U.S. patent application number 11/930528 was filed with the patent office on 2009-01-15 for endothelial cell expression patterns.
This patent application is currently assigned to The John Hopkins University. Invention is credited to Kenneth W. Kinzler, Brad St.Croix, Bert Vogelstein.
Application Number | 20090017030 11/930528 |
Document ID | / |
Family ID | 27397112 |
Filed Date | 2009-01-15 |
United States Patent
Application |
20090017030 |
Kind Code |
A1 |
St.Croix; Brad ; et
al. |
January 15, 2009 |
Endothelial Cell Expression Patterns
Abstract
To gain a better understanding of tumor angiogenesis, new
techniques for isolating endothelial cells (ECs) and evaluating
gene expression patterns were developed. When transcripts from ECs
derived from normal and malignant colorectal tissues were compared
with transcripts from non-endothelial cells, over 170 genes
predominantly expressed in the endothelium were identified.
Comparison between normal- and tumor-derived endothelium revealed
79 differentially expressed genes, including 46 that were
specifically elevated in tumor-associated endothelium. Experiments
with representative genes from this group demonstrated that most
were similarly expressed in the endothelium of primary lung,
breast, brain, and pancreatic cancers as well as in metastatic
lesions of the liver. These results demonstrate that neoplastic and
normal endothelium in humans are distinct at the molecular level,
and have significant implications for the development of
anti-angiogenic therapies in the future.
Inventors: |
St.Croix; Brad; (Frederick,
MD) ; Vogelstein; Bert; (Baltimore, MD) ;
Kinzler; Kenneth W.; (Bel Air, MD) |
Correspondence
Address: |
BANNER & WITCOFF, LTD.
1100 13th STREET, N.W., SUITE 1200
WASHINGTON
DC
20005-4051
US
|
Assignee: |
The John Hopkins University
Baltimore
MD
|
Family ID: |
27397112 |
Appl. No.: |
11/930528 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10979159 |
Nov 3, 2004 |
7358351 |
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11930528 |
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09918715 |
Aug 1, 2001 |
7402660 |
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10979159 |
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60222599 |
Aug 2, 2000 |
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60224360 |
Aug 11, 2000 |
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60282850 |
Apr 11, 2001 |
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Current U.S.
Class: |
424/138.1 ;
424/139.1; 424/184.1; 424/185.1; 435/325; 435/6.16; 435/7.21;
514/1.1; 530/350; 530/387.3; 530/387.9; 530/391.3; 530/391.7;
536/23.5 |
Current CPC
Class: |
A61P 19/02 20180101;
A61P 13/12 20180101; A61P 9/00 20180101; A61P 9/10 20180101; A61P
29/00 20180101; A61P 35/00 20180101; A61P 27/02 20180101; C07K
16/30 20130101; A61P 17/06 20180101; A61K 2039/505 20130101 |
Class at
Publication: |
424/138.1 ;
530/387.9; 530/387.3; 530/391.7; 530/391.3; 424/139.1; 435/325;
424/184.1; 424/185.1; 435/7.21; 530/350; 514/12; 536/23.5;
435/6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/18 20060101 C07K016/18; C12N 5/00 20060101
C12N005/00; G01N 33/567 20060101 G01N033/567; A61K 38/16 20060101
A61K038/16; C12Q 1/68 20060101 C12Q001/68; C07H 21/04 20060101
C07H021/04; C07K 14/435 20060101 C07K014/435; A61K 39/00 20060101
A61K039/00; C07K 17/00 20060101 C07K017/00 |
Goverment Interests
[0002] The U.S. government retains certain rights in the invention
by virtue of the provisions of National Institutes of Heath grants
CA57345 and CA43460, which supported this work.
Claims
1. An isolated molecule comprising an antibody variable region
which specifically binds to an extracellular domain of a TEM
protein selected from the group consisting of: 1, 9, 17, 19, and
44, as shown in SEQ ID NO: 196, 212, 230, 232, and 271,
respectively.
2. The isolated molecule of claim 1 which is an in tact antibody
molecule.
3. The isolated molecule of claim 1 which is a single chain
variable region (ScFv).
4. The isolated molecule of claim 1 which is a monoclonal
antibody.
5. The isolated molecule of claim 1 which is a humanized
antibody.
6. The isolated molecule of claim 1 which is a human antibody.
7. The isolated molecule of claim 1 which is bound to a cytotoxic
moiety.
8. The isolated molecule of claim 1 which is bound to a therapeutic
moiety.
9. The isolated molecule of claim 1 which is bound to a detectable
moiety.
10. The isolated molecule of claim 1 which is bound to an
anti-tumor agent.
11. A method of inhibiting neoangiogenesis, comprising:
administering to a subject in need thereof an effective amount of
an isolated molecule comprising an antibody variable region which
specifically binds to an extracellular domain of a TEM protein
selected from the group consisting of: 1, 9, 17, 19, 22, and 44, as
shown in SEQ ID NO: 196, 212, 230, 232, 238, and 271, respectively,
whereby neoangiogenesis is inhibited.
12. The method of claim 11 wherein the subject bears a vascularized
tumor.
13. The method of claim 11 wherein the subject has polycystic
kidney disease.
14. The method of claim 11 wherein the subject has diabetic
retinopathy.
15. The method of claim 11 wherein the subject has rheumatoid
arthritis.
16. The method of claim 11 wherein the subject has psoriasis.
17. A method of inhibiting tumor growth, comprising: administering
to a human subject bearing a tumor an effective amount of an
isolated molecule comprising an antibody variable region which
specifically binds to an extracellular domain of a TEM protein
selected from the group consisting of: 1, 9, 17, 19, 22, and 44, as
shown in SEQ ID NO: 196, 212, 230, 232, 238, and 271, respectively,
whereby growth of the tumor is inhibited.
18. An isolated molecule comprising an antibody variable region
which specifically binds to a TEM protein selected from the group
consisting of: 9, 17, 19, and 44, as shown in SEQ ID NO: 212, 230,
232, and 271, respectively.
19. The isolated molecule of claim 18 which is a single chain
variable region (ScFv).
20. The isolated molecule of claim 18 which is a monoclonal
antibody.
21. The isolated molecule of claim 18 which is a humanized
antibody.
22. The isolated molecule of claim 18 which is a human
antibody.
23. The isolated molecule of claim 18 which is bound to a cytotoxic
moiety.
24. The isolated molecule of claim 18 which is bound to a
therapeutic moiety.
25. The isolated molecule of claim 18 which is bound to a
detectable moiety.
26. The isolated molecule of claim 18 which is bound to an
anti-tumor agent.
27. The isolated molecule of claim 18 which is an in tact antibody
molecule.
28. An isolated and purified human transmembrane protein selected
from the group consisting of: TEM 9, 17, and 19 as shown in SEQ ID
NO: 212, 230, and 232, respectively.
29. An isolated and purified nucleic acid molecule comprising a
coding sequence for a transmembrane TEM selected from the group
consisting of:: TEM 9, 17, and 19 as shown in SEQ ID NO: 212, 230,
232, respectively.
30. The isolated and purified nucleic acid molecule of claim 29
which comprises a coding sequence selected from those shown in SEQ
ID NO: 211, 229, and 231.
31. A recombinant host cell which comprises a nucleic acid molecule
comprising a coding sequence for a transmembrane TEM selected from
the group consisting of: TEM 9, 17, and 19 as shown in SEQ ID NO:
212, 230, and 232, respectively.
32. The recombinant host cell of claim 31 which comprises a coding
sequence selected from those shown in SEQ ID NO: 211, 229, and
231.
33. A method of inducing an immune response in a mammal,
comprising: administering to the mammal a nucleic acid molecule
comprising a coding sequence for a human transmembrane protein
selected from the group consisting of: TEM 1, 9, 13, 17, 19, 22,
30, and 44 as shown in SEQ ID NO: 196, 212, 220, 230, 232, 238, 250
and 271, respectively, whereby an immune response to the human
transmembrane protein is induced in the mammal.
34. The method of claim 33 wherein the coding sequence is shown in
SEQ ID NO: 195, 211, 219, 229, 231, 237, 249, 270.
35. A method of inducing an immune response in a mammal,
comprising: administering to the mammal a purified human
transmembrane protein selected from the group consisting of: TEM 1,
9, 13, 17, 19, 22, 30, and 44 as shown in SEQ ID NO: 196, 212, 220,
230, 232, 238, 250 and 271, respectively, whereby an immune
response to the human transmembrane protein is induced in the
mammal.
36. A method for identification of a ligand involved in endothelial
cell regulation, comprising: contacting a test compound with an
isolated and purified human transmembrane protein selected from the
group consisting of 1, 9, 13, 17, 19, 30, and 44 as shown in SEQ ID
NO: 196, 212, 220, 230, 250, 232 and 271; contacting the isolated
and purified human transmembrane protein with a molecule comprising
an antibody variable region which specifically binds to an
extracellular domain of a TEM protein selected from the group
consisting of: 1, 9, 13, 17, 19, 30, and 44 as shown in SEQ ID NO:
196, 212, 220, 230, 250, 232 and 271, respectively; determining
binding of the molecule comprising an antibody variable region to
the human transmembrane protein, wherein a test compound which
diminishes the binding of the molecule comprising an antibody
variable region to the human transmembrane protein is identified as
a ligand involved in endothelial cell regulation.
37. A method for identification of a ligand involved in endothelial
cell regulation, comprising: contacting a test compound with a cell
comprising a human transmembrane protein selected from the group
consisting of 1, 9, 17, and 19 as shown in SEQ ID NO: 196, 212,
230, and 232; contacting the cell with a molecule comprising an
antibody variable region which specifically binds to an
extracellular domain of a TEM protein selected from the group
consisting of: 1, 9, 17, and 19 as shown in SEQ ID NO: 196, 212,
230, and 232, respectively; determining binding of the molecule
comprising an antibody variable region to the cell, wherein a test
compound which diminishes the binding of the molecule comprising an
antibody variable region to the cell is identified as a ligand
involved in endothelial cell regulation.
38. A soluble form of a human transmembrane protein selected from
the group consisting of: TEM 1, 9, 17, 19, 22, 30 and 44 as shown
in SEQ ID NO: 196, 212, 230, 232, 238, 250, and 271, respectively,
wherein the soluble forms lack transmembrane domains.
39. The soluble form of claim 38 wherein the soluble form consists
of an extracellular domain of the human transmembrane protein.
40. A method of inhibiting neoangiogenesis in a patient,
comprising: administering to the patient a soluble form of a human
transmembrane protein according to claim 38, whereby
neoangiogenesis in the patient is inhibited.
41. A method of inhibiting neoangiogenesis in a patient,
comprising: administering to the patient a soluble form of a human
transmembrane protein according to claim 39, whereby
neoangiogenesis in the patient is inhibited.
42. The method of claim 40 wherein the patient bears a vascularized
tumor.
43. The method of claim 41 wherein the patient bears a vascularized
tumor.
44. The method of claim 40 wherein the patient has polycystic
kidney disease.
45. The method of claim 40 wherein the patient has diabetic
retinopathy.
46. The method of claim 40 wherein the patient has rheumatoid
arthritis.
47. The method of claim 40 wherein the patient has psoriasis.
48. The method of claim 41 wherein the patient has polycystic
kidney disease.
49. The method of claim 41 wherein the patient has diabetic
retinopathy.
50. The method of claim 41 wherein the patient has rheumatoid
arthritis.
51. The method of claim 41 wherein the patient has psoriasis.
52. A method of identifying regions of neoangiogenesis in a
patient, comprising: administering to a patient a molecule
comprising an antibody variable region which specifically binds to
an extracellular domain of a TEM protein selected from the group
consisting of: 1, 9, 13, 17, 19, 22, 30, and 44, as shown in SEQ ID
NO: 196, 212, 220, 230, 232, 238, 250, and 271, respectively,
wherein the molecule is bound to a detectable moiety; and detecting
the detectable moiety in the patient, thereby identifying
neoangiogenesis.
53. A method of screening for neoangiogenesis in a patient,
comprising: contacting a body fluid collected from the patient with
a molecule comprising an antibody variable region which
specifically binds to an extracellular domain of a TEM protein
selected from the group consisting of: 1, 9, 17, 19, and 44, as
shown in SEQ ID NO: 196, 212, 230, 232, and 271, respectively,
wherein detection of cross-reactive material in the body fluid with
the molecule indicates neoangiogenesis in the patient.
54. A method of screening for neoangiogenesis in a patient,
comprising: contacting a body fluid collected from the patient with
a molecule comprising an antibody variable region which
specifically binds to a TEM protein selected from the group
consisting of: 4, 6, 7, 10, 12, 14, 25, 27, 31, 36, 37, 38, 39, as
shown in SEQ ID NO: 202, 206, 208, 214, 218, 223 & 224, 242,
244, 252, 257, 259, 261, and 263, respectively, wherein detection
of cross-reactive material in the body fluid with the molecule
indicates neoangiogenesis in the patient.
55. A method of promoting neoangiogenesis in a patient, comprising:
administering to a patient in need of neoangiogenesis a TEM protein
selected from the group consisting of: 4, 6, 7, 10, 12, 14, 20, 25,
27, 31, 36, 37, 38, 39, and 40, as shown in SEQ ID NO: 202, 206,
208, 214, 218, 223 & 224, 234, 242, 244, 252, 257, 259, 261.
263, and 265, whereby neoangiogenesis in the patient is
stimulated.
56. A method of promoting neoangiogenesis in a patient, comprising:
administering to a patient in need of neoangiogenesis a nucleic
acid molecule encoding a TEM protein selected from the group
consisting of: 4, 6, 7, 10, 12, 14, 20, 25, 27, 31, 36, 37, 38, 39,
and 40, as shown in SEQ ID NO: 202, 206, 208, 214, 218, 223 &
224, 234, 242, 244, 252, 257, 259, 261. 263, and 265, whereby the
TEM protein is expressed and neoangiogenesis in the patient is
stimulated.
57. A method of screening for neoangiogenesis in a patient,
comprising: detecting a TEM protein selected from the group
consisting of: 4, 6, 7, 10, 12, 14, 20, 25, 27, 31, 36, 37, 38, 39,
and 40, as shown in SEQ ID NO: 202, 206, 208, 214, 218, 223 &
224, 234, 242, 244, 252, 257, 259, 261. 263, and 265, respectively,
in a body fluid collected from the patient, wherein detection of
the TEM protein indicates neoangiogenesis in the patient.
58. A method of screening for neoangiogenesis in a patient,
comprising: detecting in a body fluid collected from the patient a
nucleic acid encoding a TEM protein selected from the group
consisting of: 4, 6, 7, 10, 12, 14, 20, 25, 27, 31, 36, 37, 38, 39,
and 40, wherein the nucleic acid is selected from the group
consisting of those shown in SEQ ID NO: 201, 205, 207, 213, 217,
221 & 222, 233, 241, 243, 251, 256, 258, 260, 262, and 264,
respectively, wherein detection of the TEM protein indicates
neoangiogenesis in the patient.
59. An isolated and purified nucleic acid molecule which encodes a
NEM protein selected from the group consisting of: 14, 22, 23, and
33 as shown in SEQ ID NO: 279, 283, 285, 286, 287, and 289.
60. The nucleic acid molecule of claim 60 wherein the nucleic acid
molecule comprises a coding sequence as shown in SEQ ID NO: 278,
282, 284, and 288.
61. A recombinant host cell which comprises a nucleic acid
according to claim 60.
62. An isolated and purified NEM protein selected from the group
consisting of: 14, 22, 23, and 33 as shown in SEQ ID NO: 279, 283,
285, 286, 287, and 289, respectively.
63. An isolated molecule comprising an antibody variable region
which specifically binds to a NEM protein selected from the group
consisting of: 14, 22, 23, and 33, as shown in SEQ ID NO: 279, 283,
285, 286, 287, and 289.
64. A method of inhibiting neoangiogenesis, comprising:
administering to a subject in need thereof an effective amount of a
NEM protein selected from the group consisting of: 14, 22, 23, and
33 as shown in SEQ ID NO: 279, 283, 285, 286, 287, and 289, whereby
neoangiogenesis is inhibited.
65. A method to identify candidate drugs for treating tumors,
comprising: contacting cells which express one or more TEM genes
selected from the group consisting of: 1, 2, 4, 5, 6, 7, 8, 9, 10,
11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 30, 31,
33, 35, 36, 37, 38, 39, 41, 42, 44, 45, and 46 as shown in SEQ ID
NO: 195, 197, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221
& 222, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,
247, 249, 251, 253, 255, 256, 258, 260, 262, 266, 268, 270, 272,
and 274, respectively, with a test compound; determining expression
of said one or more TEM genes by hybridization of mRNA of said
cells to a nucleic acid probe which is complementary to said mRNA;
and identifying a test compound as a candidate drug for treating
tumors if it decreases expression of said one or more TEM
genes.
66. The method of claim 66 wherein the cells are endothelial
cells.
67. The method of claim 66 wherein the cells are recombinant host
cells which are transfected with an expression construct which
encodes said one or more TEMs.
68. A method to identify candidate drugs for treating tumors,
comprising: contacting cells which express one or more TEM proteins
selected from the group consisting of: 2, 4, 5, 6, 7, 8, 9, 10, 11,
12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 30, 31, 33,
35, 36, 37, 38, 39, 41, 42, 44, 45, and 46 as shown in SEQ ID NO:
198, 202, 204, 206, 208, 210, 212, 214, 216, 218, 223 & 224,
226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,
252, 254, 358, 257, 259, 261, 263, 267, 269, 271, 273, and 275,
respectively, with a test compound; determining amount of said one
or more TEM proteins in said cells; and identifying a test compound
as a candidate drug for treating tumors if it decreases the amount
of one more TEM proteins in said cells.
69. The method of claim 69 wherein the cells are endothelial
cells.
70. The method of claim 69 wherein the cells are recombinant host
cells which are transfected with an expression construct which
encodes said one or more TEMs.
71. A method to identify candidate drugs for treating tumors,
comprising: contacting cells which express one or more TEM proteins
selected from the group consisting of: 2, 4, 5, 6, 7, 8, 9, 10, 11,
12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 40, 31, 33,
35, 36, 37, 38, 39, 41, 42, 44, 45, and 46 as shown in SEQ ID NO:
198, 202, 204, 206, 208, 210, 212, 214, 216, 218, 223 & 224,
226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250,
252, 254, 358, 257, 259, 261, 263, 267, 269, 271, 273, and 275
respectively, with a test compound; determining activity of said
one or more TEM proteins in said cells; and identifying a test
compound as a candidate drug for treating tumors if it decreases
the activity of one more TEM proteins in said cells.
72. The method of claim 72 wherein the cells are endothelial
cells.
73. The method of claim 72 wherein the cells are recombinant host
cells which are transfected with an expression construct which
encodes said one or more TEMs.
