U.S. patent application number 13/932485 was filed with the patent office on 2013-10-24 for stabilized angiopoietin-2 antibodies and uses thereof.
This patent application is currently assigned to MedImmune Limited. The applicant listed for this patent is Vahe Bedian, Michael Bowen, Jeffrey Brown, Andrew Buchanan, William Dall'Acqua, Ralph Minter, Chris Stannard, Herren Wu. Invention is credited to Vahe Bedian, Michael Bowen, Jeffrey Brown, Andrew Buchanan, William Dall'Acqua, Ralph Minter, Chris Stannard, Herren Wu.
Application Number | 20130280251 13/932485 |
Document ID | / |
Family ID | 40913201 |
Filed Date | 2013-10-24 |
United States Patent
Application |
20130280251 |
Kind Code |
A1 |
Bedian; Vahe ; et
al. |
October 24, 2013 |
Stabilized Angiopoietin-2 Antibodies And Uses Thereof
Abstract
Stabilized antibodies directed to Angiopoeitin-2 and uses of
such antibodies are described. Nucleic acid and amino acid
sequences, hybridomas or other cell lines for expressing such
antibodies are also provided.
Inventors: |
Bedian; Vahe; (Waltham,
MA) ; Dall'Acqua; William; (Gaithersburg, MD)
; Wu; Herren; (Gaithersburg, MD) ; Bowen;
Michael; (Gaithersburg, MD) ; Brown; Jeffrey;
(Waltham, MA) ; Stannard; Chris; (Cheshire,
GB) ; Minter; Ralph; (Cambridge, GB) ;
Buchanan; Andrew; (Cambridge, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bedian; Vahe
Dall'Acqua; William
Wu; Herren
Bowen; Michael
Brown; Jeffrey
Stannard; Chris
Minter; Ralph
Buchanan; Andrew |
Waltham
Gaithersburg
Gaithersburg
Gaithersburg
Waltham
Cheshire
Cambridge
Cambridge |
MA
MD
MD
MD
MA |
US
US
US
US
US
GB
GB
GB |
|
|
Assignee: |
MedImmune Limited
Cambridge
GB
|
Family ID: |
40913201 |
Appl. No.: |
13/932485 |
Filed: |
July 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12864544 |
Nov 3, 2010 |
8507656 |
|
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PCT/US09/32224 |
Jan 28, 2009 |
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13932485 |
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61023958 |
Jan 28, 2008 |
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61100063 |
Sep 25, 2008 |
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61142778 |
Jan 6, 2009 |
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Current U.S.
Class: |
424/134.1 ;
424/133.1; 435/320.1; 435/328; 530/387.3; 536/23.53 |
Current CPC
Class: |
A61K 38/1793 20130101;
C07K 16/22 20130101; A61K 31/573 20130101; A61P 35/00 20180101;
C07K 2317/92 20130101; A61P 31/10 20180101; C07K 2317/76 20130101;
A61P 35/04 20180101; A61K 31/4745 20130101; A61K 31/4375 20130101;
A61K 31/519 20130101; A61K 39/3955 20130101; A61P 31/12 20180101;
A61P 33/00 20180101; A61P 37/02 20180101; A61P 19/02 20180101; C07K
2317/21 20130101; A61K 39/39558 20130101; C07K 2317/94 20130101;
A61K 31/496 20130101; A61K 31/7068 20130101; A61P 29/00 20180101;
A61P 31/04 20180101; C07K 2317/73 20130101; A61K 31/282 20130101;
A61P 9/00 20180101; A61P 43/00 20180101; A61K 31/337 20130101; A61K
39/39591 20130101; C07K 2317/56 20130101; A61K 2039/505 20130101;
A61P 1/08 20180101; A61K 31/513 20130101; A61K 39/39558 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
424/134.1 ;
530/387.3; 536/23.53; 435/320.1; 435/328; 424/133.1 |
International
Class: |
C07K 16/22 20060101
C07K016/22; A61K 31/513 20060101 A61K031/513; A61K 38/17 20060101
A61K038/17; A61K 31/7068 20060101 A61K031/7068; A61K 31/282
20060101 A61K031/282; A61K 31/519 20060101 A61K031/519; A61K 39/395
20060101 A61K039/395; A61K 31/4375 20060101 A61K031/4375 |
Claims
1. An isolated antibody that binds to Ang-2, wherein said antibody
comprises a variable light chain, said light chain comprising a
sequence selected from the group consisting of SEQ ID No:3 (MEDI1);
SEQ ID No:4 (MEDI2); SEQ ID No:5 (MEDI3); SEQ ID No:6 (MEDI4); and
SEQ ID No:8 (MEDI6).
2. The antibody of claim 1, wherein said antibody is an IgG1 or an
IgG2 isotype antibody.
3. The antibody of claim 1 or 2, wherein said antibody further
comprises a variable heavy chain region comprising SEQ ID No:7
(MEDI5).
4. The antibody of any of claims 1-3, wherein said antibody, when
produced, exhibits a production efficiency in a mammalian host cell
equal to or greater than 2 times the production efficiency of the
Ang-2 antibody 3.19.3.
5. The antibody of claim 4, wherein said production efficiency is
equal to or greater than 3 times the production efficiency of the
Ang-2 antibody 3.19.3.
6. The antibody of claim 4, wherein said production efficiency is
equal to or greater than 5 times the production efficiency of the
Ang-2 antibody 3.19.3.
7. A nucleic acid encoding the antibody of any of claims 1-6.
8. A vector comprising the nucleic acid of claim 7
9. A host cell comprising the vector of claim 8.
10. A pharmaceutical composition comprising the antibody of any of
claims 1-6 and an excipient.
11. A method of preventing, treating, or managing cancer in an
animal in need thereof, said method comprising administering to
said animal a dose of an effective amount of the composition of
claim 10.
12. A method of preventing metastasis of cancer in an animal in
need thereof, said method comprising administering to said animal a
dose of an effective amount of the composition of claim 10.
13. A method of preventing recurrences of cancer in an animal in
need thereof, said method comprising administering to said animal a
dose of an effective amount of the composition of claim 10.
14. A method of preventing advancement of cancer in an animal in
need thereof, said method comprising administering to said animal a
dose of an effective amount of the composition of claim 10.
15. A method of preventing development of cancer from a
pre-cancerous state in an animal in need thereof, said method
comprising administering to said animal an effective amount of the
composition of claim 10.
16. A method of preventing symptoms of cancer in an animal in need
thereof, said method comprising administering to said animal a dose
of an effective amount of the composition of claim 10.
17. A method of promoting tumor regression of a cancer in an animal
in need thereof, said method comprising administering to said
animal a dose of an effective amount of the composition of claim
10.
18. A method of inhibiting tumor cell proliferation in an animal in
need thereof, said method comprising administering to said animal a
dose of an effective amount of the composition of claim 10.
19. A method of depleting malignant tumor cells in an animal in
need thereof, said method comprising administering to said animal a
dose of an effective amount of the composition of claim 10.
20. A method of inhibiting angiogenesis of a cancer tumor in an
animal in need thereof, said method comprising administering to
said animal a dose of an effective amount of the composition of
claim 10.
21. The method of any of claims 11-20, wherein said method
comprises an additional dosing to said animal of one or more other
cancer therapies.
22. The method of claim 21, wherein said one or more other cancer
therapies are chemotherapies, biological therapies/immunotherapies,
radiation therapies, hormonal therapies, or surgery.
23. The method of any of claims 11-22, wherein said method further
comprises the administration of another therapeutic agent that is
not a cancer therapeutic agent.
