Leu Ala Leu Ser Asp Leu Leu Ala Gly Val 85 90 95Ala Tyr
Thr Ala Asn Leu Leu Leu Ser Gly Ala Thr Thr Tyr Lys Leu 100 105
110Thr Pro Ala Gln Trp Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu
115 120 125Ser Ala Ser Val Phe Ser Leu Leu Ala Ile Ala Ile Glu Arg
Tyr Ile 130 135 140Thr Met Leu Lys Met Lys Leu His Asn Gly Ser Asn
Asn Phe Arg Leu145 150 155 160Phe Leu Leu Ile Ser Ala Cys Trp Val
Ile Ser Leu Ile Leu Gly Gly 165 170 175Leu Pro Ile Met Gly Trp Asn
Cys Ile Ser Ala Leu Ser Ser Cys Ser 180 185 190Thr Val Leu Pro Leu
Tyr His Lys His Tyr Ile Leu Phe Cys Thr Thr 195 200 205Val Phe Thr
Leu Leu Leu Leu Ser Ile Val Ile Leu Tyr Cys Arg Ile 210 215 220Tyr
Ser Leu Val Arg
Thr Arg Ser Arg Arg Leu Thr Phe Arg Lys Asn225 230 235 240Ile Ser
Lys Ala Ser Arg Ser Ser Glu Lys Ser Leu Ala Leu Leu Arg 245 250
255Thr Val Ile Ile Val Leu Ser Val Phe Ile Ala Cys Trp Ala Pro Leu
260 265 270Phe Ile Leu Leu Leu Leu Asp Val Gly Cys Lys Val Lys Thr
Cys Asp 275 280 285Ile Leu Phe Arg Ala Glu Tyr Phe Leu Val Leu Ala
Val Leu Asn Ser 290 295 300Gly Thr Asn Pro Ile Ile Tyr Thr Leu Thr
Asn Lys Glu Met Arg Arg305 310 315 320Ala Phe Ile Arg Ile Met Ser
Cys Cys Lys Cys Pro Ser Gly Asp Ser 325 330 335Ala Gly Lys Phe Lys
Arg Pro Ile Ile Ala 340 345
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