74. A method to identify candidate drugs for treating patients
bearing tumors, comprising: contacting a test compound with
recombinant host cells which are transfected with an expression
construct which encodes one or more TEM proteins selected from the
group consisting of 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,
17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 40, 31, 33, 35, 36, 37, 38,
39, 41, 42, 44, 45, and 46 as shown in SEQ ID NO: 198, 202, 204,
206, 208, 210, 212, 214, 216, 218, 223 & 224, 226, 228, 230,
232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 358,
257, 259, 261, 263, 267, 269, 271, 273, and 275, respectively;
determining proliferation of said cells; and identifying a test
compound which inhibits proliferation of said cells as a candidate
drug for treating patients bearing tumors.
75. A method to identify candidate drugs for treating tumors,
comprising: contacting cells which express one or more NEM genes
selected from the group consisting of: 14, 22, 23, and 33 as shown
in SEQ ID NO: 278, 282, 284, and 288, respectively, with a test
compound; determining expression of said one or more NEM genes by
hybridization of mRNA of said cells to a nucleic acid probe which
is complementary to said mRNA; and identifying a test compound as a
candidate drug for treating tumors if it increases expression of
said one or more NEM genes.
76. The method of claim 76 wherein the cells are endothelial
cells.
77. The method of claim 76 wherein the cells are recombinant host
cells which are transfected with an expression construct which
encodes said one or more NEMs.
78. A method to identify candidate drugs for treating tumors,
comprising: contacting cells which express one or more NEM proteins
selected from the group consisting of: 14, 22, 23, and 33 as shown
in SEQ ID NO: 279, 283, 285, 286, 287, and 289, with a test
compound; determining amount of said one or more NEM proteins in
said cells; and identifying a test compound as a candidate drug for
treating tumors if it increases the amount of one more NEM proteins
in said cells.
79. The method of claim 79 wherein the cells are endothelial
cells.
80. The method of claim 79 wherein the cells are recombinant host
cells which are transfected with an expression construct which
encodes said one or more NEMs.
81. A method to identify candidate drugs for treating tumors,
comprising: contacting cells which express one or more NEM proteins
selected from the group consisting of: 14, 22, 23, and 33 as shown
in SEQ ID NO: 279, 283, 285, 286, 287, and 289, with a test
compound; determining activity of said one or more NEM proteins in
said cells; and identifying a test compound as a candidate drug for
treating tumors if it increases the activity of one more NEM
proteins in said cells.
82. The method of claim 82 wherein the cells are endothelial
cells.
83. The method of claim 82 wherein the cells are recombinant host
cells which are transfected with an expression construct which
encodes said one or more NEMs.
84. A method to identify candidate drugs for treating patients
bearing tumors, comprising: contacting a test compound with
recombinant host cells which are transfected with an expression
construct which encodes one or more NEM proteins selected from the
group consisting of 14, 22, 23, and 33 as shown in SEQ ID NO: 279,
283, 285, 286, 287, and 289; determining proliferation of said
cells; and identifying a test compound which stimulates
proliferation of said cells as a candidate drug for treating
patients bearing tumors.
85. A method for identification of a ligand involved in endothelial
cell regulation, comprising: contacting a test compound with a
human transmembrane TEM protein selected from the group consisting
of 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21,
22, 24, 25, 27, 28, 29, 40, 31, 33, 35, 36, 37, 38, 39, 41, 42, 44,
45, and 46 as shown in SEQ ID NO: 196, 198, 202, 204, 206, 208,
210, 212, 214, 216, 218, 223 & 224, 226, 228, 230, 232, 234,
236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 358, 257, 259,
261, 263, 267, 269, 271, 273, and 275; determining binding of a
test compound to the human transmembrane protein, wherein a test
compound which binds to the protein is identified as a ligand
involved in endothelial cell regulation.
86. A method of inducing an immune response in a mammal,
comprising: administering to the mammal a cell which expresses a
transmembrane protein selected from the group consisting of: TEM 1,
9, 13, 17, 19, 22, 30, and 44 as shown in SEQ ID NO: 196, 212, 220,
230, 232, 238, 250 and 271, respectively, wherein the cell is a
recombinant cell which comprises a vector encoding said
transmembrane protein, or the cell is a of a dendritic cell and a
tumor endothelium cell, whereby an immune response to the human
transmembrane protein is induced in the mammal.
Description
[0001] This application claims the benefit of provisional
application Ser. Nos. 60/222,599 filed Aug. 2, 2000, 60/224,360
filed Aug. 11, 2000, and 60/282,850 filed Apr. 11, 2001, the
disclosures of which are expressly incorporated herein.
TECHNICAL FIELD OF THE INVENTION
[0003] This invention is related to the area of angiogenesis and
anti-angiogenesis. In particular, it relates to genes which are
characteristically expressed in tumor endothelial and normal
endothelial cells.
BACKGROUND OF THE INVENTION
[0004] It is now widely recognized that tumors require a blood
supply for expansive growth. This recognition has stimulated a
profusion of research on tumor angiogenesis, based on the idea that
the vasculature in tumors represents a potential therapeutic
target. However, several basic questions about tumor endothelium
remain unanswered. For example, are vessels of tumors qualitatively
different from normal vessels of the same tissue? What is the
relationship of tumor endothelium to endothelium of healing wounds
or other physiological or pathological forms of angiogenesis? The
answers to these questions critically impact on the potential for
new therapeutic approaches to inhibit angiogenesis in a specific
manner.
[0005] There is a continuing need in the art to characterize the
vasculature of tumors relative to normal vasculature so that any
differences can be exploited for therapeutic and diagnostic
benefits.
[0006] One technique which can be used to characterize gene
expression, or more precisely gene transcription, is termed serial
analysis of gene expression (SAGE). Briefly, the SAGE approach is a
method for the rapid quantitative and qualitative analysis of mRNA
transcripts based upon the isolation and analysis of short defined
sequence tags (SAGE Tags) corresponding to expressed genes. Each
Tag is a short nucleotide sequences (9-17 base pairs in length)
from a defined position in the transcript. In the SAGE method, the
Tags are dimerized to reduce bias inherent in cloning or
amplification reactions. (See, U.S. Pat. No. 5,695,937) SAGE is
particularly suited to the characterization of genes associated
with vasculature stimulation or inhibition because it is capable of
detecting rare sequences, evaluating large numbers of sequences at
one time, and to provide a basis for the identification of
previously unknown genes.
SUMMARY OF THE INVENTION
[0007] One embodiment of the invention provides an isolated
molecule comprising an antibody variable region which specifically
binds to an extracellular domain of a TEM protein selected from the
group consisting of: 1, 3, 9, 17, 19, and 44, as shown in SEQ ID
NO: 196, 200, 212, 230, 232, and 271, respectively. The molecule
can be, for example, an in tact antibody molecule, a single chain
variable region (ScFv), a monoclonal antibody, a humanized
antibody, or a human antibody. The molecule can optionally be bound
to a cytotoxic moiety, bound to a therapeutic moiety, bound to a
detectable moiety, or bound to an anti-tumor agent.
[0008] According to another embodiment of the invention a method of
inhibiting neoangiogenesis is provided. An effective amount of an
isolated molecule comprising an antibody variable region which
specifically binds to an extracellular domain of a TEM protein
selected from the group consisting of: 1, 3, 9, 17, 19, 22, and 44,
as shown in SEQ ID NO: 196, 200, 212, 230, 232, 238, and 271,
respectively, is administered to a subject in need thereof.
Neoangiogenesis is consequently inhibited. The subject may bear a
vascularized tumor, may have polycystic kidney disease, may have
diabetic retinopathy, may have rheumatoid arthritis, may have
psoriasis, for example.
[0009] Another aspect of the invention is a method of inhibiting
tumor growth. An effective amount of an isolated molecule
comprising an antibody variable region which specifically binds to
an extracellular domain of a TEM protein selected from the group
consisting of: 1, 3, 9, 17, 19, 22, and 44, as shown in SEQ ID NO:
196, 200, 212, 230, 232, 238, and 271, respectively, is
administered to a human subject bearing a tumor. The growth of the
tumor is consequently inhibited.
[0010] Still another aspect of the invention provides an isolated
molecule comprising an antibody variable region which specifically
binds to a TEM protein selected from the group consisting of: 3, 9,
17, 19, and 44, as shown in SEQ ID NO: 200, 212, 230, 232, and 271,
respectively. The molecule can be, for example, an in tact antibody
molecule, a single chain variable region (ScFv), a monoclonal
antibody, a humanized antibody, or a human antibody. The molecule
can optionally be bound to a cytotoxic moiety, bound to a
therapeutic moiety, bound to a detectable moiety, or bound to an
anti-tumor agent.
[0011] According to still another aspect of the invention an
isolated and purified human transmembrane protein is provided. The
protein is selected from the group consisting of: TEM 3, 9, 17, and
19 as shown in SEQ ID NO: 200, 212, 230, and 232, respectively.
[0012] Yet another aspect of the invention is an isolated and
purified nucleic acid molecule comprising a coding sequence for a
transmembrane TEM selected from the group consisting of:: TEM 3, 9,
17, and 19 as shown in SEQ ID NO: 200, 212, 230, and 232,
respectively. The isolated and purified nucleic acid molecule may
optionally comprise a coding sequence selected from those shown in
SEQ ID NO:: 199, 211, 229, and 231.
[0013] Still another aspect of the invention is a recombinant host
cell which comprises a nucleic acid molecule. The nucleic acid
molecule comprises a coding sequence for a transmembrane TEM
selected from the group consisting of:: TEM 3, 9, 17, and 19 as
shown in SEQ ID NO: 200, 212, 230, and 232, respectively. The
recombinant host cell optionally comprises a coding sequence
selected from those shown in SEQ ID NO: 199, 211, 229, and 231.
[0014] According to one embodiment of the invention a method is
provided for inducing an immune response in a mammal. A nucleic
acid molecule comprising a coding sequence for a human
transmembrane protein selected from the group consisting of: TEM 1,
3, 9, 13, 17, 19, 22, 30, and 44 as shown in SEQ ID NO:
respectively, is administered to the mammal. An immune response to
the human transmembrane protein is thereby induced in the mammal.
Optionally the coding sequence is shown in SEQ ID NO: 196, 200,
212, 220, 230, 232, 238, 250 and 271.
[0015] According to yet another embodiment of the invention a
method of inducing an immune response in a mammal is provided. A
purified human transmembrane protein selected from the group
consisting of: TEM 1, 3, 9, 13, 17, 19, 22, 30, and 44 as shown in
SEQ ID NO: 196, 200, 212, 220, 230, 232, 238, 250 and 271,
respectively, is administered to the mammal. An immune response to
the human transmembrane protein is thereby induced in the
mammal.
[0016] Another aspect of the invention is a method for
identification of a ligand involved in endothelial cell regulation.
A test compound is contacted with an isolated and purified human
transmembrane protein selected from the group consisting of 1, 3,
9, 13, 17, 30, 19, and 44 as shown in SEQ ID NO: 196, 200, 212,
220, 230, 232, 250, and 271. The isolated and purified human
transmembrane protein is also contacted with a molecule comprising
an antibody variable region which specifically binds to an
extracellular domain of a TEM protein selected from the group
consisting of: 1, 3, 9, 13, 17, 30, 19, and 44 as shown in SEQ ID
NO: 196, 200, 212, 220, 230, 232, 250, and 271 respectively.
Binding of the molecule comprising an antibody variable region to
the human transmembrane protein is determined. A test compound
which diminishes the binding of the molecule comprising an antibody
variable region to the human transmembrane protein is identified as
a ligand involved in endothelial cell regulation.
[0017] Yet another aspect of the invention is a method for
identification of a ligand involved in endothelial cell regulation.
A test compound is contacted with a cell comprising a human
transmembrane protein selected from the group consisting of 1, 3,
9, 17, and 19 as shown in SEQ ID NO: 196, 200, 212, 230, and 232.
The cell is also contacted with a molecule comprising an antibody
variable region which specifically binds to an extracellular domain
of a TEM protein selected from the group consisting of: 1, 3, 9,
17, and 19 as shown in SEQ ID NO: 196, 200, 212, 230, and 232,
respectively. Binding of the molecule comprising an antibody
variable region to the cell is determined. A test compound which
diminishes the binding of the molecule comprising an antibody
variable region to the cell is identified as a ligand involved in
endothelial cell regulation.
[0018] Yet another aspect of the invention is a method for
identification of a ligand involved in endothelial cell regulation.
A test compound is contacted with a human transmembrane protein
selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 40,
31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45, and 46 as shown in SEQ
ID NO: 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218,
223 & 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244,
246, 248, 250, 252, 254, 358, 257, 259, 261, 263, 267, 269, 271,
273, and 275. Binding of a test compound to the human transmembrane
protein is determined. A test compound which binds to the protein
is identified as a ligand involved in endothelial cell
regulation.
[0019] Another embodiment of the present invention is a soluble
form of a human transmembrane protein selected from the group
consisting of: TEM 1, 3, 9, 17, 19, 22, 30, and 44 as shown in SEQ
ID NO: 196, 200, 212, 230, 232, 238, 250, and 271 respectively. The
soluble forms lack transmembrane domains. The soluble form may
consist of an extracellular domain of the human transmembrane
protein.
[0020] Also provided by the present invention is a method of
inhibiting neoangiogenesis in a patient. A soluble form of a human
transmembrane protein is adminstered to the patient.
Neoangiogenesis in the patient is consequently inhibited. The
patient may bear a vascularized tumor, may have polycystic kidney
disease, may have diabetic retinopathy, may have rheumatoid
arthritis, or may have psoriasis, for example.
[0021] Another embodiment of the invention provides a method of
inhibiting neoangiogenesis in a patient. A soluble form of a human
transmembrane protein is administered to the patient.
Neoangiogenesis in the patient is consequently inhibited. The
patient may bear a vascularized tumor, may have polycystic kidney
disease, may have diabetic retinopathy, may have rheumatoid
arthritis, or may have psoriasis, for example.
[0022] According to still another aspect of the invention a method
of identifying regions of neoangiogenesis in a patient is provided.
A molecule comprising an antibody variable region which
specifically binds to an extracellular domain of a TEM protein
selected from the group consisting of: 1, 3, 9, 13, 17, 19, 22, 30,
and 44, as shown in SEQ ID NO: 196, 200, 212, 220, 230, 232, 238,
250, and 271, respectively, is administered to a patient. The
molecule is bound to a detectable moiety. The detectable moiety is
detected in the patient, thereby identifying neoangiogenesis.
[0023] According to another aspect of the invention a method is
provided for inducing an immune response to tumor endothelial cells
in a patient. A mouse TEM protein selected from the group
consisting of: 1, 2, 3, 9, 13, 17, 19, 22, and 30 as shown in SEQ
ID NO: 291, 293, 299, 295, 303, 297, 301, 305, and 307, is
administered to a patient in need thereof. An immune response to a
human TEM protein is consequently induced.
[0024] Still another embodiment of the invention is a method of
screening for neoangiogenesis in a patient. A body fluid collected
from the patient is contacted with a molecule comprising an
antibody variable region which specifically binds to an
extracellular domain of a TEM protein selected from the group
consisting of: 1, 3, 9, 17, 19, and 44, as shown in SEQ ID NO: 196,
200, 212, 230, 232, and 271, respectively. Detection of
cross-reactive material in the body fluid with the molecule
indicates neo-angiogenesis in the patient.
[0025] Still another embodiment of the invention provides a method
of inhibiting neoangiogenesis in a patient. A molecule comprising
an antibody variable region which specifically binds to a TEM
protein selected from the group consisting of: 4, 6, 7, 10, 12, 14,
20, 25, 27, 31, 36, 37, 38, 39, and 40 as shown in SEQ ID NO: 202,
206, 208, 214, 218, 223 and 224, 234, 242, 244, 252, 257, 259, 261.
263, and 265, is administered to the patient. Neoangiogenesis in
the patient consequently inhibited.
[0026] Yet another aspect of the invention is a method of screening
for neoangiogenesis in a patient. A body fluid collected from the
patient is contacted with a molecule comprising an antibody
variable region which specifically binds to a TEM protein selected
from the group consisting of: 4, 6, 7, 10, 12, 14, 20, 25, 27, 31,
36, 37, 38, 39, and 40, as shown in SEQ ID NO: 202, 206, 208, 214,
218, 223 & 224, 234, 242, 244, 252, 257, 259, 261. 263, and
265, respectively. Detection of cross-reactive material in the body
fluid with the molecule indicates neoangiogenesis in the
patient.
[0027] Also provided by the present invention is a method of
promoting neoangiogenesis in a patient. A TEM protein selected from
the group consisting of: 4, 6, 7, 10, 12, 14, 20, 25, 27, 31, 36,
37, 38, 39, and 40, as shown in SEQ ID NO: 202, 206, 208, 214, 218,
223 & 224, 234, 242, 244, 252, 257, 259, 261. 263, and 265, is
administered to a patient in need of neoangiogenesis.
Neoangiogenesis in the patient is consequently stimulated.
[0028] One embodiment of the invention provides a method of
promoting neoangiogenesis in a patient. A nucleic acid molecule
encoding a TEM protein selected from the group consisting of: 4, 6,
7, 10, 12, 14, 20, 25, 27, 31, 36, 37, 38, 39, and 40, as shown in
SEQ ID NO: 201, 205, 207, 213, 217, 221 & 222, 233, 241, 243,
251, 256, 258, 260, 262, and 264, is administered to a patient in
need of neoangiogenesis. The TEM protein is consequently expressed
and neoangiogenesis in the patient is stimulated.
[0029] Another embodiment of the invention provides a method of
screening for neoangiogenesis in a patient. A TEM protein selected
from the group consisting of: 4, 6, 7, 10, 12, 14, 20, 25, 27, 31,
36, 37, 38, 39, and 40, as shown in SEQ ID NO:: 202, 206, 208, 214,
218, 223 & 224, 234, 242, 244, 252, 257, 259, 261. 263, and
265, respectively, is detected in a body fluid collected from the
patient. Detection of the TEM protein indicates neoangiogenesis in
the patient.
[0030] Another aspect of the invention is a method of screening for
neoangiogenesis in a patient. A nucleic acid encoding a TEM protein
selected from the group consisting of: 4, 6, 7, 10, 12, 14, 20, 25,
27, 31, 36, 37, 38, 39, and 40 is detected in a body fluid
collected from the patient. The nucleic acid is selected from the
group consisting of those shown in SEQ ID NO: 201, 205, 207, 213,
217, 221 & 222, 233, 241, 243, 251, 256, 258, 260, 262, and
264. Detection of the TEM protein indicates neoangiogenesis in the
patient.
[0031] Yet another embodiment of the invention is an isolated and
purified nucleic acid molecule which encodes a NEM protein selected
from the group consisting of: 14, 22, 23, and 33 as shown in SEQ ID
NO: 279, 283, 285, 286, 287, and 289. The nucleic acid molecule
optionally comprises a coding sequence as shown in SEQ ID NO: 278,
282, 284, and 288. The nucleic acid may be maintained in a
recombinant host cell.
[0032] The present invention also provides an isolated and purified
NEM protein selected from the group consisting of: 14, 22, 23, and
33 as shown in SEQ ID NO: 279, 283, 285, 286, 287, and 289.