24. The method of claim 23, wherein said therapeutic agent is an
anti-emetic agent, anti-fungal agent, anti-parasitic agent,
anti-inflammatory agent, immunomodulatory agent, anti-viral agent,
or antibiotic.
25. The method of any of claims 21-24, wherein said chemotherapy is
selected from the group consisting of 5-Fluorouracil, carboplatin,
and paclitaxel.
26. The method of any of claims 21-25, wherein said immunotherapy
is the administration of bevacizumab or an antibody that competes
for the same epitope as bevacizumab.
27. The method of any of claims 11-26, wherein said cancer or tumor
is selected from the group consisting of melanoma, colon,
colorectal, lung, small cell lung carcinoma, non-small cell lung
carcinoma, breast, rectum, stomach, glioma, prostate, ovary,
testes, thyroid, blood, kidney, liver, hepatocellular carcinoma
pancreas, brain, neck, glioblastoma, endometrial cancer, and
central nervous system cancer.
28. The method of any of claims 11-27, wherein said animal has been
previously treated by administration of one or more cancer
therapies but not by administration of the composition of claim
10.
29. The method of any claims 11-27, wherein said animal has been
previously treated with chemotherapy alone, or in combination with
one or more radiation therapies, biological/immunotherapies,
hormonal therapies or surgery.
30. The method of any of claims 11-27, wherein said animal has been
previously treated with radiation therapy alone, or in combination
with one or more chemotherapies, biological
therapies/immunotherapies, hormonal therapies or surgery.
31. The method of any of claims 11-27, wherein said animal has been
previously treated with biological therapies/immunotherapies alone,
or in combination with one or more chemotherapies, radiation
therapy, hormonal therapies or surgery.
32. The method of any of claims 11-27, wherein said animal has been
previously treated with hormonal therapies alone, or in combination
with one or more chemotherapies, radiation therapy, biological
therapies/immunotherapies or surgery.
33. The method of any of claims 11-27, wherein said animal has been
previously treated with surgery alone, or in combination with one
or more chemotherapies, radiation therapy, hormonal therapies or
biological therapies/immunotherapies.
34. The method of any of claims 11-33, wherein said cancer is
refractory to chemotherapy or radiation therapy.
35. The method of any of claims 11-34, wherein said administration
is intravenously, subcutaneously, intratumorally, intramuscularly,
parenterally, or orally.
36. The method of any of claims 21-35, wherein said composition and
cancer therapy are administered by the same mode of
administration.
37. The method of any of claims 21-35, wherein said composition and
cancer therapy are administered by a different mode of
administration.
38. The method of any of claims 21-35, wherein said composition and
cancer therapy are administered in the same dosage form.
39. The method of any of claims 21-35, wherein said composition and
cancer therapy are administered in different dosage forms.
40. The method of any of claims 21-39, wherein said cancer therapy
is selected from the group consisting of radiation therapies,
biological therapies/immunotherapies, hormonal therapies and
surgery.
41. A method of preventing, treating, or managing disease-related
angiogenesis in an animal in need thereof, said method comprising
administering to said animal a dose of an effective amount of the
composition of claim 10.
42. The method of claim 41, wherein the disease-related
angiogenesis is associated with seronegative arthritis,
seropositive arthritis, arthritis related to other arthropathies,
osteoarthritis or SLE.
43. The method of claim 42, wherein the seropositive arthritis is
rheumatoid arthritis.
44. A method of preventing recurrences of disease-related
angiogenesis in an animal in need thereof, said method comprising
administering to said animal a dose of an effective amount of the
composition of claim 10.
45. A method of preventing advancement of disease-related
angiogenesis in an animal in need thereof, said method comprising
administering to said animal a dose of an effective amount of the
composition of claim 10.
46. A method of treating rheumatoid arthritis in an animal in need
thereof, said method comprising administering to said animal an
effective amount of the composition of claim 10.
47. A method of preventing symptoms of disease-related angiogenesis
in an animal in need thereof, said method comprising administering
to said animal a dose of an effective amount of the composition of
claim 10.
48. The method of any of claims 41-47, wherein said method
comprises an additional dosing to said animal of one or more other
anti-inflammatory therapies.
49. The method of claim 48, wherein said one or more other
anti-inflammatory therapies are chemotherapies, biological
therapies/immunotherapies, radiation therapies, hormonal therapies,
or surgery.
50. The method of claim 49, wherein said biological
therapy/immunotherapy is a TNF-.alpha. antagonist.
51. The method of claim 50, wherein said TNF-.alpha. is selected
from etanercept (ENBREL.RTM.), adalimumab (HUMIRA.RTM.), and
infliximab (REMICADE.RTM.).
52. The method of any of claims 41-51, wherein said method further
comprises the administration of another therapeutic agent that is
not an anti-inflammatory therapeutic agent.
53. The method of claim 52, wherein said therapeutic agent is an
anti-emetic agent, anti-fungal agent, anti-parasitic agent,
anti-cancer agent, immunomodulatory agent, anti-viral agent, or
antibiotic.
54. The method of any of claims 41-53, wherein said animal has been
previously treated by administration of one or more
anti-inflammatory therapies but not by administration of the
composition of claim 10.
55. The method of any of claims 41-53, wherein said animal has been
previously treated with chemotherapy alone, or in combination with
one or more radiation therapies, biological/immunotherapies,
hormonal therapies or surgery.
56. The method of any of claims 41-53, wherein said animal has been
previously treated with radiation therapy alone, or in combination
with one or more chemotherapies, biological
therapies/immunotherapies, hormonal therapies or surgery.
57. The method of any of claims 41-53, wherein said animal has been
previously treated with biological therapies/immunotherapies alone,
or in combination with one or more chemotherapies, radiation
therapy, hormonal therapies or surgery.
58. The method of any of claims 41-53, wherein said animal has been
previously treated with hormonal therapies alone, or in combination
with one or more chemotherapies, radiation therapy, biological
therapies/immunotherapies or surgery.
59. The method of any of claims 41-53, wherein said animal has been
previously treated with surgery alone, or in combination with one
or more chemotherapies, radiation therapy, hormonal therapies or
biological therapies/immunotherapies.
60. A method of reducing endothelial cell proliferation in an
animal, said method comprising administration of a dose of an
effective amount of the composition of claim 10.
61. A method of inhibiting Ang-2 and/or Ang-1 binding to Tie-2 in
an animal, said method comprising administration of a dose of an
effective amount of the composition of claim 10.
62. A method of inhibiting Tie-2 phosphorylation in an animal, said
method comprising administration of a dose of an effective amount
of the composition of claim 10.
63. A method of reducing levels of circulating Ang-2 and/or Ang-1
polypeptide in an animal, said method comprising administration of
a dose of an effective amount of the composition of claim 10.
64. A pharmaceutical composition comprising a combination of i) an
antagonist of the biological activity of Angiopoietin-2 and/or
Tie-2, and ii) an antagonist of the biological activity of CSF1R,
and/or CSF1.
65. The composition according to claim 64, wherein the antagonist
of Angiopoietin-2 is an antibody.
66. The composition according to claim 65, wherein the antagonist
of Angiopoietin-2 is a fully human monoclonal antibody.
67. The composition according to claim 65 or 66, wherein the
antibody binds to the same epitope as any one of fully human
monoclonal antibodies selected from the group consisting of 3.31.2,
5.16.3, 5.86.1, 5.88.3, 3.3.2, 5.103.1, 5.101.1, 3.19.3, 5.28.1,
5.78.3, MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, and MEDI4/5.
68. The composition according to claim 65, wherein the antibody is
a fully human monoclonal antibody selected from the group
consisting of: 33.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2, 5.103.1,
5.101.1, 3.19.3, 5.28.1, 5.78.3, MEDI1/5, MEDI2/5, MEDI3/5,
MEDI6/5, and MEDI4/5.