[0033] The present invention further provides an isolated molecule
comprising an antibody variable region which specifically binds to
a NEM protein selected from the group consisting of: 14, 22, 23,
and 33, as shown in SEQ ID NO: 279, 283, 285, 286, 287, and
289.
[0034] An additional embodiment of the present invention is a
method of inhibiting neoangiogenesis. An effective amount of a NEM
protein selected from the group consisting of: 14, 22, 23, and 33
as shown in SEQ ID NO: 279, 283, 285, 286, 287, and 289 is
administered to a subject in need thereof. Neoangiogenesis is
thereby inhibited.
[0035] A still further embodiment of the invention is a method to
identify candidate drugs for treating tumors. Cells which express
one or more TEM genes selected from the group consisting of: 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22,
24, 25, 27, 28, 29, 40, 31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45,
and 46 as shown in SEQ ID NO:: 195, 197, 199, 201, 203, 205, 207,
209, 211, 213, 215, 217, 221 & 222, 225, 227, 229, 231, 233,
235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 256, 258,
260, 262, 266, 268, 270, 272, and 274, respectively, are contacted
with a test compound. Expression of said one or more TEM genes is
determined by hybridization of mRNA of said cells to a nucleic acid
probe which is complementary to said mRNA. A test compound is
identified as a candidate drug for treating tumors if it decreases
expression of said one or more TEM genes. Optionally the cells are
endothelial cells. Alternatively or additionally, the cells are
recombinant host cells which are transfected with an expression
construct which encodes said one or more TEMs. Test compounds which
increase expression can be identified as candidates for promoting
wound healing.
[0036] Yet another embodiment of the invention is a method to
identify candidate drugs for treating tumors. Cells which express
one or more TEM proteins selected from the group consisting of: 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22,
24, 25, 27, 28, 29, 40, 31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45,
and 46 as shown in SEQ ID NO: 196, 198, 200, 202, 204, 206, 208,
210, 212, 214, 216, 218, 223 & 224, 226, 228, 230, 232, 234,
236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 358, 257, 259,
261, 263, 267, 269, 271, 273, and 275, respectively, are contacted
with a test compound. The amount of said one or more TEM proteins
in said cells is determined. A test compound is identified as a
candidate drug for treating tumors if it decreases the amount of
one or more TEM proteins in said cells. Optionally the cells are
endothelial cells. Alternatively or additionally, the cells are
recombinant host cells which are transfected with an expression
construct which encodes said one or more TEMs. Alternatively, a
test compound which increases the amount of one or more TEM
proteins in said cells is identified as a candidate drug for
treating wound healing.
[0037] According to another aspect of the invention a method is
provided to identify candidate drugs for treating tumors. Cells
which express one or more TEM proteins selected from the group
consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,
17, 19, 20, 21, 22, 24, 25, 27, 28, 29, 40, 31, 33, 35, 36, 37, 38,
39, 41, 42, 44, 45, and 46 as shown in SEQ ID NO: 196, 198, 200,
202, 204, 206, 208, 210, 212, 214, 216, 218, 223 & 224, 226,
228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252,
254, 358, 257, 259, 261, 263, 267, 269, 271, 273, and 275,
respectively, are contacted with a test compound. Activity of said
one or more TEM proteins in said cells is determined. A test
compound is identified as a candidate drug for treating tumors if
it decreases the activity of one more TEM proteins in said cells.
Optionally the cells are endothelial cells. Alternatively or
additionally, the cells are recombinant host cells which are
transfected with an expression construct which encodes said one or
more TEMs. Optionally the cells are endothelial cells. If a test
compound increases the activity of one more TEM proteins in said
cells it can be identified as a candidate drug for treating wound
healing.
[0038] An additional aspect of the invention is a method to
identify candidate drugs for treating patients bearing tumors. A
test compound is contacted with recombinant host cells which are
transfected with an expression construct which encodes one or more
TEM proteins selected from the group consisting of 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22, 24, 25, 27,
28, 29, 40, 31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45, and 46 as
shown in SEQ ID NO: 198, 200, 202, 204, 206, 208, 210, 212, 214,
216, 218, 223 & 224, 226, 228, 230, 232, 234, 236, 238, 240,
242, 244, 246, 248, 250, 252, 254, 358, 257, 259, 261, 263, 267,
269, 271, 273, and 275, respectively. Proliferation of said cells
is determined. A test compound which inhibits proliferation of said
cells is identified as a candidate drug for treating patients
bearing tumors. A test compound which stimulates proliferation of
said cells is identified as a candidate drug for promoting
neoangiogenesis, such as for use in wound healing.
[0039] Another embodiment of the invention provides a method to
identify candidate drugs for treating tumors. Cells which express
one or more NEM genes selected from the group consisting of: 14,
22, 23, and 33 as shown in SEQ ID NO: 278, 282, 284, and 288,
respectively, are contacted with a test compound. Expression of
said one or more NEM genes is determined by hybridization of mRNA
of said cells to a nucleic acid probe which is complementary to
said mRNA. A test compound is identified as a candidate drug for
treating tumors if it increases expression of said one or more NEM
genes. Optionally the cells are endothelial cells. Alternatively or
additionally, the cells are recombinant host cells which are
transfected with an expression construct which encodes said one or
more NEMs.
[0040] According to another aspect of the invention a method is
provided to identify candidate drugs for treating tumors. Cells
which express one or more NEM proteins selected from the group
consisting of: 14, 22, 23, and 33 as shown in SEQ ID NO: 279, 283,
285, 286, 287, and 289, are contacted with a test compound. The
amount of said one or more NEM proteins in said cells is
determined. A test compound is identified as a candidate drug for
treating tumors if it increases the amount of one more NEM proteins
in said cells. Optionally the cells are endothelial cells.
Alternatively or additionally, the cells are recombinant host cells
which are transfected with an expression construct which encodes
said one or more NEMs.
[0041] An additional aspect of the invention is a method to
identify candidate drugs for treating tumors. Cells which express
one or more NEM proteins selected from the group consisting of: 14,
22, 23, and 33 as shown in SEQ ID NO: 279, 283, 285, 286, 287, and
289, are contacted with a test compound. Activity of said one or
more NEM proteins in said cells is determined. A test compound is
identified as a candidate drug for treating tumors if it increases
the activity of said one or more NEM proteins in said cells.
Optionally the cells are endothelial cells. Alternatively or
additionally, the cells are recombinant host cells which are
transfected with an expression construct which encodes said one or
more NEMs.
[0042] Still another embodiment of the invention provides a method
to identify candidate drugs for treating patients bearing tumors. A
test compound is contacted with recombinant host cells which are
transfected with an expression construct which encodes one or more
NEM proteins selected from the group consisting of 14, 22, 23, and
33 as shown in SEQ ID NO: 279, 283, 285, 286, 287, and 289.
Proliferation of said cells is determined. A test compound which
stimulates proliferation of said cells is identified as a candidate
drug for treating patients bearing tumors.
[0043] Another aspect of the invention is a method for identifying
endothelial cells. One or more antibodies which bind specifically
to a TEM or NEM protein selected from the group consisting of TEM:
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22,
24, 25, 27, 28, 29, 30, 31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45,
and 46 as shown in SEQ ID NO: 198, 200, 202, 204, 206, 208, 210,
212, 214, 216, 218, 223 & 224, 226, 228, 230, 232, 234, 236,
238, 240, 242, 244, 246, 248, 250, 252, 254, 358, 257, 259, 261,
263, 267, 269, 271, 273, and 275 and NEM 14, 22, 23, and 33 as
shown in SEQ ID NO: 279, 283, 285, 286, 287, and 289, is contacted
with a population of cells. Cells in the population which have
bound to said antibodies are detected. Cells which are bound to
said antibodies are identified as endothelial cells. Optionally
cells which have bound to said antibodies are isolated from cells
which have not bound.
[0044] Still another aspect of the invention is a method for
identifying endothelial cells. One or more nucleic acid
hybridization probes which are complementary to a TEM or NEM gene
nucleic acid sequence selected from the group consisting of TEM: 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 19, 20, 21, 22,
24, 25, 27, 28, 29, 30, 31, 33, 35, 36, 37, 38, 39, 41, 42, 44, 45,
and 46 as shown in SEQ ID NO: 198, 200, 202, 204, 206, 208, 210,
212, 214, 216, 218, 223 & 224, 226, 228, 230, 232, 234, 236,
238, 240, 242, 244, 246, 248, 250, 252, 254, 358, 257, 259, 261,
263, 267, 269, 271, 273, and 275 and NEM 14, 22, 23, and 33 as
shown in SEQ ID NO: 279, 283, 285, 286, 287, and 289, is contacted
with nucleic acids of a population of cells. Nucleic acids which
have specifically hybridized to said nucleic acid hybridization
probes are detected. Cells whose nucleic acids specifically
hybridized are identified as endothelial cells.
[0045] Yet another embodiment of the invention is a method of
inhibiting neoangiogenesis. An effective amount of an isolated
molecule comprising an antibody variable region which specifically
binds to an extracellular domain of a mouse TEM protein selected
from the group consisting of: 1, 2, 3, 9, 17, and 19, as shown in
SEQ ID NO: 291, 293, 299, 295, 297, and 301, respectively, is
administered to a subject in need thereof. Neoangiogenesis is
thereby inhibited. The subject may be a mouse, may bear a
vascularized tumor, may have polycystic kidney disease, may have
diabetic retinopathy may have rheumatoid arthritis, or may have
psoriasis, for example.
[0046] These and other embodiments which will be apparent to those
of skill in the art upon reading the specification provide the art
with reagents and methods for detection, diagnosis, therapy, and
drug screening pertaining to neoangiogenesis and pathological
processes involving or requiring neoangiogenesis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1A-1B. vWF expression in colorectal cancers. vWF (red
stain) was detected in vessels by in situ hybridization. At low
power magnification (FIG. 1.A) vessels were often surrounded by a
perivascular cuff of viable cells (red arrows), with a ring of
necrotic cells evident at the periphery (black arrows). At high
power magnification (FIG. 1.B) the expression of vWF (red) was
clearly localized to the vessels. Sections were counterstained with
methyl green.
[0048] FIG. 2A-2D. Purification of Endothelial Cells (ECs) from
human normal and malignant tissue. (FIG. 2A) Vessels (red) of
frozen sections were stained by immunofluorescence with the P1H12
monoclonal antibody (Chemicon, Temecula, Calif.) and detected using
a biotinylated goat anti-mouse IgG secondary antibody followed by
rhodamine-linked strepavidin. The region stained is from within the
lamina propria of normal colonic mucosa. Note that the larger
vessels (arrowheads) and capillaries (arrows) are positive, and
staining of hematopoietic cells was undetectable. E-cadherin
positive epithelial cells (green) at the edge of the crypt were
simultaneously visualized using a rabbit polyclonal antibody (Santa
Cruz, Santa Cruz, Calif.), followed by a goat anti-rabbit IgG
secondary antibody labelled with alexa (Molecular Probes, Eugene,
Oreg.). Sections were imaged at 60.times. magnification using
confocal microscopy. (FIG. 2.B) To isolate pure populations from
collagenase dispersed tissues, the epithelial and hematopoietic
cell fractions were sequentially removed by negative selection with
magnetic beads. The remaining cells were stained with P1H12 and ECs
were isolated by positive selection with magnetic beads. (FIG. 2.C)
RT-PCR analysis used to assess the purity of the EC preparations.
Semiquantitative PCR analysis was performed on cDNA generated
either directly from colorectal cancer tissue (unfractionated
tumor) or from purified ECs isolated from normal colonic mucosa
(normal EC fraction) or colorectal cancer (tumor EC fraction). PCR
amplification of the epithelial specific marker cytokeratin 20
(CK20), demonstrated its expression was limited to the
unfractionated tumor. Two endothelial specific markers, vWF and
VE-cadherin (VE-Cad) showed robust amplification only in the
endothelial fractions, validating the purity and enrichment
protocol shown in (FIG. 2.B). The ubiquitous housekeeping enzyme
GAPDH was observed in all samples. No signal was detected in the
no-template (NT) control. cDNA templates were diluted 1:10, 1:100,
1:1000, 1:4000, and 1:40,000 as indicated by the declining wedge.
(FIG. 2.D) The relative expression level of select genes was
determined by measuring the tag abundance from several SAGE
libraries combined into four groups. The first was composed of
.about.193,000 tags from the two in vivo-derived EC preparations
(Endothelial Cell Fraction) while the second contained a single
library of .about.57,000 tags containing macrophages and other
leukocytes derived from the negative selection (Hematopoietic
Fraction). The fourth library contained .about.401,000 tags from
cultured HUVEC and HMVEC (Endothelial Cells in Culture), and the
fourth consisted of 748,000 tags from 6 colon cancer cell lines in
culture (Epithelial Cells). After normalization, the library with
the highest tag number for each marker was given a value of 100%,
and the corresponding relative expression levels of the remaining 3
libraries was plotted on the ordinate. Note the high level of CD31
present on hematopoietic cells, the likely cause of the impurity of
the initial endothelial selection, compared with the selectivity of
P1H12.
[0049] FIG. 3A-3E). Expression of Pan-Endothelial Markers (PEMs) is
limited to ECs. The endothelial origin of PEMs identified by SAGE
was confirmed using a highly sensitive in situ hybridization assay.
Localization of novel PEMs to the ECs was demonstrated by examining
two representative PEMs, PEM3 (FIG. 3A) and PEM6 (FIG. 3B) in lung
cancer and colon cancer, respectively. Hevin expression was readily
detected in the ECs of a colon tumor (FIG. 3C) despite its low
level of expression in cultured ECs. Expression of VEGFR2 was
readily detectable in the ECs of both normal (FIG. 3D) and
malignant colon tissue (FIG. 3E).
[0050] FIG. 4A-4J. Expression of Tumor Endothelial Markers (TEMs).
(FIG. 4A) RT-PCR analysis confirmed the tumor specific expression
of selected novel TEMs. Semiquantitative PCR analysis was performed
on cDNA generated either from purified epithelial cells as a
negative control (Control) or from purified ECs isolated from
normal colonic mucosa (Normal ECs) or colorectal cancer (Tumor ECs)
from two different patients. Two endothelial specific markers, vWF
and PEM6 showed robust amplification only in the endothelial
fractions whereas the ubiquitous housekeeping enzyme GAPDH was
observed in all samples. TEM1 (BSC-TEM1), TEM 17 (BSC-TEM7) and
TEM22 (BSC-TEM9) were specifically expressed in tumor compared to
normal ECs. The cDNA template was diluted 1:10, 1:100, 1:1000, and
1:10,000 as indicated by the declining wedge. (FIG. 4 B-4J) The
endothelial origin of TEMs identified by SAGE was confirmed using
in situ hybridization as in FIG. 3. Expression of TEM 1 (BSC-TEM1)
(FIG. 4 B) and TEM17 (BSC-TEM7) (FIG. 4 C) was demonstrated to be
highly specific to the ECs in colorectal cancers; sections were
imaged in the absence of a counterstain to show the complete lack
of detectable expression in the non-endothelial cells of the tumor.
Expression of TEM17 (BSC-TEM7) in ECs was demonstrated in a
metastatic liver lesion from a primary colorectal cancer (FIG. 4
D), a lung (FIG. 4 E), breast (FIG. 4 F), pancreatic (FIG. 4 G) and
brain cancer (FIG. 4 H), as well as in a sarcoma (FIG. 4 I). TEM 17
(BSC-TEM7) was also localized to vessels during normal
physiological angiogenesis of the corpus luteum (FIG. 4 J).
DETAILED DESCRIPTION OF THE INVENTION
[0051] We identified 46 human genes that were expressed at
significantly higher levels (>10-fold) in tumor endothelium than
in normal endothelium, and 33 genes that were expressed at
significantly lower levels in human tumor versus normal
endothelium. See Tables 2 and 4, respectively. Most of these genes
were either not expressed or expressed at relatively low levels in
Endothelial Cells (ECs) maintained in culture. Moreover, we
identified 93 genes which are expressed in both normal and tumor
human endothelium. Interestingly, the tumor endothelium genes were
expressed in all tumors tested, regardless of its tissue or organ
source. Most tumor endothelium genes were also expressed in corpus
luteum and wounds.
[0052] As the work has progressed, we have refined and classified
our original 46 tumor endothelial markers. We have named these
markers TEMs and renumbered them consecutively by the prevalence of
their tags in our SAGE analysis. Originally we had not used a
consecutive numbering system. Our non-consecutive numbering system
has been renamed as BSC-TEMs. For most of the original 46 SAGE
Tags, we now provide full-length nucleic acid and protein sequence.
In some cases, the sequences were obtained through the public
databases, in others the sequences were obtained by cloning and
through the use of gene prediction tools. In some cases, we found
SAGE Tags corresponding to genes having different splice varients
or with known polymorphisms. For example, in one case the SAGE Tag
BSC-TEM3 has been found to hybridize to an alternatively spliced
form of the transcript encoding BSC-TEM7. The proteins encoded by
the two transcripts are the same; therefore they are cumulatively
called TEM7. A highly related sequence was found via homology
searches, BSC-TEM7R. This paralog sequence is now called TEM3. See
Table 2, which follows, showing tumor endothelial markers by order
of prevalence (except for TEM 3). Column 1 indicates the prevalence
number. Column 2 indicates the original nomenclature. Column 3
indicates the short tags. Column 4 indicates the long tags. Column
5 indicates the accession number in GenBank. Column 6 indicates the
sequence identifiers for the short tag, the long tag, the full
nucleic acid, and the protein. Column 7 provides a functional
description, which is expanded below in the text.
TABLE-US-00001 TEM1 BSC-TEM1 GGGGCTGCCC GGGGCTGCCCAGCT NM020404 SEQ
ID NO: tumor endothelial marker 1 precursor A GA 94, 309, 195, 196
TEM 2 BSC-TEM2 GATCTCCGTG SEQ ID NO: sapiens tumor endothelial
marker 2 T 95, 197, (BSC-TEM2) mRNA/mouse Ras, dexa- 198
methasone-induced 1 (RASD1), mRNA TEM 3 BSC-TEM7R SEQ ID NO: human
ortholog of mouse paralog of 199, 200 mouse TEM-7 TEM 4 CTTTCTTTGA
CTTTCTTTGAGTTT AB034203 SEQ ID NO: Homo sapiens dickkopf-3 (DKK-3)
mRNA, G TAA 97, 311, 201, 202 TEM 5 BSC-TEM4 TATTAACTCT
TATTAACTCTCTTT SEQ ID NO: Tumor endothelial marker 4 C GGA 98, 312,
203, 204 TEM 6 CAGGAGACCC CAGGAGACCCCAGG X57766 SEQ ID NO: Human
stromelysin-3 mRNA. C CCC 99, 314, 205, 206 TEM 7 GGAAATGTCA
GGAAATGTCAGCAA BC002576 SEQ ID NO: matrix metalloproteinase 2 A GTA
100, 315, (gelatinase A, 72kD gelatinase, 72kD 207, 208 type IV
collagenase) TEM 8 CCTGGTTCAG SEQ ID NO: HeyL transcription factor
T 101, 316, 209, 210 TEM 9 BSC-TEM5 TTTTTAAGAA TTTTTAAGAACTCG SEQ
ID NO: C GGT 102, 317, 211, 212 TEM 10 TTTGGTTTTC TTTGGTTTTCCAAA
J03464, SEQ ID NO: Human collagen alpha-2 type I mRNA, C AGA
M18057, 103, 319 complete cds, clone pHCOL2A1. X02488 213, 214 TEM
11 ATTTTGTATG ATTTTGTATGATTT NM_002508 SEQ ID NO: nidogen/entactin
A TTA 104, 321, 215, 216 TEM 12 ACTTTAGATG ACTTTAGATGGGAA X52022
SEQ ID NO: H. sapiens RNA for type VI collagen G GCC 105, 322,
alpha3 chain. 217, 218 TEM 13 GAGTGAGACC GAGTGAGACCCAGG M11749 SEQ
ID NO: Human Thy-1 glycoprotein gene, C AGC 106, 324, complete cds.