69. The composition according to any of claims 64-68, wherein the
antagonist of the biological activity of CSF1R is a tyrosine kinase
inhibitor.
70. The composition according to claim 69, wherein the antagonist
of the biological activity of CSF1R is selected from any one of:
2-chloro-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;
2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-(trifluoromethyl)benzoyl]amino}be-
nzamide;
2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-fluoro-5-(trifluoromethyl-
)benzoyl]amino}-benzamide;
2-methyl-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;
5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-pyridin-3-ylben-
zamide;
2-chloro-5-[(3-cyclopropylbenzoyl)amino]-N-pyridin-3-ylbenzamide;
2-chloro-5-[(3-chlorobenzoyl)amino]-N-pyridin-3-ylbenzamide;
5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;
5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide-
; 5-[(3-chlorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;
5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-thia-
zol-5-yl)benzamide;
2-chloro-N-1,3-thiazol-5-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamid-
e; 2-chloro-5-[(3-chlorobenzoyl)amino]-N-1,3-thiazol-5-ylbenzamide;
2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-1,3-thiazol-5-ylbenzamide;
5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-1,3-thiazol-5-ylbe-
nzamide;
2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)ben-
zoyl]amino}benzamide;
2-chloro-5-[(3-chlorobenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamid-
e;
2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)be-
nzamide;
2-chloro-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)ben-
zoyl]amino}benzamide;
2-chloro-5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-N-(2-methyl-1,3-t-
hiazol-5-yl)benzamide;
5-[(5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methylbenzoyl)amino]-N--
methyl-1,3-thiazole-2-carboxamide;
5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-t-
hiazol-5-yl)benzamide;
5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl-
)benzamide;
5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-
-5-yl)benzamide;
5-[(3-chlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamid-
e;
5-[3,4-dichlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)ben-
zamide;
5-[(3-cyclopropylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-
-yl)benzamide;
5-[(3,5-dimethylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benz-
amide;
2-methyl-5-[(3-methylbenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)be-
nzamide;
2,6-dichloro-N-(4-methyl-3-{[(2-methyl-1,3-thiazol-5-yl)amino]car-
bonyl}phenyl)isonicotinamide;
2-methyl-5-{[(3-methylcyclohexyl)carbonyl]amino}-N-(2-methyl-1,3-thiazol--
5-yl)benzamide;
2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-(pentanoylamino)benzamide;
2-methyl-5-[(4-methylhexanoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzami-
de;
4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quino-
line-3-carboxamide;
4-[(2,3-dichlorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinolin-
e-3-carboxamide;
7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)-
quinoline-3-carboxamide;
4-[(3-chloro-2-fluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)qui-
noline-3-carboxamide;
7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)qui-
noline-3-carboxamide;
7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)qui-
noline-3-carboxamide;
4-[(2,4-difluorophenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-1--
yl)quinoline-3-carboxamide;
4-[(2-fluoro-4-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperaz-
in-1-yl)quinoline-3-carboxamide;
4-[(2-fluoro-5-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperaz-
in-1-yl)quinoline-3-carboxamide;
4-[(2-fluoro-4-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)-7-(2-met-
hoxyethoxy)quinoline-3-carboxamide;
7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-methylpiperidin-4-yl)qui-
noline-3-carboxamide;
4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-methylpiperidin-4-yl)quinolin-
e-3-carboxamide;
4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-isopropylpiperidin-4-yl)quino-
line-3-carboxamide;
7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-
quinoline-3-carboxamide;
4-[(2-fluoro-4-methylphenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)qu-
inoline-3-carboxamide;
4-[(3-chloro-2-fluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)qu-
inoline-3-carboxamide;
4-[(2,4-difluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)quinoli-
ne-3-carboxamide;
4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methox-
yquinoline-3-carboxamide;
4-[(2,4-difluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxyquin-
oline-3-carboxamide; and
4-[(3-chloro-2-fluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methox-
yquinoline-3-carboxamide;
7-Ethoxy-4-[(2-fluoro-4-methyl-phenyl)amino]-6-(4-methylpiperazin-1-yl)ci-
nnoline-3-carboxamide;
4-(2-Fluoro-4-methylphenylamino)-7-methoxy-6-(4-methylpiperazin-1-yl)cinn-
oline-3-carboxamide;
4-[(2,4-Difluorophenyl)amino]-7-methoxy-6-(4-methylpiperazin-1-yl)cinnoli-
ne-3-carboxamide;
6-[(3R,5S)-3,5-Dimethylpiperazin-1-yl]-4-[(2-fluoro-4-methylphenyl)amino]-
-7-methoxycinnoline-3-carboxamide;
4-[(2-Fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazin-1-yl]-7-
-methoxycinnoline-3-carboxamide;
7-Ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazi-
n-1-yl]cinnoline-3-carboxamide;
4-[(3-Chloro-2-fluorophenyl)amino]-6-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-
-7-methoxycinnoline-3-carboxamide;
4-[(2-Fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methox-
ycinnoline-3-carboxamide hydrochloride;
4-[(2-Fluoro-4-methylphenyl)amino]-6-[1-(2-hydroxyethyl)piperidin-4-yl]-7-
-methoxycinnoline-3-carboxamide; and
4-[(2-Fluoro-4-methylphenyl)amino]-6-{4-[(2R)-2-hydroxypropanoyl]piperazi-
n-1-yl}-7-methoxycinnoline-3-carboxamide; or a pharmaceutically
acceptable salt thereof.
71. A pharmaceutical composition comprising a combination according
to any one of claims 62 to 70, in association with a
pharmaceutically-acceptable excipient or carrier.
72. A pharmaceutical composition according to claim 71, for use in
the treatment of disease-related angiogenesis or inflammation.
73. A pharmaceutical composition according to claim 71, for use in
the treatment of cancer.
74. A method of treating disease-related angiogenesis or
inflammation in an animal in need thereof with a combination
according to any one of claims 62-73.
75. A method of treating cancer in an animal in need thereof with a
combination according to any one of claims 62-73.
76. A composition comprising an antagonist of the biological
activity of Angiopoietin-2, and/or Tie-2; and a chemotherapeutic
agent.
77. The composition according to claim 76, wherein the antagonist
of Angiopoietin-2 is an antibody.
78. The composition according to claim 77, wherein the antagonist
of Angiopoietin-2 is a fully human monoclonal antibody.
79. The composition according to any one of claim 77 or 78, wherein
the antibody binds to the same epitope as an antibody selected from
the group consisting of 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2,
5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3, MEDI1/5, MEDI2/5,
MEDI3/5, MEDI6/5, and MEDI4/5.
80. The composition according to claim 78, wherein the antibody is
a fully human monoclonal antibody selected from the group
consisting of 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2, 5.103.1,
5.101.1, 3.19.3, 5.28.1, 5.78.3, MEDI1/5, MEDI2/5, MEDI3/5,
MEDI6/5, and MEDI4/5.
81. The composition according to any of claims 76-80, wherein the
chemotherapeutic agent is selected from the group consisting of
docetaxel, AZD4877, vincristine, vinblastine, vindesine and
vinorelbine, taxol, taxotere, 5-fluorouracil, gemcitabine,
fluoropyrimidines tegafur, raltitrexed, capecitabine, methotrexate,
pemetrexed, cytosine arabinoside, hydroxyurea; irinotecan,
etoposide topotecan, camptothecin teniposide, amsacrine,
oxaliplatin, cisplatin oxaliplatin, 5-fluorouracil, irinotecan,
gemcitabine and carboplatin.