219, 220 TEM 14 GTACACACAC GTACACACACCCCC SEQ ID NO: Cystatin SN C
ACC 107, 325, 221, 223 TEM 14 GTACACACAC GTACACACACCCCC X54667 SEQ
ID NO: H. sapiens mRNA for cystatin S. C ACC 107, 325, 222, 224 TEM
15 CCACAGGGGA CCACAGGGGATTCT NM_000090 SEQ ID NO: Human mRNA 3'
region for pro-alpha1 T CCT 108, 327, (III) collagen. 225, 226 TEM
16 BSC-TEM6 TTAAAAGTCA TTAAAAGTCACTGT SEQ ID NO: AC GCA 109, 328,
227, 228 TEM 17 BSC-TEM7 ACAGACTGTT ACAGACTGTTAGCC AF279144 SEQ ID
NO: Human Tumor endothelial marker 7 A AAG 110, 329, 229, 230 TEM
18 CCACTGCAAC SEQ ID NO: C 111 TEM 19 BSC-TEM8 CTATAGGAGA SEQ ID
NO: C 112, 330, 231, 232 TEM 20 GTTCCACAGA NM_000089 SEQ ID NO:
collagen, type I, alpha 2 (COL1A2 A 113, 233, 234 TEM 21 TACCACCTCC
TACCACCTCCCTTT SEQ ID NO: Homo sapiens mRNA; cDNA DKFZp762B245 C
CCT 114, 331, (from clone DKFZp762B245); 235, 236 TEM 22 BSC-TEM9
GCCCTTTCTC GCCCTTTCTCTGTA NM_006039 SEQ ID NO: endocytic receptor
(macrophage T GTT 115, 334, mannose receptor family) (KIAA0709),
237, 238 TEM 23 TTAAATAGCA TTAAATAGCACCTT SEQ ID NO: no match C TAG
116, 335 TEM 24 AGACATACTG AGACATACTGACAG NM_022648 SEQ ID NO: Homo
sapiens mRNA; cDNA DKFZp434G162 A AAT 117, 336, (from clone
DKFZp434G162); 239, 240 TEM 25 TCCCCCAGGA TCCCCCAGGAGCCA L35279,
SEQ ID NO: Homo sapiens (clone KT2) bone morpho- G CCG NM_006129
118, 338, genetic protein-1 (BMP-1) mRNA 241, 242 TEM 26 AGCCCAAAGT
SEQ ID NO: No Match G 119 TEM 27 ACTACCATAA NM_003062 SEQ ID NO:
Homo sapiens mRNA for MEGF5, partial C 120, 243. cds. 244 TEM 28
TACAAATCGT TACAAATCGTTGTC NM_014859 SEQ ID NO: Homo sapiens mRNA
for KIAA0672 T AAA 121, 339, protein, complete cds. 245, 246 TEM 29
TTGGGTGAAA SEQ ID NO: ESTs (2 unigene clusters) A 122, 247, 248 TEM
30 CATTATCCAA CATTATCCAAAAAC THC534029, SEQ ID NO: integrin, alpha
1 A AAT X68742, 123, 340, AI262158, 249, 250 AI88747, AI394565,
AA679721 TEM 31 AGAAACCACG AGAAACCACGGAAA NM_001845 SEQ ID NO:
hypothetical protein KIAA1164 G TGG 124, 341, 251, 252 TEM 32
ACCAAAACCA SEQ ID NO: no match C 125 TEM 33 TGAAATAAAC NM_000255
SEQ ID NO: methylmalonyl Coenzyme A mutase 126, 253, 254 TEM 34
TTTGGTTTCC SEQ ID NO: no match 127 TEM 35 GTGGAGACGG GTGGAGACGGACTC
ESTAI186535 SEQ ID NO: est A TGT 128, 345, 255, 358 TEM 36
TTTGTGTTGT TTTGTGTTGTATAT NM_004370 SEQ ID NO: est A TTA 129, 346,
256, 257 TEM 37 TTATGTTTAA TTATGTTTAATAGT NM_002345 SEQ ID NO:
Human lumican mRNA, complete cds. T TGA 130, 347, 258, 259 TEM 38
TGGAAATGAC TGGAAATGACCCAA NM_000088 SEQ ID NO: collagen type1
alpha1 AAA 131, 348, 260, 261 TEM 39 TGCCACACAG TGCCACACAGTGAC
NM_003239 SEQ ID NO: Human transforming growth factor-beta T TTG
132, 350, 3 (TGF-beta3) mRNA, complete 262, 263 TEM 40 GATGAGGAGA
GATGAGGAGACTGG SEQ ID NO: collagen, type I, alpha 2 C CAA 133, 351,
264, 265 TEM 41 ATCAAAGGTT ATCAAAGGTTTGAT SEQ ID NO: est T TTA 134,
352, 266, 267 TEM 42 AGTCACTAGT AGTCACATAGTACA NM_025226 SEQ ID NO:
ESTs AA 135, 353, 268, 269 TEM 43 TTCGGTTGGT TTCGGTTGGTCAAA SEQ ID
NO: No match C GAT 136, 354 TEM 44 CCCCACACGG CCCCACACGGGCAA
NM_018354v SEQ ID NO: Homo sapiens cDNA FLJ11190 fis, clone G GCA
137, 355, PLACE1007583. 270, 271 TEM 45 GGCTTGCCTT GGCTTGCCTTTTTG
NM_000366 SEQ ID NO: est T TAT 138, 356, 272, 273 TEM 46 ATCCCTTCCC
ATCCCTTCCCGCCA NM_002688 SEQ ID NO: Homo sapiens mRNA for
peanut-like G CAC 139, 357, protein 1, PNUTL1 (hCDCreI-1). 274,
275
[0053] The studies described below provide the first definitive
molecular characterization of ECs in an unbiased and general
manner. They lead to several important conclusions that have direct
bearing on long-standing hypotheses about angiogenesis. First, it
is clear that normal and tumor endothelium are highly related,
sharing many endothelial cell specific markers. Second, it is
equally clear that the endothelium derived from tumors is
qualitatively different from that derived from normal tissues of
the same type and is also different from primary endothelial
cultures. Third, these genes are characteristically expressed in
tumors derived from several different tissue types, documenting
that tumor endothelium, in general, is different from normal
endothelium. Fourth, the genes expressed differentially in tumor
endothelium are also expressed during other angiogenic processes
such as corpus luteum formation and wound healing. It is therefore
more appropriate to regard the formation of new vessels in tumors
as "neoangiogenesis" rather than "tumor angiogenesis" per se. This
distinction is important from a variety of perspectives, and is
consistent with the idea that tumors recruit vasculature using much
of, or basically the same signals elaborated during other
physiologic or pathological processes. That tumors represent
"unhealed wounds" is one of the oldest ideas in cancer biology.
[0054] The nature and precise biological function of many of the
Tumor Endothelial Markers (TEMs) identified here are unknown. Of
the previously characterized genes shown in Table 2, it is
intriguing that several encode proteins involved in extracellular
matrix formation or remodelling (TEM 6, TEM 6, TEM 10, TEM 7, TEM
11, TEM 12, TEM 14, TEM 20, TEM 24, TEM 25, TEM 27, TEM 37, TEM 38,
and TEM 40,) Deposition of extracellular matrix is likely critical
to the growth of new vessels. Finally, it is perhaps not surprising
that so many of the endothelial-specific transcripts identified
here, whether expressed only in neovasculature or in endothelium in
general, have not been previously characterized, and some are not
even represented in EST databases. In part, this may be due to the
fact that the EST databases are heavily biased toward certain
tissues, but moreover, may be due to the fact that even in highly
vascularized tissues endothelial cells are still a relatively small
proportion of the population. Thus, the sensitivity of the SAGE
method is a particularly appropriate tool.
[0055] Sequence and literature study has permitted the following
identifications to be made among the family of TEM proteins. TEM
proteins have been identified which contain transmembrane regions.
These include TEM 1, TEM 3, TEM 9, TEM 13, TEM 17, TEM 19, TEM 22,
TEM 30, and TEM 44. TEM proteins have been identified which are
secreted proteins, including TEM 4, TEM 6, TEM 7, TEM 10, TEM 12,
TEM 14, TEM 20, TEM 25, TEM 27, TEM 31, TEM 36, TEM 37, TEM 38, and
TEM 39. HeyL (TEM 8) is a transcription factor which may be
involved in regulating TEMs as one or more groups. The protein
corresponding to the tag for TEM44 was found in the public
databases, but no biological function has yet been ascribed to
it.
[0056] TEM 1 has been named endosialin in the literature. It has a
signal sequence at amino acids 1-17 and a transmembrare domain at
amino acids 686-708. Thus it is a cell surface protein. Its
extracellular domain is at resiudes 1-685. Endosialin may be
involved in endocytosis. The mouse ortholog is predicted to have a
signal peptide at residues 1-21.
[0057] TEM 2 is a dexamethasone induced, ras related protein
homolog of 266 amino acids. It has neither a signal sequence nor a
transmembrane domain. Thus it is neither a cell surface nor a
secreted protein. TEM 2 plays a role in signal transduction. It
regulates alterations in cell morphology, proliferation, and
cell-extracellular matrix interactions.
[0058] TEM 3 (originally termed TEM 7R) has both a signal sequence
(at residues 1-24 or 1-30) and a transmembrane domain (at residues
456-477). Thus it is a cell surface protein. The portion of the
protein which is extracellular is at amino acids 1-455. TEM 3 has
domains with homology to integrins, plexin, and adhesion molecules.
TEM 3 may regulate GTPases that control signal transduction
pathways linking plasma membrane receptors to the actin
cytoskeleton. In the mouse ortholog, the signal peptide is
predicted to be residues 1-30.
[0059] TEM 4 is also known as DKK-3. It has a signal sequence
(residues1-16), suggesting that is a secreted protein. TEM 4
regulates wnt signaling, and it may be involved in vasculogenesis
and wnt-dependent signaling for endothelial growth. TEM 4 is an
inhibitor of Wnt oncogene and such inhibition can be determined by
assay. Tsuji et al., Biochem. Biophys. Res. Comm. 268:20-4,
2000.
[0060] TEM 5 appears to be neither secreted nor a cell surface
protein. TEM 5 appears to be a component of a G protein--GTPase
signaling pathway.
[0061] TEM 6 is also known as stromelysin-3/Matrix
metalloproteinase 11 (MMP-11). It has a signal sequence at residues
1-31, but no transmembrane domain. It has an alternative signal
peptide splice site at residues 108-109. Thus it appears to be a
secreted protein. TEM 6 belongs to the zinc metalloprotease family,
also known as the matrixin subfamily. TEM 6 is expressed in most
invasive carcinomas. Alpha 1-protease inhibitor is a natural
substrate of MMP 11. TEM 6 degrades extracellular matrix proteins
such as collagen and is involved in extracellular matrix remodeling
and cell migration. Stromelysin can be assayed using a
casein-resorufin substrate, for example. See Tortorella and Amer,
Inflammation Research 46 Supp. 2:S 122-3, 1997.
[0062] TEM 7 is a protein of many names, also being known as matrix
metalloproeinase 2, gelatinase A, and 72 KD type IV collagenase.
TEM 7 has a signal sequence at residues 1-26 and is a secreted
protein. Like TEM 6, TEM 7 belongs to the matrixin subfamily (zinc
metalloproteinases). TEM 7 cleaves gelatin type I, collagen type I,
IV, V VII and X. TEM 7 associates with integrin on the surface of
endothelial cells and promotes vascular invasion. TEM 7 is involved
in tissue remodeling. TEM 7 can be assayed using zymography or
quenched fluorescent substrate hydrolysis, for example. Garbett, et
al., Molecular Pathology 53:99-106, 2000. A fluorogenic matrix
metalloproteinase substrate assay can also be used which employs
methoxycoumarin containing septapeptide analog of the alpha2(I)
collagen cleavage site. See Bhide et al., J. Periodontology
71:690-700, 2000.
[0063] TEM 8 is HEYL protein. It has neither a signal sequence nor
a transmembrane domain. It is related to the hairy/Enhancer of
split genes. TEM 8 is likely a nuclear protein, having a role as a
transcription factor. TEM 8 belongs to a new class of Notch signal
transducers and plays a key role in various developmental
processes, such as vascular development, somatogenesis and
neurogenesis. SNP's at residues 615 and 2201 have Cytosine bases.
Notch 3 mutations underlie the CADASIL vascular disorder. See Mech
Dev 2000 November; 98 (1-2):175
[0064] TEM 9 is a G-protein coupled receptor homolog, having both a
signal sequence at residues 1-26 and 7 transmembrane domains. Thus
it is a cell surface protein. Its extracellular region resides in
amino acids 1-769. Its transmembrane domains are at residues
817-829 (TM2 and TM3), residues 899-929 (TM4 and TM5), and residues
1034-1040 (TM6 and TM7). TEM 9 acts as a G-protein coupled receptor
with extracellular domains characteristic of cell adhesion
proteins. One of its splice variants may function as a soluble
receptor. TEM 9 may regulate cell polarity and cell migration. It
may be involved in exocytosis based on latrophilin function. The
mouse ortholog has a predicted signal peptide at residues 1-29.
[0065] TEM 10 is collagen type I, alpha2 (COL1A2), which has a
signal sequence at residues 1-22. It is an extracellular matrix
(ECM) protein which is secreted subsequent to synthesis. TEM 10
interacts with a number of proteins including other ECM proteins,
certain growth factors, and matrix metalloproteases. TEM 10 is
required for the induction of endothelial tube formation and is
involved in tissue remodeling. A variant at nucleotide 3233 which
substitutes an A, is associated with osteogenesis imperfecta type
IV. A variant at nucleotide 4321 substituting an A retains a wild
type phenotype. Nucleotide 715 is a site of a polymorphism.
Nucleotides 695-748 are deleted in Ehlers-Danos syndrome. Other
mutations are associated with idiopathic osteoporosis, and atypical
Marfan syndrome. Variants are known at nucleotides 226(T,C),
314(A,C), 385(T,C), 868 (G,A), 907(C,T), 965(A,G), 970(T,A), 1784
(G,C), 2017(T,G), 2172(C,A), 2284(T,C), 2308(T,C), 2323(T,G),
2344(T,G), 2604(G,A), 2974(A,T), 2903(A,G), 2995(C,T), 3274(C,T),
3581(A,C), 3991(A,C), 4201(G,T), 4434(C,T), 4551(A,C), 4606(C,A),
4947(T,C), 4978(C,T), 4982(G,T), 5051(G,T). PolyA sites are located
at nucleotides 4450, 4550, 4885, and 5082. PolyA signals are
located at 4420-4424, 4515-4520, 4529-4534, 4866-4871, 5032-5037,
5053-5058. TEM 10, 20, and 40 derive from the same gene but are
different isoforms having different lengths.
[0066] TEM 11 is Nidogen/Entactin. It is a secreted protein which
has a signal sequence at residues 1-28. TEM 11 is an extracellular
matrix protein which is a component of a basement membrane. TEM 11
binds to laminin and collagen IV and other extracellular matrix
proteins. TEM 11 regulates capillary formation and is involved in
tissue remodelling. Variations have been observed at nucleotides
4265(T,C), 4267(G,C,T), and 4738(T,G). Nidogen can be assayed by
its effect on the morphology of astrocytes. See Grimpe et al., GLIA
28:138-49, 1999.
[0067] TEM 12 is the alpha 3 chain of collagen type VI. It has a
signal sequence at residues 1-25. A secreted protein, TEM 12 is an
extracellular matrix protein. TEM 12 has a splice variant. TEM 12
is a major constituent of vascular subendothelium and is involved
in tissue remodeling. It regulates platelet activation and
aggregation. Alternatively spliced domains are located at
nucleotides 347-964, 965-1567, 2153-3752, and 4541-5041.
[0068] TEM 13 is also known as Thy-1 glycoprotein. It has both a
signal sequence (at residues 1-19) and a transmembrane domain (at
residues 143-159). Residues 131-161 are removed in a matured form
of the protein. The extracellular region of the protein is residues
1-142 or residues 1-130. TEM 13 has a glycosyl phosphatidylinositol
(GPI) anchor at residue 130 anchoring it to the membrane. TEM 13 is
detectable in its soluble form in human serum. TEM 13 is reported
to be a marker for activated endothelial cells (a marker of adult
but not embryonic angiogenesis). TEM 13 on vascular endothelial
cells may function as a possible vascular permeability modulator.
Antibody to Thy-1 is a mitogenic signal for the CD4+CD45+ and
CD8+CD45+ cells, but fails to induce proliferation in the CD45- T
cells. Pingel et al., International Immunology 6:169-78, 1994.
Thy-1 can be assayed as an inhibitor of such signal.
[0069] TEM 14 is also known as cystatin S. It is a secreted protein
with a signal sequence at residues 1-20 and an extracellular region
at residues 1-141. It is a cysteine protease inhibitor. TEM 14 may
regulate cysteine protease function involved in angiogenesis and
tissue remodeling. TEM14 is an inhibitor of the activity of papain
and such inhibition can be assayed. Hiltke et al., J. Dental
Research 78:1401-9, 1999.
[0070] TEM 15 is collagen type III, alpha 1 (COL3A1). It has a
signal sequence (residues 1-23) and is secreted. Type III collagen
binds to von Willebrand factor. It is involved in cell-cell
adhesion, proliferation, and migration activities. Variants at
nucloetides 2104(C,A), 2194(G,A), 2346(C,T), 2740(C,T), 3157(T),
3468(G), 3652(T), 3666(C), 3693(C), 3755(G), 3756(T), 3824(C),
4546(A, G), 4661(G), 4591(C,T), 4665(C), 5292(C), 5293(C), and 5451
(A) have been observed.
[0071] TEM 16 is a tensin homolog which is apparently an
intracellular protein. It may have splice variants or isoforms. One
form with 1704 amino acids has a region at the N-terminal domain
which is similar to a tumor suppressor protein, phosphatase and
tensin homolog (PTEN). Tensin is a focal adhesion molecule that
binds to actins and phosphorylated proteins. It is involved in cell
migration linking signal tranduction pathways to the cytoskeleton.
PTEN regulates tumor induced angiogenesis.
[0072] TEM 17 (BSC-TEM 7) has a signal sequence which includes
residues 1-18 and a transmembrane domain at residues 427-445. It is
a cell surface marker with an extracellular region comprising
residues 1-426. It has homologs in both mouse and C. elegans.
Residues 137-244 share weak homology with nidogen; residues 280-344
share homology to PSI domains found in plexin, semaphorins and
integrin beta subunits. Variants have been observed at nucleotides
1893(A,G), 1950(C,G), 2042(A,G), and 2220(G,A). In mouse TEM 17 the
signal sequence includes residues 1-19.
[0073] TEM 19 was originally reported to be tumor endothelial
marker 8, i.e., BSC-TEM 8. It has a signal sequence at residues
1-27 and a transmembrane domain at residues 322-343. It is a cell
surface protein having an extracellular region at residues 1-321.
TEM 19 has a von Willebrand Factor (vWF) A domain at residues
44-216; a domain at residues 34-253 which is found in leukointegrin
alpha D chain; and a domain at residues 408-560 found in PRAM-1 or
adaptor molecule-1 of the vinculin family. TEM 19's function is
adhesion related. von Willebrand Factor domains are typically
involved in a variety of functions including vascular processes.
TEM 19 may play a role in the migration of vascular endothelial
cells. The mouse ortholog has a predicted signal peptide at
residues 1-27.
[0074] TEM 20 is collagen type I, alpha 2 (COL1A2). It has a signal
sequence at residues 1-22 and is a secreted extracellular matrix
protein. TEM 20 induces endothelial tube formation in vitro and is
involved in tissue remodeling. Variants have been observed at
nucleotides 226(T,C), 314(A,C), 385(T,C), 868 (G,A), 907(C,T),
965(A,G), 970(T,A), 1784(G,C), 2017(T,G), 2172(C,A), 2284(T,C),
2308(T,C), 2323(T,G), 2344(T,G), 2604(G,A), 2794(A,T), 2903(A,G),
2995(C,T), 3274(C,T), 3581(A,C), 3991(A,C), 4201(G,T), 4434(C,T),
4551(A,C), 4606(C,A), 4895-4901 (--, GGACAAC), 4947(T,C),
4978(C,T), 4982(G,T), 5051(G,T).
[0075] TEM 21 is a Formin-like protein homolog which is an
intracellular protein. Formin related proteins interact with Rho
family small GTPases, profilin, and other actin associated
proteins. Formin-binding proteins bind to FH1 domains with their WW
domains. TEM 21 has a proline rich FH1 domain at residues 221-449.
Formin related proteins play crucial roles in morphogenesis, cell
polarity, cytokinesis and reorganization of the actin cytoskeleton.
They may also regulate apoptosis, cell adhesion and migration.
[0076] TEM 22 is an endocytic receptor in the macrophage mannose
receptor family. It has both a signal sequence at residues 1-30 and
a transmembrane domain at residues 1415-1435, and resides on the
cell surface. Its extracellular domain is amino acids 1-1414. TEM
22 may be present as a soluble (secreted) form and act as an
inhibitor. It may bind secreted phopholipase A2 (sPLA2) and mediate
biological responses elicited by sPLA2. TEM 22 may have endocytic
properties for sPLA2 and mediate endocytosis for endothelial
related proteins. It may promote cell adhesion and be involved in
cell-cell communication. Variations have been observed at
nucleotide 5389 (A, G). TEM 22 mediates uptake of micro-organisms
and host-derived glycoproteins. Groger et al., J. Immunology
165:5428-34, 2000.
[0077] TEM 24 is tensin, an intracellular protein. It is a focal
adhesion molecule that binds to actin filaments and interacts with
phosphotyrosine containing proteins. It may mediate kinase
signaling activities and regulate cellular transformation.
Variations have been observed at nucleotides 2502 (A, G), 2622(A,
G), 6027(A, G). TEM24 binds to actin filaments and interacts with
phosphotyrosine-containing proteins. Chen et al., Biochem. J. 351
Pt 2:403-11, 2000. TEM24 also binds to phosphoinositide3-kinase.
Auger et al., J. Bio. Chem. 271:23452-7, 1996 TEM 24 also binds to
nuclear protein p130. Lo et al., Bioassays 16:817-23, 1994.
[0078] TEM 25 is Bone morphogenic protein 1 (BMP-1) which has a
signal sequence at residues 1-22. It is a secreted protein. There
are at least 6 isoforms of BMP-1 as well as splice variants which
add carboxy terminal CUB domains and an additional EGF domain. TEM
25 is a metalloprotease enzyme. It cleaves the C-terminal
propeptide of collagen type I, II and III and laminin 5 gamma 2,
proteins that are important for vascular processes. It is involved
in cartilage formation. Variations have been observed at
nucleotides 3106(C,T), 3248(G,A), 3369(G,A). TEM 25 cleave
probiglycan at a single site, removing the propeptide and producing
a biglycan molecule with an NH(2) terminus identical to that of the
mature form found in tissues. Sctt et al., J. Biol. Chem.
275:30504-11, 2000. Laminin alpha 3 and gamma2 short chains are
substrates of TEM 25. Amano et al., J. Biol. Chem. 275:22728-35,
2000.
[0079] TEM 27 is known as Slit homolog 3, a secreted protein with a
signal sequence at residues 1-27. TEM 27 is a secreted guide
protein involved in migration, repulsion and patterning. It
interacts with "round about" receptors (Robo receptors). TEM 27 may
interact with extracellular matrix (ECM) proteins and is involved
in cell adhesion. Variations have been observed at nucleotides 4772
(C,T)
[0080] TEM 28 is similar to mouse nadrin (neuron specific GTPase
activating protein). TEM 28 is an intracellular protein with a
RhoGAP domain. The RhoGAP domain activates RhoA, Rac1, and Cdc42
GTPases. It is involved in the reorganization of actin filaments
and enhancing exocytosis. It may also be involved in cell
signalling. Variations have been observed at nucleotide 3969
(A,C),
[0081] TEM 29 is protein tyrosine phosphatase type IVA, member 3,
isoform 1, an intracellular protein. It has alternate splice
variants. TEM 29 belongs to a small class of prenylated protein
tyrosine phosphatases (PTPs). It may be membrane associated by
prenylation. PTPs are cell signaling molecules and play regulatory
roles in a variety of cellular processes and promote cell
proliferation. PTP PRL-3 regulates angiotensin-II induced signaling
events.
[0082] TEM 30 is integrin alpha 1, a cell surface protein having
both a signal sequence (residues 1-28) and a transmembrane domain
(residues 1142-1164). Its extracellular region includes amino acids
1-1141. TEM 30 is a receptor for laminin and collagen. It mediates
a variety of adhesive interactions. TEM 30 is abundantly expressed
on microvascular endothelial cells. It stimulates endothelial cell
proliferation and vascularization. TEM 30 may regulate angiostatin
production. Variations have been observed at nucleotide 418 (C,T).
TEM 30 activates the Ras/Shc/mitogen-activated protein kinase
pathway promoting fibroblast cell proliferation. It also acts to
inhibit collagen and metalloproteinase synthesis. Pozzi et al.,
Proc. Nat. Acad. Sci. USA 97:2202-7, 2000,
[0083] TEM 31 is Collagen IV alpha 1 (COL4A1) a secreted protein
with a at residues 1-27. TEM 31 is a component of the basement
membrane. It binds to alpha3 beta lintegrin and promotes integrin
mediated cell adhesion. Non-collagenous domains of type IV subunits
are involved in tumoral angiogenesis. TEM 31 is involved in tissue
remodeling. Variations have been observed at nucleotide 4470
(C,T)
[0084] TEM 33 is methylmalonyl Co-A Mutase a protein which is
localized in the mitochondrial matrix. It degrades several amino
acids, odd-numbered-acid fatty acids, and cholesterol to the
tricarboylic acid cycle. A defect in TEM 33 causes a fatal disorder
in organic acid metabolism termed methylmalonic acidurea.
Variations have been observed at nucleotides 1531(G,A), 1671(G,A),
2028(T,C), 2087(G,A), 2359(A,G), 2437(C,A), 2643(G,C), 2702(G,C).
TEM 33 converts L-methylmalonyl CoA to succinyl CoA. This reaction
can be assayed as is known in the art. See, e.g., Clin. Chem. 41(8
Pt I): 1164-70, 1995.
[0085] TEM 36 is collagen type XII, alpha1 (COL12A1), an
extracellular matrix protein having a signal sequence at residues
1-23 or 24. TEM 36 has von Willebrand Factor (vWF) type A domains,
Fibronectin type III domains, and thrombospondin N-terminal like
domain. TEM 36 is expressed in response to stress environment. TEM
36 may organize extracellular matrix architecture and be involved
in matrix remodeling. There are two isoforms of the protein, a long
form and a short form. The short form is missing amino acids
25-1188, and therefore nucleotides 73 to 3564. Both forms share the
signal sequence and are therefore both secreted.
[0086] TEM 37 is lumican, an extracellular matrix sulfated
proteoglycan having a signal sequence at residues 1-18. Lumican
interacts with proteins that are involved in matrix assembly such
as collagen type I and type VI; it is involved in cell
proliferation and tissue morphogenesis. Lumican plays an important
role in the regulation of collagen fiber assembly. Variations have
been observed at nucleotides 1021(G,T), 1035(A,G), 1209(A,G),
1259(A,C), 1418(C,A), 1519(T,A). TEM 37 is a binding partner of
TGF-.beta.. See FASEB J. 15:559-61, 2000. One assay that can be
used to determine TEM 37 activity is a collagen fibril
formation/sedimentation assay. Svensson et al., FEBS Letters
470:178-82, 2000.
[0087] TEM 38 is collagen type I, alpha 1 (COL1A1), an
extracellular matrix protein having a signal sequence at residues
1-22. Type I collagen promotes endothelial cell migration and
vascularization and induces tube formation and is involved in
tissue remodelling. Telopeptide derivative is used as a marker for
malignancy and invasion for certain cancer types. Variations have
been observed at nucleotides 296(T,G), 1810(G,A), 1890(G,A),
2204(T,A), 3175(G,C), 3578(C,T), 4298(C,T), 4394(A,T), 4410(A,C),
4415(C.A), 4419 (A,T), 4528(C,A), 4572(G,T), 4602(T,C), 5529(T,C),
5670(C,T), 5985(C,T), 6012(C,T).
[0088] TEM 39 is transforming growth factor .beta.-3 (TGF-beta3).
It has a signal sequence at residues 1-23. It is a secreted
protein. TEM 39 regulates cell growth and differentiation. TGF-beta
isoforms play a major role in vascular repair processes and
remodeling. Variations have been observed at nucleotide
2020(G,T).
[0089] TEM 41 is similar to Olfactomedin like protein. It appears
to be an intracellular protein, having no obvious predicted signal
sequence. Olfactomedin is the major glycoprotein of the
extracellular mucous matrix of olfactory neuroepithelium. TEM 41
shares homology with latrophilin (extracellular regions) which has
cell-adhesive type domains. TEM 41 may be involved in adhesive
function.
[0090] TEM 42 is MSTP032 protein, a cell surface protein having a
transmembrane domain at residues 42-61. Its function is unknown and
it shares little homology with other proteins. Variations have been
observed at nucleotides 418(A,T), 724(C,A).
[0091] TEM 44 is a hypothetical protein FLJ1190 (NM.sub.--018354)
which has two predicted transmembrane domains at residues 121-143
and 176-197. Residues 144-175 may form an extracellular region. TEM
44's function is not known and shares no homology to other known
proteins.
[0092] TEM 45 is tropomyosin 1 (alpha), a protein which is
intracellular. It forms dimers with a beta subunit. It influences
actin function. TEM 45 may be involved in endothelial cell
cytoskeletal rearrangement. Variations have been observed at
nucleotides 509(A,C), 621(A,C), 635(T,G), 642(C,G), 1059(G,T).
[0093] TEM 46 is peanut-like 1 protein/septin 5, which belongs to
the septin family. Proteins in the septin family bind to GTP and
phosphatidylinositol 4,5-bisphosphate. They are involved in the
signal tranduction cascades controlling cytokinesis and cell
division.
[0094] NEM 4 is a member of the small inducible cytokine subfamily
A (cys-cys), member 14 (SCYA14). NEM4 is a secreted protein
characterized by two adjacent cysteine residues. One isoform lacks
internal 16 amino acids compared to isoform 2.
[0095] NEM 22 shares homology with guanylate kinase-interacting
protein 1Maguin-1. It is a membrane associated protein.
[0096] NEM 23 is human signaling lymphocytic activation molecule
(SLAM). It has a signal sequence at residues 1-20. The
extracellular domain may reside at residues 21-237. There is a
secreted isoform of the protein.
[0097] NEM33 is netrin 4. It induces neurite outgrowth and promotes
vascular development. At higher concentration, neurite outgrowth is
inhibited.
[0098] ECs represent only a minor fraction of the total cells
within normal or tumor tissues, and only those EC transcripts
expressed at the highest levels would be expected to be represented
in libraries constructed from unfractionated tissues. The genes
described in the current study should therefore provide a valuable
resource for basic and clinical studies of human angiogenesis in
the future. Genes which have been identified as tumor endothelial
markers (TEMs) correspond to tags shown in SEQ ID NOS: 94-139,
173-176, 180-186. Genes which have been identified as normal
endothelial markers (NEMs) correspond to tags shown in SEQ ID NOS:
140-172. Genes which have been identified as pan-endothelial
markers (PEMs) i.e., expressed in both tumor and normal endothelial
cells correspond to tags shown in SEQ ID NOS: 1-93. Genes which
have been previously identified as being expressed predominantly in
the endothelium correspond to PEM tags shown in SEQ ID NOS: 1-6, 8,
10-15. Markers in each class can be used interchangeably for some
purposes.
[0099] Isolated and purified nucleic acids, according to the
present invention are those which are not linked to those genes to
which they are linked in the human genome. Moreover, they are not
present in a mixture such as a library containing a multitude of
distinct sequences from distinct genes. They may be, however,
linked to other genes such as vector sequences or sequences of
other genes to which they are not naturally adjacent. Tags
disclosed herein, because of the way that they were made, represent
sequences which are 3' of the 3' most restriction enzyme
recognition site for the tagging enzyme used to generate the SAGE
tags. In this case, the tags are 3' of the most 3' most NlaIII site
in the cDNA molecules corresponding to mRNA. Nucleic acids
corresponding to tags may be RNA, cDNA, or genomic DNA, for
example. Such corresponding nucleic acids can be determined by
comparison to sequence databases to determine sequence identities.
Sequence comparisons can be done using any available technique,
such as BLAST, available from the National Library of Medicine,
National Center for Biotechnology Information. Tags can also be
used as hybridization probes to libraries of genomic or cDNA to
identify the genes from which they derive. Thus, using sequence
comparisons or cloning, or combinations of these methods, one
skilled in the art can obtain full-length nucleic acid sequences.
Genes corresponding to tags will contain the sequence of the tag at
the 3' end of the coding sequence or of the 3' untranslated region
(UTR), 3' of the 3' most recognition site in the cDNA for the
restriction endonuclease which was used to make the tags. The
nucleic acids may represent either the sense or the anti-sense
strand. Nucleic acids and proteins although disclosed herein with
sequence particularity, may be derived from a single individual.
Allelic variants which occur in the population of humans are
including within the scope of such nucleic acids and proteins.
Those of skill in the art are well able to identify allelic
variants as being the same gene or protein Given a nucleic acid,
one of ordinary skill in the art can readily determine an open
reading frame present, and consequently the sequence of a
polypeptide encoded by the open reading frame and, using techniques
well known in the art, express such protein in a suitable host.
Proteins comprising such polypeptides can be the naturally
occurring proteins, fusion proteins comprising exogenous sequences
from other genes from humans or other species, epitope tagged
polypeptides, etc. Isolated and purified proteins are not in a
cell, and are separated from the normal cellular constituents, such
as nucleic acids, lipids, etc. Typically the protein is purified to
such an extent that it comprises the predominant species of protein
in the composition, such as greater than 50, 60 70, 80, 90, or even
95% of the proteins present.
[0100] Using the proteins according to the invention, one of
ordinary skill in the art can readily generate antibodies which
specifically bind to the proteins. Such antibodies can be
monoclonal or polyclonal. They can be chimeric, humanized, or
totally human. Any functional fragment or derivative of an antibody
can be used including Fab, Fab', Fab2, Fab'2, and single chain
variable regions. So long as the fragment or derivative retains
specificity of binding for the endothelial marker protein it can be
used. Antibodies can be tested for specificity of binding by
comparing binding to appropriate antigen to binding to irrelevant
antigen or antigen mixture under a given set of conditions. If the
antibody binds to the appropriate antigen at least 2, 5, 7, and
preferably 10 times more than to irrelevant antigen or antigen
mixture then it is considered to be specific.
[0101] Techniques for making such partially to fully human
antibodies are known in the art and any such techniques can be
used. According to one particularly preferred embodiment, fully
human antibody sequences are made in a transgenic mouse which has
been engineered to express human heavy and light chain antibody
genes. Multiple strains of such transgenic mice have been made
which can produce different classes of antibodies. B cells from
transgenic mice which are producing a desirable antibody can be
fused to make hybridoma cell lines for continuous production of the
desired antibody. See for example, Nina D. Russel, Jose R. F.
Corvalan, Michael L. Gallo, C. Geoffrey Davis, Liise-Anne Pirofski.
Production of Protective Human Antipneumococcal Antibodies by
Transgenic Mice with Human Immunoglobulin Loci Infection and
Immunity April 2000, p. 1820-1826; Michael L. Gallo, Vladimir E.
Ivanov, Aya Jakobovits, and C. Geoffrey Davis. The human
immunoglobulin loci introduced into mice: V (D) and J gene segment
usage similar to that of adult humans European Journal of
Immunology 30: 534-540, 2000; Larry L. Green. Antibody engineering
via genetic engineering of the mouse: XenoMouse strains are a
vehicle for the facile generation of therapeutic human monoclonal
antibodies Journal of Immunological Methods 231 11-23, 1999; Yang
X-D, Corvalan JRF, Wang P, Roy CM-N and Davis CG. Fully Human
Anti-interleukin-8 Monoclonal Antibodies: Potential Therapeutics
for the Treatment of Inflammatory Disease States. Journal of
Leukocyte Biology Vol. 66, pp 401-410 (1999); Yang X-D, Jia X-C,
Corvalan JRF, Wang P, CG Davis and Jakobovits A. Eradication of
Established Tumors by a Fully Human Monoclonal Antibody to the
Epidermal Growth Factor Receptor without Concomitant Chemotherapy.