82. The composition according to any of claims 76 to 81 in
association with a pharmaceutically acceptable excipient or
carrier.
83. A method of antagonizing the biological activity of
Angiopoietin-2, and/or Tie-2 comprising administering the
composition according to any of claims 76 to 82.
84. A method of producing an anti-cancer effect in a patient
comprising administering a therapeutically effective amount of a
composition of any one of claims 76 to 82.
85. A method of reducing tumor growth in an animal comprising
administering a therapeutically effective amount of a composition
of any one of claims 76 to 82.
Description
I. FIELD
[0001] The invention relates to stabilized monoclonal antibodies
against Angiopoietin-2 (Ang-2) and uses of such antibodies. Aspects
of the invention also relate to hybridomas or other cell lines
expressing such antibodies. The described antibodies are useful as
diagnostics and for the treatment of diseases associated with the
activity of Ang-2 and/or Ang-1.
II. BACKGROUND
[0002] Angiogenesis is the process of forming new capillaries from
preexisting blood vessels and is an essential component of
embryogenesis, normal physiological growth, repair, and tumor
expansion. Although a variety of factors can modulate endothelial
cell (EC) responses in vitro and blood vessel growth in vivo, only
vascular endothelial growth factor (VEGF) family members and the
angiopoietins are believed to act almost exclusively on vascular
ECs (Yancopoulos et al., Nature 407:242-48 (2000)).
[0003] The angiopoietins were discovered as ligands for the Ties, a
family of tyrosine kinases that is selectively expressed within the
vascular endothelium (Yancopoulos et al., Nature 407:242-48
(2000)). There are now four definitive members of the angiopoietin
family: Angiopoietin-3 and -4 (Ang-3 and Ang-4) may represent
widely diverged counterparts of the same gene locus in mouse and
man (Kim et al., FEBS Let, 443:353-56 (1999); Kim et al., J Biol
Chem 274:26523-28 (1999)). Ang-1 and Ang-2 were originally
identified in tissue culture experiments as agonist and antagonist,
respectively (Davis et al., Cell 87:1161-69 (1996); Maisonpierre et
al., Science 277:55-60 (1997)). All of the known angiopoietins bind
primarily to Tie2, and both Ang-1 and -2 bind to Tie2 with an
affinity of 3 nM (Kd) (Maisonpierre et al., Science 277:55-60
(1997)). Ang-1 was shown to support EC survival and to promote
endothelium integrity, (Davis et al., Cell 87:1161-69 (1996); Kwak
et al., FEBS Lett 448:249-53 (1999); Suri et al., Science
282:468-71 (1998); Thurston et al., Science 286: 2511-14 (1999);
Thurston et al., Nat. Med. 6:460-63 (2000)), whereas Ang-2 had the
opposite effect and promoted blood vessel destabilization and
regression in the absence of the survival factors VEGF or basic
fibroblast growth factor (Maisonpierre et al., Science 277:55-60
(1997)). However, many studies of Ang-2 function have suggested a
more complex situation. Ang-2 might be a complex regulator of
vascular remodeling that plays a role in both vessel sprouting and
vessel regression. Supporting such roles for Ang-2, expression
analyses reveal that Ang-2 is rapidly induced, together with VEGF,
in adult settings of angiogenic sprouting, whereas Ang-2 is induced
in the absence of VEGF in settings of vascular regression (Holash
et al., Science 284:1994-98 (1999); Holash et al., Oncogene
18:5356-62 (1999)). Consistent with a context-dependent role, Ang-2
binds to the same endothelial-specific receptor, Tie-2, which is
activated by Ang-1, but has context-dependent effects on its
activation (Maisonpierre et al., Science 277:55-60 (1997)).
[0004] Corneal angiogenesis assays have shown that both Ang-1 and
Ang-2 had similar effects, acting synergistically with VEGF to
promote growth of new blood vessels (Asahara et al., Circ. Res.
83:233-40 (1998)). The possibility that there was a dose-dependent
endothelial response was raised by the observation that in vitro at
high concentration, Ang-2 can also be pro-angiogenic (Kim et al.,
Oncogene 19:4549-52 (2000)). At high concentration, Ang-2 acts as
an apoptosis survival factor for endothelial cells during serum
deprivation apoptosis through activation of Tie2 via PI-3 kinase
and Akt pathway (Kim et al., Oncogene 19:4549-52 (2000)).
[0005] Other in vitro experiments suggested that during sustained
exposure, the effects of Ang-2 may progressively shift from that of
an antagonist to an agonist of Tie2, and at later time points, it
may contribute directly to vascular tube formation and neovessel
stabilization (Teichert-Kuliszewska et al., Cardiovasc. Res.
49:659-70 (2001)). Furthermore, if ECs were cultivated on fibrin
gel, activation of Tie2 with Ang-2 was also observed, perhaps
suggesting that the action of Ang-2 could depend on EC
differentiation state (Teichert-Kuliszewska et al., Cardiovasc.
Res. 49:659-70 (2001)). In microvascular EC cultured in a
three-dimensional collagen gel, Ang-2 can also induce Tie2
activation and promote formation of capillary-like structures
(Mochizuki et al., J. Cell. Sci. 115:175-83 (2002)). Use of a 3-D
spheroidal coculture as an in vitro model of vessel maturation
demonstrated that direct contact between ECs and mesenchymal cells
abrogates responsiveness to VEGF, whereas the presence of VEGF and
Ang-2 induced sprouting (Korff et al., Faseb J. 15:447-57 (2001)).
Etoh et al. demonstrated that ECs that constitutively express Tie2,
the expression of MMP-1, -9 and u-PA were strongly up-regulated by
Ang-2 in the presence of VEGF (Etoh, et al., Cancer Res. 61:2145-53
(2001)). With an in vivo pupillary membrane model, Lobov et al.
showed that Ang-2 in the presence of endogenous VEGF promotes a
rapid increase in capillary diameter, remodeling of the basal
lamina, proliferation and migration of endothelial cells, and
stimulates sprouting of new blood vessels (Lobov et al., Proc.
Natl. Acad. Sci. USA 99:11205-10 (2002)). By contrast, Ang-2
promotes endothelial cell death and vessel regression without
endogenous VEGF (Lobov et al., Proc. Natl. Acad. Sci. USA
99:11205-10 (2002)). Similarly, with an in vivo tumor model,
Vajkoczy et al. demonstrated that multicellular aggregates initiate
vascular growth by angiogenic sprouting via the simultaneous
expression of VEGFR-2 and Ang-2 by host and tumor endothelium
(Vajkoczy et al., J. Clin. Invest. 109:777-85 (2002)). This model
illustrated that the established microvasculature of growing tumors
is characterized by a continuous remodeling, putatively mediated by
the expression of VEGF and Ang-2.