Cancer Research Vol. 59, Number 6, pp 1236-1243 (1999); Jakobovits
A. Production and selection of antigen-specific fully human
monoclonal antibodies from mice engineered with human Ig loci.
Advanced Drug Delivery Reviews Vol. 31, pp: 33-42 (1998); Green L
and Jakobovits A. Regulation of B cell development by variable gene
complexity in mice reconstituted with human immunoglobulin yeast
artificial chromosomes. J. Exp. Med. Vol. 188, Number 3, pp:
483-495 (1998); Jakobovits A. The long-awaited magic bullets:
therapeutic human monoclonal antibodies from transgenic mice. Exp.
Opin. Invest. Drugs Vol. 7(4), pp: 607-614 (1998); Tsuda H,
Maynard-Currie K, Reid L, Yoshida T, Edamura K, Maeda N, Smithies
O, Jakobovits A. Inactivation of Mouse HPRT locus by a 203-bp
retrotransposon insertion and a 55-kb gene-targeted deletion:
establishment of new HPRT-Deficient mouse embryonic stem cell
lines. Genomics Vol. 42, pp: 413-421 (1997); Sherman-Gold, R.
Monoclonal Antibodies: The Evolution from '80s Magic Bullets To
Mature, Mainstream Applications as Clinical Therapeutics. Genetic
Engineering News Vol. 17, Number 14 (August 1997); Mendez M, Green
L, Corvalan J, Jia X-C, Maynard-Currie C, Yang X-d, Gallo M, Louie
D, Lee D, Erickson K, Luna J, Roy C, Abderrahim H, Kirschenbaum F,
Noguchi M, Smith D, Fukushima A, Hales J, Finer M, Davis C, Zsebo
K, Jakobovits A. Functional transplant of megabase human
immunoglobulin loci recapitulates human antibody response in mice.
Nature Genetics Vol. 15, pp: 146-156 (1997); Jakobovits A. Mice
engineered with human immunoglobulin YACs: A new technology for
production of fully human antibodies for autoimmunity therapy.
Weir's Handbook of Experimental Immunology, The Integrated Immune
System Vol. IV, pp: 194.1-194.7 (1996); Jakobovits A. Production of
fully human antibodies by transgenic mice. Current Opinion in
Biotechnology Vol. 6, No. 5, pp: 561-566 (1995); Mendez M,
Abderrahim H, Noguchi M, David N, Hardy M, Green L, Tsuda H, Yoast
S, Maynard-Currie C, Garza D, Gemmill R, Jakobovits A, Klapholz S.
Analysis of the structural integrity of YACs comprising human
immunoglobulin genes in yeast and in embryonic stem cells. Genomics
Vol. 26, pp: 294-307 (1995); Jakobovits A. YAC Vectors: Humanizing
the mouse genome. Current Biology Vol. 4, No. 8, pp: 761-763
(1994); Arbones M, Ord D, Ley K, Ratech H, Maynard-Curry K, Otten
G, Capon D, Tedder T. Lymphocyte homing and leukocyte rolling and
migration are impaired in L-selectin-deficient mice. Immunity Vol.
1, No. 4, pp: 247-260 (1994); Green L, Hardy M, Maynard-Curry K,
Tsuda H, Louie D, Mendez M, Abderrahim H, Noguchi M, Smith D, Zeng
Y, et. al. Antigen-specific human monoclonal antibodies from mice
engineered with human Ig heavy and light chain YACs. Nature
Genetics Vol. 7, No. 1, pp: 13-21 (1994); Jakobovits A, Moore A,
Green L, Vergara G, Maynard-Curry K, Austin H, Klapholz S.
Germ-line transmission and expression of a human-derived yeast
artificial chromosome. Nature Vol. 362, No. 6417, pp: 255-258
(1993); Jakobovits A, Vergara G, Kennedy J, Hales J, McGuinness R,
Casentini-Borocz D, Brenner D, Otten G. Analysis of homozygous
mutant chimeric mice: deletion of the immunoglobulin heavy-chain
joining region blocks B-cell development and antibody production.
Proceedings of the National Academy of Sciences USA Vol. 90, No. 6,
pp: 2551-2555 (1993); Kucherlapati et al., U.S. Pat. No.
6,1075,181.
[0102] Antibodies can also be made using phage display techniques.
Such techniques can be used to isolate an initial antibody or to
generate variants with altered specificity or avidity
characteristics. Single chain Fv can also be used as is convenient.
They can be made from vaccinated transgenic mice, if desired.
Antibodies can be produced in cell culture, in phage, or in various
animals, including but not limited to cows, rabbits, goats, mice,
rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys,
chimpanzees, apes.
[0103] Antibodies can be labeled with a detectable moiety such as a
radioactive atom, a chromophore, a fluorophore, or the like. Such
labeled antibodies can be used for diagnostic techniques, either in
vivo, or in an isolated test sample. Antibodies can also be
conjugated, for example, to a pharmaceutical agent, such as
chemotherapeutic drug or a toxin. They can be linked to a cytokine,
to a ligand, to another antibody. Suitable agents for coupling to
antibodies to achieve an anti-tumor effect include cytokines, such
as interleukin 2 (IL-2) and Tumor Necrosis Factor (TNF);
photosensitizers, for use in photodynamic therapy, including
aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and
phthalocyanine; radionuclides, such as iodine-131 (.sup.131I),
yttrium-90 (.sup.90Y), bismuth-212 (.sup.212Bi), bismuth-213
(.sup.213Bi), technetium-99m (.sup.99mTc), rhenium-186 (.sup.186
Re), and rhenium-188 (.sup.188Re); antibiotics, such as
doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin,
neocarzinostatin, and carboplatin; bacterial, plant, and other
toxins, such as diphtheria toxin, pseudomonas exotoxin A,
staphylococcal enterotoxin A, abrin-A toxin, ricin A
(deglycosylated ricin A and native ricin A), TGF-alpha toxin,
cytotoxin from chinese cobra (naja naja atra), and gelonin (a plant
toxin); ribosome inactivating proteins from plants, bacteria and
fungi, such as restrictocin (a ribosome inactivating protein
produced by Aspergillus restrictus), saporin (a ribosome
inactivating protein from Saponaria officinalis), and RNase;
tyrosine kinase inhibitors; ly207702 (a difluorinated purine
nucleoside); liposomes containing antitumor agents (e.g., antisense
oligonucleotides, plasmids which encode for toxins, methotrexate,
etc.); and other antibodies or antibody fragments, such as
F(ab).
[0104] Those of skill in the art will readily understand and be
able to make such antibody derivatives, as they are well known in
the art. The antibodies may be cytotoxic on their own, or they may
be used to deliver cytotoxic agents to particular locations in the
body. The antibodies can be administered to individuals in need
thereof as a form of passive immunization.
[0105] Characterization of extracellular regions for the cell
surface and secreted proteins from the protein sequence is based on
the prediction of signal sequence, transmembrane domains and
functional domains. Antibodies are preferably specifically
immunoreactive with membrane associated proteins, particularly to
extracellular domains of such proteins or to secreted proteins.
Such targets are readily accessible to antibodies, which typically
do not have access to the interior of cells or nuclei. However, in
some applications, antibodies directed to intracellular proteins
may be useful as well. Moreover, for diagnostic purposes, an
intracellular protein may be an equally good target since cell
lysates may be used rather than a whole cell assay.
[0106] Computer programs can be used to identify extracellular
domains of proteins whose sequences are known. Such programs
include SMART software (Schultz et al., Proc. Natl. Acad. Sci. USA
95: 5857-5864, 1998) and Pfam software (Bateman et al., Nucleic
acids Res. 28: 263-266, 2000) as well as PSORTII. Typically such
programs identify transmembrane domains; the extracellular domains
are identified as immediately adjacent to the transmembrane
domains. Prediction of extracellular regions and the signal
cleavage sites are only approximate. It may have a margin of error
+ or -5 residues. Signal sequence can be predicted using three
different methods (Nielsen et al, Protein Engineering 10: 1-6,
1997, Jagla et. al, Bioinformatics 16: 245-250, 2000, Nakai, K and
Horton, P. Trends in Biochem. Sci. 24:34-35, 1999) for greater
accuracy. Similarly transmembrane (TM) domains can be identified by
multiple prediction methods. (Pasquier, et. al, Protein Eng.
12:381-385, 1999, Sonnhammer et al., In Proc. of Sixth Int. Conf.
on Intelligent Systems for Molecular Biology, p. 175-182, Ed J.
Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C.
Sensen Menlo Park, Calif.: AAAI Press, 1998, Klein, et. al,
Biochim. Biophys. Acta, 815:468, 1985, Nakai and Kanehisa Genomics,
14: 897-911, 1992). In ambiguous cases, locations of functional
domains in well characterized proteins are used as a guide to
assign a cellular localization.
[0107] Putative functions or functional domains of novel proteins
can be inferred from homologous regions in the database identified
by BLAST searches (Altschul et. al. Nucleic Acid Res. 25:
3389-3402, 1997) and/or from a conserved domain database such as
Pfam (Bateman et. al, Nucleic Acids Res. 27:260-262 1999) BLOCKS
(Henikoff, et. al, Nucl. Acids Res. 28:228-230, 2000) and SMART
(Ponting, et. al, Nucleic Acid Res. 27,229-232, 1999).
Extracellular domains include regions adjacent to a transmembrane
domain in a single transmembrane domain protein (out-in or type I
class). For multiple transmembrane domains proteins, the
extracellular domain also includes those regions between two
adjacent transmembrane domains (in-out and out-in). For type II
transmembrane domain proteins, for which the N-terminal region is
cytoplasmic, regions following the transmembrane domain is
generally extracellular. Secreted proteins on the other hand do not
have a transmembrane domain and hence the whole protein is
considered as extracellular.
[0108] Membrane associated proteins can be engineered to delete the
transmembrane domains, thus leaving the extracellular portions
which can bind to ligands. Such soluble forms of transmembrane
receptor proteins can be used to compete with natural forms for
binding to ligand. Thus such soluble forms act as inhibitors. and
can be used therapeutically as anti-angiogenic agents, as
diagnostic tools for the quantification of natural ligands, and in
assays for the identification of small molecules which modulate or
mimic the activity of a TEM:ligand complex.
[0109] Alternatively, the endothelial markers themselves can be
used as vaccines to raise an immune response in the vaccinated
animal or human. For such uses, a protein, or immunogenic fragment
of such protein, corresponding to the intracellular, extracellular
or secreted TEM of interest is administered to a subject. The
immunogenic agent may be provided as a purified preparation or in
an appropriately expressing cell. The administration may be direct,
by the delivery of the immunogenic agent to the subject, or
indirect, through the delivery of a nucleic acid encoding the
immunogenic agent under conditions resulting in the expression of
the immunogenic agent of interest in the subject. The TEM of
interest may be delivered in an expressing cell, such as a purified
population of tumor endothelial cells or a populations of fused
tumor endothelial and dendritic cells. Nucleic acids encoding the
TEM of interest may be delivered in a viral or non-viral delivery
vector or vehicle. Non-human sequences encoding the human TEM of
interest or other mammalian homolog can be used to induce the
desired immunologic response in a human subject. For several of the
TEMs of the present invention, mouse, rat or other ortholog
sequences are described herein or can be obtained from the
literature or using techniques well within the skill of the
art.
[0110] Endothelial cells can be identified using the markers which
are disclosed herein as being endothelial cell specific. These
include the human markers identified by SEQ ID NOS: 1-172, i.e.,
the normal, pan-endothelial, and the tumor endothelial markers.
Homologous mouse markers include tumor endothelial markers of SEQ
ID NO: 182-186 and 190-194. Antibodies specific for such markers
can be used to identify such cells, by contacting the antibodies
with a population of cells containing some endothelial cells. The
presence of cross-reactive material with the antibodies identifies
particular cells as endothelial. Similarly, lysates of cells can be
tested for the presence of cross-reactive material. Any known
format or technique for detecting cross-reactive material can be
used including, immunoblots, radioimmunoassay, ELISA,
immunoprecipitation, and immunohistochemistry. In addition, nucleic
acid probes for these markers can also be used to identify
endothelial cells. Any hybridization technique known in the art
including Northern blotting, RT-PCR, microarray hybridization, and
in situ hybridization can be used.
[0111] One can identify tumor endothelial cells for diagnostic
purposes, testing cells suspected of containing one or more TEMs.
One can test both tissues and bodily fluids of a subject. For
example, one can test a patient's blood for evidence of
intracellular and membrane associated TEMs, as well as for secreted
TEMs. Intracellular and/or membrane associated TEMs may be present
in bodily fluids as the result of high levels of expression of
these factors and/or through lysis of cells expressing the
TEMs.
[0112] Populations of various types of endothelial cells can also
be made using the antibodies to endothelial markers of the
invention. The antibodies can be used to purify cell populations
according to any technique known in the art, including but not
limited to fluorescence activated cell sorting. Such techniques
permit the isolation of populations which are at least 50, 60, 70,
80, 90, 92, 94, 95, 96, 97, 98, and even 99% the type of
endothelial cell desired, whether normal, tumor, or
pan-endothelial. Antibodies can be used to both positively select
and negatively select such populations. Preferably at least 1, 5,
10, 15, 20, or 25 of the appropriate markers are expressed by the
endothelial cell population.
[0113] Populations of endothelial cells made as described herein,
can be used for screening drugs to identify those suitable for
inhibiting the growth of tumors by virtue of inhibiting the growth
of the tumor vasculature.
[0114] Populations of endothelial cells made as described herein,
can be used for screening candidate drugs to identify those
suitable for modulating angiogenesis, such as for inhibiting the
growth of tumors by virtue of inhibiting the growth of endothelial
cells, such as inhibiting the growth of the tumor or other
undesired vasculature, or alternatively, to promote the growth of
endothelial cells and thus stimulate the growth of new or
additional large vessel or microvasculature.
[0115] Inhibiting the growth of endothelial cells means either
regression of vasculature which is already present, or the slowing
or the absence of the development of new vascularization in a
treated system as compared with a control system. By stimulating
the growth of endothelial cells, one can influence development of
new (neovascularization) or additional vasculature development
(revascularization). A variety of model screen systems are
available in which to test the angiogenic and/or anti-angiogenic
properties of a given candidate drug. Typical tests involve assays
measuring the endothelial cell response, such as proliferation,
migration, differentiation and/or intracellular interaction of a
given candidate drug. By such tests, one can study the signals and
effects of the test stimuli. Some common screens involve
measurement of the inhibition of heparanase, endothelial tube
formation on Matrigel, scratch induced motility of endothelial
cells, platelet-derived growth factor driven proliferation of
vascular smooth muscle cells, and the rat aortic ring assay (which
provides an advantage of capillary formation rather than just one
cell type).
[0116] Drugs can be screened for the ability to mimic or modulate,
inhibit or stimulate, growth of tumor endothelium cells and/or
normal endothelial cells. Drugs can be screened for the ability to
inhibit tumor endothelium growth but not normal endothelium growth
or survival. Similarly, human cell populations, such as normal
endothelium populations or tumor endothelial cell populations, can
be contacted with test substances and the expression of tumor
endothelial markers and/or normal endothelial markers determined.
Test substances which decrease the expression of tumor endothelial
markers (TEMs) are candidates for inhibiting angiogenesis and the
growth of tumors. Conversely, markers which are only expressed in
normal endothelium but not in tumor endothelium (NEMs) can be
monitored. Test substances which increase the expression of such
NEMs in tumor endothelium and other human cells can be identified
as candidate antitumor or anti-angiogenic drugs In cases where the
activity of a TEM or NEM is known, agents can be screened for their
ability to decrease or increase the activity.
[0117] For those tumor endothelial markers identified as containing
transmembrane regions, it is desirable to identify drug candidates
capable of binding to the TEM receptors found at the cell surface.
For some applications, the identification of drug candidates
capable of blocking the TEM receptor from its native ligand will be
desired. For some applications, the identification of a drug
candidate capable of binding to the TEM receptor may be used as a
means to deliver a therapeutic or diagnostic agent. For other
applications, the identification of drug candidates capable of
mimicking the activity of the native ligand will be desired. Thus,
by manipulating the binding of a transmembrane TEM receptor:ligand
complex, one may be able to promote or inhibit further development
of endothelial cells and hence, vascularization.
[0118] For those tumor endothelial markers identified as being
secreted proteins, it is desirable to identify drug candidates
capable of binding to the secreted TEM protein. For some
applications, the identification of drug candidates capable of
interfering with the binding of the secreted TEM it is native
receptor. For other applications, the identification of drug
candidates capable of mimicking the activity of the native receptor
will be desired. Thus, by manipulating the binding of the secreted
TEM:receptor complex, one may be able to promote or inhibit further
development of endothelial cells, and hence, vascularization.
[0119] Expression can be monitored according to any convenient
method. Protein or mRNA can be monitored. Any technique known in
the art for monitoring specific genes' expression can be used,
including but not limited to ELISAs, SAGE, microarray
hybridization, Western blots. Changes in expression of a single
marker may be used as a criterion for significant effect as a
potential pro-angiogenic, anti-angiogenic or anti-tumor agent.
However, it also may be desirable to screen for test substances
which are able to modulate the expression of at least 5, 10, 15, or
20 of the relevant markers, such as the tumor or normal endothelial
markers. Inhibition of TEM protein activity can also be used as a
drug screen. Human and mouse TEMS can be used for this purpose.
[0120] Test substances for screening can come from any source. They
can be libraries of natural products, combinatorial chemical
libraries, biological products made by recombinant libraries, etc.
The source of the test substances is not critical to the invention.
The present invention provides means for screening compounds and
compositions which may previously have been overlooked in other
screening schemes. Nucleic acids and the corresponding encoded
proteins of the markers of the present invention can be used
therapeutically in a variety of modes. NEMs, can be used to
restrict, diminish, reduce, or inhibit proliferation of tumor or
other abnormal or undesirable vasculature. TEMs can be used to
stimulate the growth of vasculature, such as for wound healing or
to circumvent a blocked vessel. The nucleic acids and encoded
proteins can be administered by any means known in the art. Such
methods include, using liposomes, nanospheres, viral vectors,
non-viral vectors comprising polycations, etc. Suitable viral
vectors include adenovirus, retroviruses, and sindbis virus.
Administration modes can be any known in the art, including
parenteral, intravenous, intramuscular, intraperitoneal, topical,
intranasal, intrarectal, intrabronchial, etc.
[0121] Specific biological antagonists of TEMs can also be used to
therapeutic benefit. For example, antibodies, T cells specific for
a TEM, antisense to a TEM, and ribozymes specific for a TEM can be
used to restrict, inhibit, reduce, and/or diminish tumor or other
abnormal or undesirable vasculature growth. Such antagonists can be
administered as is known in the art for these classes of
antagonists generally. Anti-angiogenic drugs and agents can be used
to inhibit tumor growth, as well as to treat diabetic retinopathy,
rheumatoid arthritis, psoriasis, polycystic kidney disease (PKD),
and other diseases requiring angiogenesis for their
pathologies.
[0122] Mouse counterparts to human TEMS can be used in mouse cancer
models or in cell lines or in vitro to evaluate potential
anti-angiogenic or anti-tumor compounds or therapies. Their
expression can be monitored as an indication of effect. Mouse TEMs
are disclosed in SEQ ID NO: 182-186 and 190-194. Mouse TEMs can be
used as antigens for raising antibodies which can be tested in
mouse tumor models. Mouse TEMs with transmembrane domains are
particularly preferred for this purpose. Mouse TEMs can also be
used as vaccines to raise an immunological response in a human to
the human ortholog.
[0123] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
EXAMPLE 1
Visualization of Vasculature of Colorectal Cancers
[0124] The endothelium of human colorectal cancer was chosen to
address the issues of tumor angiogenesis, based on the high
incidence, relatively slow growth, and resistance to
anti-neoplastic agents of these cancers. While certain less common
tumor types, such as glioblastomas, are highly vascularized and are
regarded as good targets for anti-angiogenic therapy, the
importance of angiogenesis for the growth of human colorectal
cancers and other common solid tumor types is less well
documented.
[0125] We began by staining vessels in colorectal cancers using von
Willebrand Factor (vWF) as a marker. In each of 6 colorectal
tumors, this examination revealed a high density of vessels
throughout the tumor parenchyma (Examples in FIGS. 1A and B).
Interestingly, these analyses also substantiated the importance of
these vessels for tumor growth, as endothelium was often surrounded
by a perivascular cuff of viable cells, with a ring of necrotic
cells evident at the periphery (Example in FIG. 1A). Although these
preliminary studies suggested that colon tumors are
angiogenesis-dependent, reliable markers that could distinguish
vessels in colon cancers from the vessels in normal colon are
currently lacking. One way to determine if such markers exist is by
analyzing gene expression profiles in endothelium derived from
normal and neoplastic tissue.
EXAMPLE 2
Purification of Endothelial Cells
[0126] Global systematic analysis of gene expression in tumor and
normal endothelium has been hampered by at least three experimental
obstacles. First, endothelium is enmeshed in a complex tissue
consisting of vessel wall components, stromal cells, and neoplastic
cells, requiring highly selective means of purifying ECs for
analysis. Second, techniques for defining global gene expression
profiles were not available until recently. And third, only a small
fraction of the cells within a tumor are endothelial, mandating the
development of methods that are suitable for the analysis of global
expression profiles from relatively few cells.
[0127] To overcome the first obstacle, we initially attempted to
purify ECs from dispersed human colorectal tissue using CD31, an
endothelial marker commonly used for this purpose. This resulted in
a substantial enrichment of ECs but also resulted in contamination
of the preparations by hematopoietic cells, most likely due to
expression of CD31 by macrophages. We therefore developed a new
method for purifying ECs from human tissues using P1H12, a recently
described marker for ECs. Unlike CD31, P1H12 was specifically
expressed on the ECs of both colorectal tumors and normal
colorectal mucosa. Moreover, immunofluorescence staining of normal
and cancerous colon with a panel of known cell surface endothelial
markers (e.g. VE-cadherin, CD31 and CD34) revealed that P1H12 was
unique in that it stained all vessels including microvessels (see
FIG. 2A and data not shown). In addition to selection with P1H12,
it was necessary to optimize the detachment of ECs from their
neighbors without destroying their cell surface proteins as well as
to employ positive and negative affinity purifications using a
cocktail of antibodies (FIG. 2B). The ECs purified from normal
colorectal mucosa and colorectal cancers were essentially free of
epithelial and hematopoietic cells as judged by RT-PCR (FIG. 2C)
and subsequent gene expression analysis (see below).
EXAMPLE 3
Comparison of Tumor and Normal Endothelial Cell Expression
Patterns
[0128] To overcome the remaining obstacles, a modification of the
Serial Analysis of Gene Expression (SAGE) technique was used. SAGE
associates individual mRNA transcripts with 14 base pair tags
derived from a specific position near their 3' termini. The
abundance of each tag provides a quantitative measure of the
transcript level present within the mRNA population studied.
[0129] SAGE is not dependent on pre-existing databases of expressed
genes, and therefore provides an unbiased view of gene expression
profiles. This feature is particularly important in the analysis of
cells that constitute only a small fraction of the tissue under
study, as transcripts from these cells are unlikely to be well
represented in extant EST databases. We adapted the SAGE protocol
so that it could be used on small numbers of purified ECs obtained
from the procedure outlined in FIG. 2B. A library of 100,000 tags
from the purified ECs of a colorectal cancer, and a similar library
from the ECs of normal colonic mucosa from the same patient were
generated. These 193,000 tags corresponded to over 32,500 unique
transcripts. Examination of the expression pattern of
hematopoietic, epithelial and endothelial markers confirmed the
purity of the preparations (FIG. 2D).
EXAMPLE 4
Markers of Normal and Tumor Endothelium
[0130] We next sought to identify Pan Endothelial Markers (PEMs),
that is, transcripts that were expressed at significantly higher
levels in both normal and tumor associated endothelium compared to
other tissues. To identify such PEMs, tags expressed at similar
levels in both tumor and normal ECs were compared to 1.8 million
tags from a variety of cell lines derived from tumors of
non-endothelial origin. This simple comparison identified 93
transcripts that were strikingly EC-specific, i.e. expressed at
levels at least 20-fold higher in ECs in vivo compared to
non-endothelial cells in culture. The 15 tags corresponding to
characterized genes which were most highly and specifically
expressed in endothelium are shown in Table 1A. Twelve of these 15
most abundant endothelial transcripts had been previously shown to
be preferentially expressed in endothelium, while the other 3 genes
had not been associated with endothelium in the past (Table 1A).
These data sets also revealed many novel PEMs, which became
increasingly prevalent as tag expression levels decreased (Table
1B). For many of the transcripts, their endothelial origin was
confirmed by SAGE analysis of 401,000 transcripts derived from
primary cultures of human umbilical vein endothelial cells (HUVEC)
and human dermal microvascular endothelial cells (HMVEC) (Table 1A
and B). To further validate the expression of these PEMs in vivo,
we developed a highly sensitive non-radioactive in situ
hybridization method that allowed the detection of transcripts
expressed at relatively low levels in frozen sections of human
tissues. Two uncharacterized markers, PEM3 and PEM6, were chosen
for this analysis. In each case, highly specific expression was
clearly limited to vascular ECs in both normal and neoplastic
tissues (FIGS. 3 A and B and data not shown). These data also
suggest that ECs maintained in culture do not completely
recapitulate expression patterns observed in vivo. For example,
Hevin and several other PEM's were expressed at high levels in both
tumor and normal ECs in vivo, but few or no transcripts were
detected in cultured HUVEC or HMVEC (Table 1). The source of the
Hevin transcripts was confirmed to be endothelium by in situ
hybridization in normal and malignant colorectal tissue (FIG.
3C).
[0131] Many of the markers reported in Table 1 were expressed at
significantly higher levels than previously characterized genes
commonly associated with ECs. For example, the top 25 markers were
all expressed at greater than 200 copies per cell. In contrast, the
receptors for VEGF (VEGFR-1 and VEGFR-2) were expressed at less
than 20 copies per cell. Interestingly, VEGFR2 (KDR), which had
previously been reported to be up-regulated in vessels during colon
cancer progression, was found to be expressed in both normal and
neoplastic colorectal tissue (FIGS. 3 D and E). The lack of
specificity of this gene was in accord with the SAGE data, which
indicated that the VEGFR was expressed at 12 copies per cell in
both normal and tumor endothelium.
EXAMPLE 5
Tumor Versus Normal Endothelium
[0132] We next attempted to identify transcripts that were
differentially expressed in endothelium derived from normal or
neoplastic tissues. This comparison revealed 33 tags that were
preferentially expressed in normal-derived endothelium at levels at
least 10-fold higher than in tumor-derived endothelium. Conversely,
46 tags were expressed at 10-fold or higher levels in tumor
vessels. Because those transcripts expressed at higher levels in
tumor endothelium are most likely to be useful in the future for
diagnostic and therapeutic purposes, our subsequent studies
focussed on this class. Of the top 25 tags most differentially
expressed, 12 tags corresponded to 11 previously identified genes,
one with an alternative polyadenylation site (see Table 2). Of
these 10 genes, 6 have been recognized as markers associated with
angiogenic vessels. The remaining 14 tags corresponded to
uncharacterised genes, most of which have only been deposited as
ESTs (Table 2).
[0133] To validate the expression patterns of these genes, we chose
to focus on 9 Tumor Endothelial Markers (BSC-TEM 1-9; TEM 1, 2, 5,
9, 16, 17, 19, and 22) for which EST sequences but no other
information was available (Table 2). These tags were chosen simply
because they were among the most differentially expressed on the
list and because we were able to obtain suitable probes. In many
cases, this required obtaining near full-length sequences through
multiple rounds of sequencing and cDNA walking (See accession
numbers in Table 2). RT-PCR analysis was then used to evaluate the
expression of the corresponding transcripts in purified ECs derived
from normal and tumor tissues of two patients different from the
one used to construct the SAGE libraries. As shown in FIG. 4 A, the
vWF gene, expected to be expressed in both normal and tumor
endothelium on the basis of the SAGE data as well as previous
studies, was expressed at similar levels in normal and tumor ECs
from both patients, but was not expressed in purified tumor
epithelial cells. As expected, PEM2 displayed a pattern similar to
vWF. In contrast, all 9 TEMs chosen for this analysis were
prominently expressed in tumor ECs, but were absent or barely
detectable in normal ECs (Table 3 and examples in FIG. 4A). It is
important to note that these RT-PCR assays were extremely sensitive
indicators of expression, and the absence of detectable transcripts
in the normal endothelium, combined with their presence in tumor
endothelial RNAs even when diluted 100-fold, provides compelling
confirmatory evidence for their differential expression. These
results also show that these transcripts were not simply expressed
differentially in the ECs of the original patient, but were
characteristic of colorectal cancer endothelium in general.
[0134] It could be argued that the results noted above were
compromised by the possibility that a small number of
non-endothelial cells contaminated the cell populations used for
SAGE and RT-PCR analyses, and that these non-endothelial cells were
responsible for the striking differences in expression of the noted
transcripts. To exclude this possibility, we performed in situ
hybridization on normal and neoplastic colon tissue. In every case
where transcripts could be detected (BSC-TEM 1, 3, 4, 5, 7, 8, and
9; TEM 1, 5, 9, 17, and 19), they were specifically localized to
ECs (Table 3 and examples in FIGS. 4 B and C). Although caution
must be used when interpreting negative in situ hybridization
results, none of the TEMs were expressed in vascular ECs associated
with normal colorectal tissue even though vWF and Hevin were
clearly expressed (Table 3).
EXAMPLE 6
Tumor Endothelium Markers are Expressed in Multiple Tumor Types
[0135] Were these transcripts specifically expressed in the
endothelium within primary colorectal cancers, or were they
characteristic of tumor endothelium in general? To address this
question, we studied the expression of a representative TEM
(BSC-TEM7; TEM 17) in a liver metastasis from a colorectal cancer,
a sarcoma, and in primary cancers of the lung, pancreas, breast and
brain. As shown in FIG. 4, the transcript was found to be expressed
specifically in the endothelium of each of these cancers, whether
metastatic (FIG. 4D) or primary (FIG. 4E-1). Analysis of the other
six TEMs, (BSC-TEM 1, 3, 4, 5, 7, 8 and 9; TEM 1, 5, 9, 17, and 19)
revealed a similar pattern in lung tumors, brain tumors, and
metastatic lesions of the liver (see Table 3).
EXAMPLE 7
Tumor Endothelium Markers are Neo-Angiogenic
[0136] Finally, we asked whether these transcripts were expressed
in angiogenic states other than that associated with tumorigenesis.
We thus performed in situ hybridizations on corpus luteum tissue as
well as healing wounds. Although there were exceptions, we found
that these transcripts were generally expressed both in the corpus
luteum and in the granulation tissue of healing wounds (Table 3 and
example in FIG. 4J). In all tissues studied, expression of the
genes was either absent or exclusively confined to the EC
compartment.
REFERENCES AND NOTES
[0137] The disclosure of each reference cited is expressly
incorporated herein. [0138] 1. J. Folkman, in Cancer Medicine J.
Holland, Bast Jr, R C, Morton D L, Frei III, E, Kufe, D W,
Weichselbaum, R R, Ed. (Williams & Wilkins, Baltimore, 1997)
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[0140] 3. P. Wesseling, D. J. Ruiter, P. C. Burger, J Neurooncol
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W. Hewett, J. L. Brough, C. J. Hawkey, Gastroenterology 111, 1230
(1996). [0144] 7. G. Haraldsen, et al., Gut 37, 225 (1995). [0145]
8. The original EC isolation protocol was the same as that shown in
FIG. 2B except that dispersed cells were stained with anti-CD31
antibodies instead of anti-P1H12, and magnetic beads against CD64
and CD14 were not included in the negative selection. After
generating 120,000 SAGE tags from these two EC preparations,
careful analysis of the SAGE data revealed that, in addition to
endothelial-specific markers, several macrophage-specific markers
were also present. [0146] 9. A. Solovey, et al., N Engl J Med 337,
1584 (1997). [0147] 10. V. E. Velculescu, L. Zhang, B. Vogelstein,
K. W. Kinzler, Science 270, 484-487 (1995). [0148] 11. In order to
reduce the minimum amount of starting material required from
.about.50 million cells to .about.50,000 cells (i.e.
.about.1000-fold less) we and others (38) have introduced several
modifications to the original SAGE protocol. A detailed version of
our modified "MicroSAGE" protocol is available from the authors
upon request. [0149] 12. 96,694 and 96,588 SAGE tags were analyzed
from normal and tumor derived ECs, respectively, and represented
50,298 unique tags. A conservative estimate of 32,703 unique
transcripts was derived by considering only those tags observed
more than once in the current data set or in the 134,000
transcripts previously identified in human transcriptomes (39).
[0150] 13. To identify endothelial specific transcripts, we
normalized the number of tags analyzed in each group to 100,000,
and limited our analysis to transcripts that were expressed at
levels at least 20-fold higher in ECs than in non-endothelial cell
lines in culture and present at fewer than 5 copies per 100,000
transcripts in non-endothelial cell lines and the hematopoietic
fraction (.about.57,000 tags)(41). Non-endothelial cell lines
consisted of 1.8.times.106 tags derived from a total of 14
different cancer cell lines including colon, breast, lung, and
pancreatic cancers, as well as one non-transformed keratinocyte
cell line, two kidney epithelial cell lines, and normal monocytes.
A complete list of PEMs is available at
www.sagenet.org\angio\table1.htm. [0151] 14. M. Tucci, et al., J
Endocrinol 157, 13 (1998). [0152] 15. T. Oono, et al., J Invest
Dermatol 100, 329 (1993). [0153] 16. K. Motamed, Int J Biochem Cell
Biol 31, 1363 (1999). [0154] 17. N. Bardin, et al., Tissue Antigens
48, 531 (1996). [0155] 18. D. M. Bradham, A. Igarashi, R. L.
Potter, G. R. Grotendorst, J Cell Biol 114, 1285 (1991). [0156] 19.
K. Akaogi, et al., Proc Natl Acad Sci USA 93,8384 (1996). [0157]
20. Y. Muragaki, et al., Proc Natl Acad Sci USA 92, 8763 (1995).
[0158] 21. M. L. Iruela-Arispe, C. A. Diglio, E. H. Sage,
Arterioscler Thromb 11, 805 (1991). [0159] 22. J. P. Girard, T. A.
Springer, Immunity 2, 113 (1995). [0160] 23. E. A. Jaffe, et al., J
Immunol 143, 3961 (1989). [0161] 24. J. P. Girard, et al., Am J
Pathol 155, 2043 (1999). [0162] 25. H. Ohtani, N. Sasano, J
Electron Microsc 36, 204 (1987). [0163] 26. For non-radioactive in
situ hybridization, digoxigenin (DIG)-labelled sense and anti-sense
riboprobes were generated through PCR by amplifying 500-600 bp
products and incorporating a T7 promoter into the anti-sense
primer. In vitro transcription was performed using DIG RNA
labelling reagents and T7 RNA polymerase (Roche, Indianapolis,
Ind.). Frozen tissue sections were fixed with 4% paraformaldehyde,
permeabilized with pepsin, and incubated with 200 ng/ml of
riboprobe overnight at 55.degree. C. For signal amplification, a
horseradish peroxidase (HRP) rabbit anti-DIG antibody (DAKO,
Carpinteria, Calif.) was used to catalyse the deposition of
Biotin-Tyramide (from GenPoint kit, DAKO). Further amplification
was achieved by adding HRP rabbit anti-biotin (DAKO),
biotin-tyramide, and then alkaline-phosphatase (AP) rabbit
anti-biotin (DAKO). Signal was detected using the AP substrate Fast
Red TR/Napthol AS-MX (Sigma, St. Louis, Mo.), and cells were
counterstained with hematoxylin unless otherwise indicated. A
detailed protocol including the list of primers used to generate
the probes can be obtained from the authors upon request. [0164]
27. Transcript copies per cell were calculated assuming an average
cell contains 300,000 transcripts. [0165] 28. R. S. Warren, H.
Yuan, M. R. Matli, N. A. Gillett, N. Ferrara, J Clin Invest 95,
1789 (1995). [0166] 29. Y. Takahashi, Y. Kitadai, C. D. Bucana, K.
R. Cleary, L. M. Ellis, Cancer Res 55, 3964 (1995). [0167] 30. L.
F. Brown, et al., Cancer Res 53, 4727 (1993). [0168] 31.
Endothelial-specific transcripts were defined as those expressed at
levels at least 5-fold higher in ECs in vivo than in
non-endothelial cell lines in culture (13), and present at no more
than 5 copies per 100,000 transcripts in non-endothelial cell lines
and the hematopoietic cell fraction (41). Transcripts showing
statistically different levels of expression (P<0.05) were then
identified using Monte Carlo analysis as previously described (40).
Transcripts preferentially expressed in normal endothelium were
then defined as those expressed at levels at least 10-fold higher
in normal endothelium than in tumor endothelium. Conversely, tumor
endothelial transcripts were at least 10-fold higher in tumor
versus normal endothelium. See www.sagenet.org\angio\table2.htm and
www.sagenet.org\angio\table3.htm for a complete list of
differentially expressed genes. [0169] 32. M. Turlaro, et al., Eur
J Clin Invest 29, 793 (1999). [0170] 33. W. S. Lee, et al., Circ
Res 82, 845 (1998). [0171] 34. J. Niquet, A. Represa, Brain Res Dev
Brain Res 95, 227 (1996). [0172] 35. L. Fouser, L. Iruela-Arispe,
P. Bornstein, E. H. Sage, J Biol Chem 266, 18345 (1991). [0173] 36.
M. L. Iruela-Arispe, P. Hasselaar, H. Sage, Lab Invest 64, 174
(1991). [0174] 37. H. F. Dvorak, N Engl J Med 315, 1650 (1986).