[0006] Knock-out mouse studies of Tie-2 and Angiopoietin-1 show
similar phenotypes and suggest that Angiopoietin-1 stimulated Tie-2
phosphorylation mediates remodeling and stabilization of developing
vessel, promoting blood vessel maturation during angiogenesis and
maintenance of endothelial cell-support cell adhesion (Dumont et
al., Genes & Development, 8:1897-1909 (1994); Sato, Nature,
376:70-74 (1995); (Thurston, G. et al., 2000 Nature Medicine: 6,
460-463)). The role of Angiopoietin-1 is thought to be conserved in
the adult, where it is expressed widely and constitutively
(Hanahan, Science, 277:48-50 (1997); Zagzag, et al., Exp Neurology,
159:391-400 (1999)). In contrast, Angiopoietin-2 expression is
primarily limited to sites of vascular remodeling where it is
thought to block the constitutive stabilizing or maturing function
of Angiopoietin-1, allowing vessels to revert to, and remain in, a
plastic state which may be more responsive to sprouting signals
(Hanahan, 1997; Holash et al., Oncogene 18:5356-62 (1999);
Maisonpierre, 1997). Studies of Angiopoietin-2 expression in
pathological angiogenesis have found many tumor types to show
vascular Angiopoietin-2 expression (Maisonpierre et al., Science
277:55-60 (1997)). Functional studies suggest Angiopoietin-2 is
involved in tumor angiogenesis and associate Angiopoietin-2
overexpression with increased tumor growth in a mouse xenograft
model (Ahmad, et al., Cancer Res., 61:1255-1259 (2001)). Other
studies have associated Angiopoietin-2 overexpression with tumor
hypervascularity (Etoh, et al., Cancer Res. 61:2145-53 (2001);
Tanaka et al., Cancer Res. 62:7124-29 (2002)).
[0007] In recent years Angiopoietin-1, Angiopoietin-2 and/or Tie-2
have been proposed as possible anti-cancer therapeutic targets
(See, for example, U.S. Pat. Nos. 6,166,185, 5,650,490, 5,814,464,
US Patent Publication No. 20060018909 and PCT publication Nos.
WO2006/068953 and WO2007/068895).
[0008] Ang-2 is expressed during development at sites where blood
vessel remodeling is occurring (Maisonpierre et al., Science
277:55-60 (1997)). In adult individuals, Ang-2 expression is
restricted to sites of vascular remodeling as well as in highly
vascularized tumors, including glioma (Osada et al., Int. J. Oncol.
18:305-09 (2001); Koga et al., Cancer Res. 61:6248-54 (2001)),
hepatocellular carcinoma, (Tanaka et al, J. Clin. Invest.
103:341-45 (1999)), gastric carcinoma, (Etoh, et al., Cancer Res.
61:2145-53 (2001); Lee et al, Int. J. Oncol. 18:355-61 (2001)),
thyroid tumor (Bunonc et al., Am J Pathol 155:1967-76 (1999)),
non-small cell lung cancer (Wong et al., Lung Cancer 29:11-22
(2000)), cancer of colon (Ahmad et al., Cancer 92:1138-43 (2001)),
and prostate Wurmbach et al., Anticancer Res. 20:5217-20 (2000)).
Some tumor cells are found to express Ang-2. For example, Tanaka et
al. (1999) detected Ang-2 mRNA in 10 out of 12 specimens of human
hepatocellular carcinoma (HCC). Ellis' group reported that Ang-2 is
expressed ubiquitously in tumor epithelium (Ahmad et al., Cancer
92:1138-43 (2001)). Other investigators reported similar findings
(Chen et al., J. Tongji Med. Univ. 21:228-30, 235 (2001)). By
detecting Ang-2 mRNA levels in archived human breast cancer
specimens, Sfilogoi et al. (Int. J. Cancer 103:466-74 (2003))
reported that Ang-2 mRNA is significantly associated with auxiliary
lymph node invasion, short disease-free time and poor overall
survival. Tanaka et al. (Cancer Res. 62:7124-29 (2002) reviewed a
total of 236 patients of non-small cell lung cancer (NSCLC) with
pathological stage-I to -IIIA, respectively. Using
immunohistochemistry, they found that 16.9% of the NSCLC patients
were Ang-2 positive. The microvessel density for Ang-2 positive
tumor is significantly higher than that of Ang-2 negative. Such an
angiogenic effect of Ang-2 was seen only when VEGF expression was
high. Moreover, positive expression of Ang-2 was a significant
factor to predict a poor postoperative survival. However, they
found no significant correlation between Ang-1 expression and the
microvessel density (Tanaka et al., Cancer Res. 62:7124-29 (2002)).
These results suggest that Ang-2 is an indicator of poor prognosis
patients with several types of cancer.
[0009] The development of antibody therapeutics for the treatment
of disease is a complex process in which candidate molecules must
pass through multiple tests to ensure suitability in every
application. In most cases, the initial candidates are developed
based on a pre-determined group of desired characteristics, such as
antigen affinity, antibody format, and others. Once a candidate
molecule is chosen, the suitability for large scale production and
stability are considered. Often, the candidate molecule, although
highly applicable based on initial desired characteristics, needs
to be refined to ensure the prolonged stability and high production
efficiency required for feasibility as a commercial
therapeutic.
[0010] Disulfide bond formation in proteins is a complex process,
which is determined by the redox potential of the environment and
specialized thiol-disulfide exchanging enzymes (Creighton, Methods
Enzymol. 107, 305-329, 1984; Houee-Levin, Methods Enzymol. 353,
35-44, 2002). The disulfides are formed in cells during or shortly
after secretion of the nascent chains into the endoplasmic
reticulum. Several conformational isoforms of the same protein, but
with different disulfide structures, can be generated during
recombinant protein production in mammalian cells due to the
failing disulfide formation process, close proximity of cysteine
residues in the protein structure or surface exposure of unpaired
cysteine residues.
[0011] In general, cysteine residues in proteins (for example,
antibodies specific for Ang-2) are either engaged in
cysteine-cysteine disulfide bonds or sterically protected from the
disulfide bond formation when they are a part of folded protein
region. When a cysteine residue does not have a pair in protein
structure and is not sterically protected by folding, it can form a
disulfide bond with a free cysteine from solution in a process
known as disulfide shuffling. In another process known as disulfide
scrambling, free cysteines may also interfere with naturally
occurring disulfide bonds (such as those present in antibody
structures) and lead to low binding, low biological activity and/or
low stability.
[0012] Glycosylation of immunoglobulins has also been shown to have
significant effects on their binding characteristics, effector
functions, structural stability, and rate of secretion from
antibody-producing cells (Leatherbarrow et al., Mol. Immunol.
22:407 (1985)). In particular, glycosylation of the variable region
of antibodies may influence the interaction of the antibody with
its cognate antigen. It has been shown that glycosylation in the
variable region can have a negative effect on antibody binding
affinity, likely due to steric hindrance (Co, M. S., et al., Mol.
Immunol. (1993) 30:1361-1367). The heterogeneity of the
glycosylation process may also lead to a number of antibody species
with altered binding properties. As such, it is desirable to remove
or alter the interfering glycosylation site to ensure a consistent
antigen binding profile.
[0013] Thus, there is a need to develop highly stable antibodies
specific for Ang-2 for a variety of therapeutic and diagnostic
applications.
[0014] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
III. SUMMARY
[0015] One aspect of the invention provides certain antibodies
directed to Angiopoietin-2 (hereinafter referred to as "antibodies
of the invention") which are stable and do not readily aggregate in
certain pharmaceutical formulations.
[0016] In one embodiment, an Ang-2 antibody is provided which
comprises a light chain shown as MEDI1, MEDI2, MEDI3, MEDI6 or
MEDI4 and/or a heavy chain shown as MEDI5.
[0017] Antibodies of the invention have the ability to specifically
bind Ang-2 and inhibit tumor angiogenesis and reduce tumor growth.
Mechanisms by which this can be achieved can include, but are not
limited to, either inhibition of binding of Ang-2 and/or Ang1 to
its receptor Tie2, inhibition of Ang-2 and/or Ang-1 induced Tie2
signaling, inhibition of Ang-2 and/or Ang-1 induced Tie2
phosphorylation, or increased clearance of Ang-2 and/or Ang1,
therein reducing the effective concentration of Ang-2 and/or
Ang-1.