[0175] 38. B. Virlon, et al., Proc Natl Acad Sci USA 96, 15286
(1999). [0176] 39. V. E. Velculescu, et al., Nat Genet 23, 387
(1999). [0177] 40. L. Zhang, et al., Science 276, 1268 (1997).
[0178] 41. Human colon tissues were obtained within 1/2 hour after
surgical removal from patients. Sheets of epithelial cells were
peeled away from normal tissues with a glass slide following
treatment with 5 mM DDT, then 10 mM EDTA, leaving the lamina
propria intact. After a 2 h incubation in collagenase at 37.degree.
C., cells were filtered sequentially through 400 um, 100 um, 50 um
and 25 um mesh, and spun through a 30% pre-formed Percoll gradient
to pellet RBCs. Epithelial cells (Epithelial Fraction), which were
found to non-specifically bind magnetic beads, were removed using
Dynabeads coupled to BerEP4 (Dynal, Lake Success, N.Y.).
Subsequently, macrophages and other leukocytes (Hematopoietic
Fraction) were removed using a cocktail of beads coupled to
anti-CD45, anti-CD 14 and anti-CD64 (Dynal). The remaining cells
were stained with P1H12 antibody, purified with anti-mouse
IgG-coupled magnetic beads, and lysed in mRNA lysis buffer. A
detailed protocol can be obtained from the authors upon request.
[0179] 42. H. Sheikh, H. Yarwood, A. Ashworth, C. M. Isacke, J Cell
Sci 113, 1021-32 (2000).
TABLE-US-00002 [0179] SEQ ID SEQ ID Sequence name NO: NO: Sequence
name PEM 1 1 1 PEM 1 PEM 2 2 2 PEM 2 PEM 3 3 3 PEM 3 PEM 4 4 4 PEM
4 PEM 5 5 5 PEM 5 PEM 6 6 6 PEM 6 PEM 7 7 7 PEM 7 PEM 8 8 8 PEM 8
PEM 9 9 9 PEM 9 PEM 10 10 10 PEM 10 PEM 11 11 11 PEM 11 PEM 12 12
12 PEM 12 PEM 13 13 13 PEM 13 PEM 14 14 14 PEM 14 PEM 15 15 15 PEM
15 PEM 16 16 16 PEM 16 PEM 17 17 17 PEM 17 PEM 18 18 18 PEM 18 PEM
19 19 19 PEM 19 PEM 20 20 20 PEM 20 PEM 21 21 21 PEM 21 PEM 22 22
22 PEM 22 PEM 23 23 23 PEM 23 PEM 24 24 24 PEM 24 PEM 25 25 25 PEM
25 PEM 26 26 26 PEM 26 PEM 27 27 27 PEM 27 PEM 28 28 28 PEM 28 PEM
29 29 29 PEM 29 PEM 30 30 30 PEM 30 PEM 31 31 31 PEM 31 PEM 32 32
32 PEM 32 PEM 33 33 33 PEM 33 PEM 34 34 34 PEM 34 PEM 35 35 35 PEM
35 PEM 36 36 36 PEM 36 PEM 37 37 37 PEM 37 PEM 38 38 38 PEM 38 PEM
39 39 39 PEM 39 PEM 40 40 40 PEM 40 PEM 41 41 41 PEM 41 PEM 42 42
42 PEM 42 PEM 43 43 43 PEM 43 PEM 44 44 44 PEM 44 PEM 45 45 45 PEM
45 PEM 46 46 46 PEM 46 PEM 47 47 47 PEM 47 PEM 48 48 48 PEM 48 PEM
49 49 49 PEM 49 PEM 50 50 50 PEM 50 PEM 51 51 51 PEM 51 PEM 52 52
52 PEM 52 PEM 53 53 53 PEM 53 PEM 54 54 54 PEM 54 PEM 55 55 55 PEM
55 PEM 56 56 56 PEM 56 PEM 57 57 57 PEM 57 PEM 58 58 58 PEM 58 PEM
59 59 59 PEM 59 PEM 60 60 60 PEM 60 PEM 61 61 61 PEM 61 PEM 62 62
62 PEM 62 PEM 63 63 63 PEM 63 PEM 64 64 64 PEM 64 PEM 65 65 65 PEM
65 PEM 66 66 66 PEM 66 PEM 67 67 67 PEM 67 PEM 68 68 68 PEM 68 PEM
69 69 69 PEM 69 PEM 70 70 70 PEM 70 PEM 71 71 71 PEM 71 PEM 72 72
72 PEM 72 PEM 73 73 73 PEM 73 PEM 74 74 74 PEM 74 PEM 75 75 75 PEM
75 PEM 76 76 76 PEM 76 PEM 77 77 77 PEM 77 PEM 78 78 78 PEM 78 PEM
79 79 79 PEM 79 PEM 80 80 80 PEM 80 PEM 81 81 81 PEM 81 PEM 82 82
82 PEM 82 PEM 83 83 83 PEM 83 PEM 84 84 84 PEM 84 PEM 85 85 85 PEM
85 PEM 86 86 86 PEM 86 PEM 87 87 87 PEM 87 PEM 88 88 88 PEM 88 PEM
89 89 89 PEM 89 PEM 90 90 90 PEM 90 PEM 91 91 91 PEM 91 PEM 92 92
92 PEM 92 PEM 93 93 93 PEM 93 TEM 1 94 94 TEM 1 TEM 2 95 95 TEM 2
TEM 3 96 96 TEM 3 TEM 4 97 97 TEM 4 TEM 5 98 98 TEM 5 TEM 6 99 99
TEM 6 TEM 7 100 100 TEM 7 TEM 8 101 101 TEM 8 TEM 9 102 102 TEM 9
TEM 10 103 103 TEM 10 TEM 11 104 104 TEM 11 TEM 12 105 105 TEM 12
TEM 13 106 106 TEM 13 TEM 14 107 107 TEM 14 TEM 15 108 108 TEM 15
TEM 16 109 109 TEM 16 TEM 17 110 110 TEM 17 TEM 18 111 111 TEM 18
TEM 19 112 112 TEM 19 TEM 20 113 113 TEM 20 TEM 21 114 114 TEM 21
TEM 22 115 115 TEM 22 TEM 23 116 116 TEM 23 TEM 24 117 117 TEM 24
TEM 25 118 118 TEM 25 TEM 26 119 119 TEM 26 TEM 27 120 120 TEM 27
TEM 28 121 121 TEM 28 TEM 29 122 122 TEM 29 TEM 30 123 123 TEM 30
TEM 31 124 124 TEM 31 TEM 32 125 125 TEM 32 TEM 33 126 126 TEM 33
TEM 34 127 127 TEM 34 TEM 35 128 128 TEM 35 TEM 36 129 129 TEM 36
TEM 37 130 130 TEM 37 TEM 38 131 131 TEM 38 TEM 39 132 132 TEM 39
TEM 40 133 133 TEM 40 TEM 41 134 134 TEM 41 TEM 42 135 135 TEM 42
TEM 43 136 136 TEM 43 TEM 44 137 137 TEM 44 TEM 45 138 138 TEM 45
TEM 46 139 139 TEM 46 NEM 1 140 140 NEM 1 NEM 2 141 141 NEM 2 NEM 3
142 142 NEM 3 NEM 4 143 143 NEM 4 NEM 5 144 144 NEM 5 NEM 6 145 145
NEM 6 NEM 7 146 146 NEM 7 NEM 8 147 147 NEM 8 NEM 9 148 148 NEM 9
NEM 10 149 149 NEM 10 NEM 11 150 150 NEM 11 NEM 12 151 151 NEM 12
NEM 13 152 152 NEM 13 NEM 14 153 153 NEM 14 NEM 15 154 154 NEM 15
NEM 16 155 155 NEM 16 NEM 17 156 156 NEM 17 NEM 18 157 157 NEM 18
NEM 19 158 158 NEM 19 NEM 20 159 159 NEM 20 NEM 21 160 160 NEM 21
NEM 22 161 161 NEM 22 NEM 23 162 162 NEM 23 NEM 24 163 163 NEM 24
NEM 25 164 164 NEM 25 NEM 26 165 165 NEM 26 NEM 27 166 166 NEM 27
NEM 28 167 167 NEM 28 NEM 29 168 168 NEM 29 NEM 30 169 169 NEM 30
NEM 31 170 170 NEM 31 NEM 32 171 171 NEM 32 NEM 33 172 172 NEM 33
TEM 1 DNA 173 173 TEM 1 DNA TEM 2 DNA 174 174 TEM 2 DNA TEM 7 DNA
175 175 TEM 7 DNA TEM 8 DNA 176 176 TEM 8 DNA TEM 1 Protein 177 177
TEM 1 Protein TEM 2 Protein 178 178 TEM 2 Protein TEM 8 Protein 179
179 TEM 8 Protein TEM 5 DNA 180 180 TEM 5 DNA TEM 7B DNA 181 181
TEM 7B DNA mTEM 1 DNA 182 182 mTEM 1 DNA mTEM 5 DNA 183 183 mTEM 5
DNA mTEM 7 DNA 184 184 mTEM 7 DNA mTEM 7B DNA 185 185 mTEM 7B DNA
mTEM 8 DNA 186 186 mTEM 8 DNA TEM 8 Protein 187 187 TEM 8 Protein
TEM 5 Protein 188 188 TEM 5 Protein TEM 7B Protein 189 189 TEM 7B
Protein mTEM 1 Protein 190 190 mTEM 1 Protein mTEM 5 Protein 191
191 mTEM 5 Protein mTEM 7 Protein 192 192 mTEM 7 Protein mTEM 7b
Protein 193 193 mTEM 7b Protein mTEM 8 Protein 194 194 mTEM 8
Protein TEM 1 DNA 195 195 TEM 1 DNA TEM 1 Protein 196 196 TEM 1
Protein TEM 2 DNA 197 197 TEM 2 DNA TEM 2 Protein 198 198 TEM 2
Protein TEM 3 DNA 199 199 TEM 3 DNA TEM 3 Protein 200 200 TEM 3
Protein TEM 4 DNA 201 201 TEM 4 DNA TEM 4 Protein 202 202 TEM 4
Protein TEM 5 DNA 203 203 TEM 5 DNA TEM 5 Protein 204 204 TEM 5
Protein TEM 6 DNA 205 205 TEM 6 DNA TEM 6 Protein 206 206 TEM 6
Protein TEM 7 DNA 207 207 TEM 7 DNA TEM 7 Protein 208 208 TEM 7
Protein TEM 8 DNA 209 209 TEM 8 DNA TEM 8 Protein 210 210 TEM 8
Protein TEM 9 DNA 211 211 TEM 9 DNA TEM 9 Protein 212 212 TEM 9
Protein TEM 10 DNA 213 213 TEM 10 DNA TEM 10 Protein 214 214 TEM 10
Protein TEM 11 DNA 215 215 TEM 11 DNA TEM 11 Protein 216 216 TEM 11
Protein TEM 12 DNA 217 217 TEM 12 DNA TEM 12 Protein 218 218 TEM 12
Protein TEM 13 DNA 219 219 TEM 13 DNA TEM 13 Protein 220 220 TEM 13
Protein TEM 14a DNA 221 221 TEM 14a DNA TEM 14b DNA 222 222 TEM 14b
DNA TEM 14a Protein 223 223 TEM 14a Protein TEM 14b Protein 224 224
TEM 14b Protein TEM 15 DNA 225 225 TEM 15 DNA TEM 15 Protein 226
226 TEM 15 Protein TEM 16 DNA 227 227 TEM 16 DNA TEM 16 Protein 228
228 TEM 16 Protein TEM 17 DNA 229 229 TEM 17 DNA TEM 17 Protein 230
230 TEM 17 Protein TEM 19 DNA 231 231 TEM 19 DNA TEM 19 Protein 232
232 TEM 19 Protein TEM 20 DNA 233 233 TEM 20 DNA TEM 20 Protein 234
234 TEM 20 Protein TEM 21 DNA 235 235 TEM 21 DNA TEM 21 Protein 236
236 TEM 21 Protein TEM 22 DNA 237 237 TEM 22 DNA TEM 22 Protein 238
238 TEM 22 Protein TEM 24 DNA 239 239 TEM 24 DNA TEM 24 Protein 240
240 TEM 24 Protein TEM 25 DNA 241 241 TEM 25 DNA TEM 25 Protein 242
242 TEM 25 Protein TEM 27 DNA 243 243 TEM 27 DNA TEM 27 Protein 244
244 TEM 27 Protein TEM 28 DNA 245 245 TEM 28 DNA
TEM 28 Protein 246 246 TEM 28 Protein TEM 29 DNA 247 247 TEM 29 DNA
TEM 29 Protein 248 248 TEM 29 Protein TEM 30 DNA 249 249 TEM 30 DNA
TEM 30 Protein 250 250 TEM 30 Protein TEM 31 DNA 251 251 TEM 31 DNA
TEM 31 Protein 252 252 TEM 31 Protein TEM 33 DNA 253 253 TEM 33 DNA
TEM 33 Protein 254 254 TEM 33 Protein TEM 35 DNA 255 255 TEM 35 DNA
TEM 35 Protein 358 256 TEM 36 DNA TEM 36 DNA 256 257 TEM 36 Protein
TEM 36 Protein 257 258 TEM 37 DNA TEM 37 DNA 258 259 TEM 37 Protein
TEM 37 Protein 259 260 TEM 38 DNA TEM 38 DNA 260 261 TEM 38 Protein
TEM 38 Protein 261 262 TEM 39 DNA TEM 39 DNA 262 263 TEM 39 Protein
TEM 39 Protein 263 264 TEM 40 DNA TEM 40 DNA 264 265 TEM 40 Protein
TEM 40 Protein 265 266 TEM 41 DNA TEM 41 DNA 266 267 TEM 41 Protein
TEM 41 Protein 267 268 TEM 42 DNA TEM 42 DNA 268 269 TEM 42 Protein
TEM 42 Protein 269 270 TEM 44 DNA TEM 44 DNA 270 271 TEM 44 Protein
TEM 44 Protein 271 272 TEM 45 DNA TEM 45 DNA 272 273 TEM 45 Protein
TEM 45 Protein 273 274 TEM 46 DNA TEM 46 DNA 274 275 TEM 46 Protein
TEM 46 Protein 275 276 NEM 4 DNA NEM 4 DNA 276 277 NEM 4 Protein
NEM 4 Protein 277 278 NEM 14 DNA NEM 14 DNA 278 279 NEM 14 Protein
NEM 14 Protein 279 280 NEM 17 DNA NEM 17 DNA 280 281 NEM 17 Protein
NEM 17 Protein 281 282 NEM 22 DNA NEM 22 DNA 282 283 NEM 22 Protein
NEM 22 Protein 283 284 NEM 23 DNA NEM 23 DNA 284 285 NEM 23 Protein
NEM 23 Protein 285 286 NEM 23 Secreted NEM 23 Secreted 286 287 NEM
23 Short NEM 23 Short 287 288 NEM 33 DNA NEM 33 DNA 288 289 NEM 33
Protein NEM 33 Protein 289 290 mTEM 1 DNA mTEM 1 DNA 290 291 mTEM 1
Protein mTEM 1 Protein 291 292 mTEM 2 DNA mTEM 2 DNA 292 293 mTEM 2
Protein mTEM 2 Protein 293 294 mTEM 9 DNA mTEM 3 DNA 298 295 mTEM 9
Protein mTEM 3 Protein 299 296 mTEM 17 DNA mTEM 9 DNA 294 297 mTEM
17 Protein mTEM 9 Protein 295 298 mTEM 3 DNA mTEM 13 DNA 302 299
mTEM 3 Protein mTEM 13 Protein 303 300 mTEM 19 DNA mTEM 17 DNA 296
301 mTEM 19 Protein mTEM 17 Protein 297 302 mTEM 13 DNA mTEM 19 DNA
300 303 mTEM 13 Protein mTEM 19 Protein 301 304 mTEM 22 DNA mTEM 22
DNA 304 305 mTEM 22 Protein mTEM 22 Protein 305 306 mTEM 30 DNA
mTEM 30 DNA 306 307 mTEM 30 Protein mTEM 30 Protein 307 308 TEM 2
tag TEM 2 tag 308 309 TEM 1 long tag TEM 1 long tag 309 310 TEM 3
long tag TEM 3 long tag 310 311 TEM 4 long tag TEM 4 long tag 311
312 TEM 5 long tag TEM 5 long tag 312 313 TEM 5 long tag TEM 5 long
tag 313 314 TEM 6 long tag TEM 6 long tag 314 315 TEM 7 long tag
TEM 7 long tag 315 316 TEM 8 long tag TEM 8 long tag 316 317 TEM 9
long tag TEM 9 long tag 317 318 TEM 10 long tag TEM 10 long tag 318
319 TEM 10 long tag TEM 10 long tag 319 320 TEM 10 long tag TEM 10
long tag 320 321 TEM 11 long tag TEM 11 long tag 321 322 TEM 12
long tag TEM 12 long tag 322 323 TEM 13 long tag TEM 13 long tag
323 324 TEM 13 long tag TEM 13 long tag 324 325 TEM 14 long tag TEM
14 long tag 325 326 TEM 15 long tag TEM 15 long tag 326 327 TEM 15
long tag TEM 15 long tag 327 328 TEM 16 long tag TEM 16 long tag
328 329 TEM 17 long tag TEM 17 long tag 329 330 TEM 19 long tag TEM
19 long tag 330 331 TEM 21 long tag TEM 21 long tag 331 332 TEM 21
long tag TEM 21 long tag 332 333 TEM 22 long tag TEM 22 long tag
333 334 TEM 22 long tag TEM 22 long tag 334 335 TEM 23 long tag TEM
23 long tag 335 336 TEM 24 long tag TEM 24 long tag 336 337 TEM 25
long tag TEM 25 long tag 337 338 TEM 25 long tag TEM 25 long tag
338 339 TEM 28 long tag TEM 28 long tag 339 340 TEM 30 long tag TEM
30 long tag 340 341 TEM 31 long tag TEM 31 long tag 341 342 TEM 32
long tag TEM 32 long tag 342 343 TEM 33 long tag TEM 33 long tag
343 344 TEM 33 long tag TEM 33 long tag 344 345 TEM 35 long tag TEM
35 long tag 345 346 TEM 36 long tag TEM 36 long tag 346 347 TEM 37
long tag TEM 37 long tag 347 348 TEM 38 long tag TEM 38 long tag
348 349 TEM 38 long tag TEM 38 long tag 349 350 TEM 39 long tag TEM
39 long tag 350 351 TEM 40 long tag TEM 40 long tag 351 352 TEM 41
long tag TEM 41 long tag 352 353 TEM 42 long tag TEM 42 long tag
353 354 TEM 43 long tag TEM 43 long tag 354 355 TEM 44 long tag TEM
44 long tag 355 356 TEM 45 long tag TEM 45 long tag 356 357 TEM 46
long tag TEM 46 long tag 357 358 TEM 35 Protein
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090017030A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090017030A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References