[0018] In one embodiment, the antibodies of the invention exhibit
enhanced stability as compared to the control Ang-2 specific
antibody 3.19.3. In another embodiment, the antibodies of the
invention exhibit enhanced production yields as compared to a
control Ang-2 specific antibody. In one embodiment, the antibody of
the invention is a human Ang-2 antibody which comprises a Val
substitution at position 37 of the heavy chain as defined by the EU
numbering system ((Kabat et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, NIH Publication 91-3242,
Bethesda Md. (1991), vols. 1-3.), and a substitution of Asp, Thr,
Asn, or Ala at position 49 of the light chain based on the Kabat
numbering system.
[0019] In another embodiment, the antibodies of the invention may
comprise a variable light chain amino acid sequence selected from
the group consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4),
MEDI3 (SEQ ID No.:5), MEDI6 (SEQ ID No.:8) and MEDI4 (SEQ ID
No.:6). In another embodiment, the antibodies of the invention may
comprise the heavy chain variable amino acid sequence MEDI5 (SEQ ID
No.:7). In another embodiment, the antibodies of the invention may
comprise a variable light chain amino acid sequence selected from
the group consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4),
MEDI3 (SEQ ID No.:5), MEDI6 (SEQ ID No.:8) and MEDI4 (SEQ ID No.:6)
as well as a heavy chain variable sequence defined as MEDI5 (SEQ ID
No.:7).
[0020] In another aspect, the invention also provides nucleic acid
sequences, vectors and cell lines for expression of the antibodies
of the invention.
[0021] The invention further provides methods for assaying the
level of Angiopoietin-2 (Ang-2) in a patient sample, comprising
contacting an anti-Ang-2 antibody with a biological sample from a
patient, and detecting the level of binding between said antibody
and Ang-2 in said sample. In more specific embodiments, the
biological sample is blood.
[0022] In other embodiments the invention provides compositions,
including an antibody or functional fragment thereof, and a
pharmaceutically acceptable carrier.
[0023] Further embodiments include methods of effectively treating
an animal suffering from an angiogenesis-related disease, including
selecting an animal in need of treatment for a neoplastic or
non-neoplastic disease, and administering to said animal a
therapeutically effective dose of a monoclonal antibody of the
invention.
[0024] Treatable angiogenesis-related diseases can include
neoplastic diseases, such as, melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, thyroid tumor, gastric (stomach) cancer, prostate
cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer,
glioblastoma, endometrial cancer, kidney cancer, colon cancer,
pancreatic cancer, esophageal carcinoma, head and neck cancers,
mesothelioma, sarcomas, biliary (cholangiocarcinoma), small bowel
adenocarcinoma, pediatric malignancies and epidermoid
carcinoma.
[0025] Additional embodiments include methods of inhibiting
Angiopoietin-2 (Ang-2) induced angiogenesis in an animal. These
methods include selecting an animal in need of treatment for Ang-2
induced angiogenesis, and administering to said animal a
therapeutically effective dose of an antibody of the invention.
[0026] Further embodiments include the use of an antibody of the
invention in the preparation of medicament for the treatment of
angiogenesis-related diseases in an animal, wherein said antibody
specifically binds to Angiopoietin-2 (Ang-2). Treatable
angiogenesis-related diseases can include neoplastic diseases, such
as, melanoma, small cell lung cancer, non-small cell lung cancer,
glioma, hepatocellular (liver) carcinoma, thyroid tumor, gastric
(stomach) cancer, prostate cancer, breast cancer, ovarian cancer,
bladder cancer, lung cancer, glioblastoma, endometrial cancer,
kidney cancer, colon cancer, pancreatic cancer, esophageal
carcinoma, head and neck cancers, mesothelioma, sarcomas,
cholangiocarcinoma, small bowel adenocarcinoma, pediatric
malignancies and epidermoid carcinoma.
[0027] Embodiments of the invention described herein relate to
monoclonal antibodies that bind Ang-2 and affect Ang-2 and/or Ang1
function. Other embodiments relate to fully human anti-Ang-2
antibodies and anti-Ang-2 antibody preparations with desirable
properties from a therapeutic perspective, including high binding
affinity for Ang-2, the ability to neutralize Ang-2 and/or Ang1 in
vitro and in vivo, and the ability to inhibit Ang-2 and/or Ang-1
induced angiogenesis.
[0028] Another embodiment of the invention is a fully human
antibody that binds to other Angiopoietin-2 family members
including, but not limited to, Angiopoietin-1, Angiopoietin-3, and
Angiopoietin-4. A further embodiment herein is an antibody that
cross-competes for binding to Tie2 with Ang-2 with the fully human
antibodies of the invention. In one embodiment of the invention,
the antibody binds to and neutralizes Angiopoietin-2, and also
binds to and neutralizes, Angiopoietin-1.
[0029] It will be appreciated that embodiments of the invention are
not limited to any particular form of an antibody or method of
generation or production. For example, the anti-Ang-2 antibody may
be a full-length antibody (e.g., having an intact human Fc region)
or an antibody fragment (e.g., a Fab, Fab' or F(ab').sub.2). In
addition, the antibody may be manufactured from a hybridoma that
secretes the antibody, or from a recombinantly produced cell that
has been transformed or transfected with a gene or genes encoding
the antibody.
[0030] Other embodiments of the invention include isolated nucleic
acid molecules encoding any of the antibodies described herein or
portions thereof, vectors having isolated nucleic acid molecules
encoding anti-Ang-2 antibodies or a host cell transformed with any
of such nucleic acid molecules. In addition, one embodiment of the
invention is a method of producing an anti-Ang-2 antibody by
culturing host cells under conditions wherein a nucleic acid
molecule is expressed to produce the antibody followed by
recovering the antibody. It should be realized that embodiments of
the invention also include any nucleic acid molecule which encodes
an antibody or fragment of an antibody of the invention including
nucleic acid sequences optimized for increasing yields of
antibodies or fragments thereof when transfected into host cells
for antibody production.
[0031] Another embodiment of the invention includes a method of
diagnosing diseases or conditions in which an antibody prepared as
described herein is utilized to detect the level of Ang-2 in a
patient sample. In one embodiment, the patient sample is blood or
blood serum. In further embodiments, methods for the identification
of risk factors, diagnosis of disease, and staging of disease is
presented which involves the identification of the overexpression
of Ang-2 using anti-Ang-2 antibodies.
[0032] Another embodiment of the invention includes a method for
diagnosing a condition associated with the expression of Ang-2 in a
cell by contacting the serum or a cell with an anti-Ang-2 antibody,
and thereafter detecting the presence of Ang-2. Selected conditions
include angiogenesis-related diseases including, but not limited
to, neoplastic diseases, such as, melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, glioblastoma, and carcinoma of the thyroid, stomach,
prostate, breast, ovary, bladder, lung, uterus, kidney, colon, and
pancreas, salivary gland, and colorectum.
[0033] In another embodiment, the invention includes an assay kit
for detecting Angiopoietin-2 and Angiopoietin family members in
mammalian tissues, cells, or body fluids to screen for
angiogenesis-related diseases. The kit includes an antibody that
binds to Angiopoietin-2 and a means for indicating the reaction of
the antibody with Angiopoietin-2, if present. In one embodiment,
the antibody that binds Ang-2 is labeled. In another embodiment the
antibody is an unlabeled primary antibody and the kit further
includes a means for detecting the primary antibody. In one
embodiment, the means includes a labeled second antibody that is an
anti-immunoglobulin. In other embodiments, the antibody is labeled
with a marker selected from the group consisting of a fluorochrome,
an enzyme, a radionuclide and a radiopaque material.
[0034] Yet another embodiment includes methods for treating
diseases or conditions associated with the expression of Ang-2 in a
patient, by administering to the patient an effective amount of an
antibody of the invention. The antibody of the invention can be
administered alone, or can be administered in combination with
chemotherapies, biological/immunological therapies, radiation
therapies, hormonal therapies, or surgery. For example, a
monoclonal, oligoclonal or polyclonal mixture of Ang-2 antibodies
that block angiogenesis can be administered in combination with a
drug shown to inhibit tumor cell proliferation directly. The method
can be performed in vivo and the patient, in some embodiments, is a
human patient. In one embodiment, the method concerns the treatment
of angiogenesis-related diseases including, but not limited to,
neoplastic diseases, such as, melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, glioblastoma, and carcinoma of the thyroid, stomach,
prostate, breast, ovary, bladder, lung, uterus, kidney, colon, and
pancreas, salivary gland, and colorectum.
[0035] In another embodiment, the invention provides an article of
manufacture including a container. The container includes a
composition containing an antibody of the invention, and a package
insert or label indicating that the composition can be used to
treat angiogenesis-related diseases characterized by the
overexpression of Ang-2.
[0036] In some embodiments, the anti-Ang-2 antibody is administered
to a patient, followed by administration of a clearing agent to
remove excess circulating antibody from the blood.
[0037] Yet another embodiment is the use of an antibody of the
invention in the preparation of a medicament for the treatment of
diseases such as angiogenesis-related diseases. In one embodiment,
the angiogenesis-related diseases include carcinoma, such as
breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, colorectum, esophageal, thyroid, pancreatic,
prostate and bladder cancer. In another embodiment, the
angiogenesis-related diseases include, but are not limited to,
neoplastic diseases, such as, melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, sarcoma, head and neck cancers, mesothelioma, biliary
(cholangiocarcinoma), small bowel adenocarcinoma, pediatric
malignancies and glioblastoma. In other embodiments,
angiogenesis-related disease include, but are not limited to
non-neoplastic diseases, such as psoriasis, arthritis (rheumatoid,
osteo, and the like), macular degeneration, restenosis, and
others.
[0038] Ang-2 is an important "on-switch" of angiogenesis.
Accordingly, antagonizing this molecule is expected to inhibit
pathophysiological procedures, and thereby act as a potent therapy
for various angiogenesis-dependent diseases. Besides solid tumors
and their metastases, hematological malignancies, such as
leukemias, lymphomas and multiple myeloma, are also
angiogenesis-dependent. Excessive vascular growth contributes to
numerous non-neoplastic disorders. These non-neoplastic
angiogenesis-dependent diseases include: atherosclerosis,
hemangioma, hemangioendothelioma, angiofibroma, vascular
malformations (e.g. Hereditary Hemorrhagic Teleangiectasia (HHT),
or Osler-Weber syndrome), warts, pyogenic granulomas, excessive
hair growth, Kaposi's sarcoma, scar keloids, allergic edema,
psoriasis, dysfunctional uterine bleeding, follicular cysts,
ovarian hyperstimulation, endometriosis, respiratory distress,
ascites, peritoneal sclerosis in dialysis patients, adhesion
formation result from abdominal surgery, obesity, rheumatoid
arthritis, synovitis, osteomyelitis, pannus growth, osteophyte,
hemophilic joints, inflammatory and infectious processes (e.g.
hepatitis, pneumonia, glomerulonephritis), asthma, nasal polyps,
liver regeneration, pulmonary hypertension, retinopathy of
prematurity, diabetic retinopathy, age-related macular
degeneration, leukomalacia, neovascular glaucoma, corneal graft
neovascularization, trachoma, thyroiditis, thyroid enlargement, and
lymphoproliferative disorders.
[0039] In other embodiments, the invention provides methods of
using antibodies of the invention in combination with other agents,
such as anti-angiogenic or anti-inflammatory agents to treat
diseases and/or conditions in a mammal. In one embodiment, methods
of the invention comprise the combination of anti-Ang-2 antibodies
with antagonists of the biological activity of Colony Stimulating
Factor 1 (CSF1) and/or CSF1 receptor (CSF1R) useful to treat
disease.
[0040] In other embodiments, the invention provides methods of
treatment of cancer in a patient. More specifically the methods of
the invention may comprise administration of an antagonist of the
biological activity of Angiopoietin-2, and/or Tie-2, in combination
with a chemotherapeutic agent; a pharmaceutical composition
comprising an antagonist of the biological activity of
Angiopoietin-2, and/or Tie-2, and a chemotherapeutic agent; a
combination product comprising an antagonist of the biological
activity of Angiopoietin-2, and/or Tie-2, and a chemotherapeutic
agent for use in a method of treatment of a patient; a kit
comprising an antagonist of the biological activity of
Angiopoietin-2, and/or Tie-2, and a chemotherapeutic agent; to the
use of an antagonist of the biological activity of Angiopoietin-2,
and/or Tie-2, and a chemotherapeutic agent in the manufacture of a
medicament for use in the production of an anti-cancer effect in a
patient. Such combinations are also useful for the treatment of
other diseases associated with the activity of Angiopoietin-2,
and/or Tie-2.
IV. BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1 represents the results from a Differential Scanning
calorimetry experiment of the wild type antibody 3.19.3 (WT) as
well as antibodies comprising a VL corresponding to MEDI1 and a VH
corresponding to MEDI5 in both an IgG1 and an IgG2 format. The
figure depicts the relative increase of melting temperature of the
two MEDI1/5 antibodies of the to WT control antibody.
[0042] FIG. 2 represents a chromatograph of various preparations of
Ang-2 antibodies. In (A) the 3.19.3 antibody demonstrates a
heterogeneity of sizes corresponding to various adducts formed with
the antibody. (B) and (C) represent the Ang-2 specific antibody
MEDI1/5 in the IgG1 (B) and IgG2 (C) formats. These antibodies do
not exhibit the heterogeneity in sizes displayed by the wild type
3.19.3 antibody.
[0043] FIG. 3 represents the results from a competition ELISA based
Ang-2 binding assay performed on wild type 3.19.3 antibodies as
well as MEDI1/5 antibodies in an IgG1 or an IgG2 format. The figure
depicts that the MEDI1/5 antibodies exhibit a similar binding
profile for Ang-2 as compared to the 3.19.3 antibody as measured by
a competition assay with immobilized Tie-2.
[0044] FIG. 4a demonstrates combination efficacy following
treatment with mAb 3.19.3 and AZD6495 in mice bearing MCF7
xenograft tumors. The y axis shows the Tumor volume in mm.sup.3,
against the Days of treatment, in which the square points represent
vehicle; circular points represent mAb 3.19.3; triangular points
represent AZD6495; diamond points represent mAb 3.19.3 and AZD6495
combination.
[0045] FIG. 4b demonstrates effects on host body weight changes
following combination treatment with mAb 3.19.3 and AZD6495 in mice
bearing MCF7 xenograft tumors. The y axis shows the Body weight in
g, against the Days of treatment, in which the square points
represent vehicle; circular points represent mAb 3.19.3; triangular
points represent AZD6495; diamond points represent mAb 3.19.3 and
AZD6495 combination.
[0046] FIG. 5a demonstrates combination efficacy following
treatment with mAb 3.19.3 and AZD6495 in mice bearing MDA-MB-231
xenograft tumors. The y axis shows the Tumor volume in mm.sup.3,
against the Days of treatment, in which the square points represent
vehicle; circular points represent mAb 3.19.3; triangular points
represent AZD6495; diamond points represent mAb 3.19.3 and AZD6495
combination.
[0047] FIG. 5b. Shows effects on host body weight changes following
combination treatment with mAb 3.19.3 and AZD6495 in mice bearing
MDA-MB-231 xenograft tumors. The y axis shows the Body weight in g,
against the Days of treatment, in which the square points represent
vehicle; circular points represent mAb 3.19.3; triangular points
represent AZD6495; diamond points represent mAb 3.19.3 and AZD6495
combination.
[0048] FIG. 6a. Shows combination efficacy following treatment with
mAb 3.19.3 and 5-fluorouracil in mice bearing LoVo xenograft
tumors.
[0049] FIG. 6b. Shows effects on host body weight changes following
combination treatment with mAb 3.19.3 and 5-fluoruracil in mice
bearing LoVo xenograft tumors.
[0050] FIG. 7a. Shows combination efficacy following treatment with
mAb 3.19.3 and Irinotecan in mice bearing HT-29 xenograft
tumors.
[0051] FIG. 7b. Shows effects on host body weight changes following
combination treatment with mAb 3.19.3 and Irinotecan in mice
bearing HT29 xenograft tumors.
[0052] FIG. 8a. Shows combination efficacy following treatment with
mAb 3.19.3 and Gemcitabine in mice bearing Colo205 xenograft
tumors.
[0053] FIG. 8b. Shows effects on host body weight changes following
combination treatment with mAb 3.19.3 and Gemcitabine in mice
bearing Colo205 xenograft tumors.
[0054] FIG. 9a. Shows combination efficacy following treatment with
mAb 3.19.3 and Docetaxel in mice bearing Calu6 xenograft
tumors.
[0055] FIG. 9b. Shows effects on host body weight changes following
combination treatment with mAb 3.19.3 and Docetaxel in mice bearing
Calu6 xenograft tumors.
[0056] FIG. 10a. Shows combination efficacy following treatment
with mAb 3.19.3 and Oxaliplatin in mice bearing H460 xenograft
tumors.
[0057] FIG. 10b. Shows effects on host body weight changes
following combination treatment with mAb 3.19.3 and Oxaliplatin in
mice bearing H460 xenograft tumors.
[0058] FIG. 11a. Shows combination efficacy following treatment
with mAb 3.19.3 and AZD4877 in mice bearing H460 xenograft
tumors.
[0059] FIG. 11b. Shows effects on host body weight changes
following combination treatment with mAb 3.19.3 and AZD4877 in mice
bearing H460 xenograft tumors.
[0060] FIG. 12a. Effect of 3.19.3 treatment on clinical disease
progression in the collagen induced arthritis disease model.
Collagen induced arthritis was induced in male DBA/1 mice and
animals dosed therapeutically with test treatments. FIG. 12a shows
the Arthritic score mean (+/-standard error of the mean) against
Days from disease onset (i.e. Days of treatment). Closed squares
represent PBS vehicle treated animals (n=15), Open triangles
represent human IgG isotype control treated animals (n=15), closed
circles represent 3.19.3 10 mg/kg treated animals (n=15) and open
squares represent Prednisolone 3 mg/kg treated animals (n=10).
[0061] FIG. 12b: Effect on mean animal body weights. FIG. 12b shows
that no significant changes in mean body weight (g) were observed
between each treatment group throughout time course of collagen
induced arthritis, suggesting the 3.19.3 therapy was well
tolerated. FIG. 12b shows Body weight in grams against Days from
disease onset (i.e. Days of treatment). Closed squares represent
PBS vehicle treated animals (n=15), Open triangles represent human
IgG isotype control treated animals (n=15), closed circles
represent 3.19.3 10 mg/kg treated animals (n=15) and open squares
represent Prednisolone 3 mg/kg treated animals (n=10).
[0062] FIG. 13: Anti-Ang-2 antibodies inhibit retinal angiogenesis.
FIG. 13 represents alterations in angiogenesis of murine retinas in
control pups (a) and in pups treated with 0.3 mg/kg MEDI1/5 (b),
1.0 mg/kg MEDI1/5 (c), and 10 mg/kg MEDI1/5 (d). These panels
demonstrate that murine retinal angiogenesis is inhibited in a
dose-dependent fashion with MEDI1/5 anti-Ang-2 antibodies, as
compared to animals treated with control antibodies.
[0063] FIG. 14. Anti-Ang-2 antibodies inhibit FGF2 mediated
angiogenesis. FIG. 14 represents results demonstrating the
inhibition of FGF2 mediated angiogenesis in mice by the
administration of the anti-Ang-2 antibody, MEDI1/5. Briefly,
Matrigel.TM. was mixed with FGF2 and implanted subcutaneously into
athymic nude mice. MEDI1/5 was dosed intraperitoneally at 1, 10 or
20 mg/kg, on days 1, 4 and 8 of implant. On day 11 post-implant,
mice were intravenously dosed with FITC-dextran and Matrigel.TM.
plugs were harvested. Plugs were quantitated for FITC-Dextran
content (a) all three doses of MEDI1/5 resulted in significant
reduction in angiogenesis (*p<0.05). Plugs were also prepared
hematoxylin and eosin staining (b) which showed a lower level of
vascularization as compared to the control FGF2 treated sample.
[0064] FIG. 15: Anti-Ang-2 antibodies inhibit arthritis disease
progression. FIG. 15A represents the global arthritic score of
arthritis-induced animals treated with various agents including the
anti-Ang-2 antibody, 3.19.3 and Prednisolone (open squares=PBS,
open triangles=isotype control, closed squares=0.1 mg/kg 3.19.3,
closed triangles=1 mg/kg 3.19.3, closed circles=10 mg/kg 3.19.3 and
open circles=prednisolone). Dose-dependent reductions in clinical
signs of disease progression (arthritic score) and were observed.
There was a significant reduction at doses of 1 and 10 mg/kg of
3.19.3. Area under the curve (AUC) for clinical disease progression
was calculated for each animal from disease onset and presented in
FIG. 15B. FIGS. 15C-H further demonstrate the ameliorative effect
of treatment with the anti-Ang-2 antibody 3.19.3.
Histolopathological evaluation of CIA model showed evidence of a
dose-dependent anti-arthritic effect following administration of
3.19.3 on all parameters evaluated including synovial hyperplasmia
(FIG. 15 C), synovitis (FIG. 15 D), pannus (FIG. 15 E), synovial
fibrosis (FIG. 15F), and periostitis (FIG. 15G). Histologically,
there were no significant differences between the isotype
control-treated group and the PBS vehicle group (FIGS. 15C-G).
Further, investigation into the microvessel density using CD31
staining showed a significant reduction in the presence of
microvessels in the synovium at doses of 1 and 10 mg/kg as well as
with prednisolone. There was no effect with 0.1 mg/kg 3.19.3
treatment (FIG. 15 H).
[0065] FIG. 16: Combinations of Anti-Ang-2 and Anti-TNF.alpha.
agents demonstrate efficacy in prophylactic treatment of arthritis.
FIGS. 16A+B represent the arthritic score of arthritis-induced
animals prophylactically treated with a combination of
MEDI1/5+etanercept (closed circles=isotype control, closed
diamond=10 mg/kg MEDI1/5, open diamond=1 mg/kg etanercept, grey
diamond=combination of 10 mg/kg MEDI1/5 with 1 mg/kg etanercept,
open square=4 mg/kg enbrel, grey square=combination of 10 mg/kg
MEDI1/5 with 4 mg/kg etanercept). A reduction was observed with
either etanercept or MEDI1/5 treatment. There was a further
reduction in clincal score when MEDI1/5 was administered in
combination with the lower dose of etanercept. Histological
assessment of synovitis and joint destruction (FIG. 16C) supported
the clinical score results as did the protection from loss of bone
mineral density (FIG. 16D).
[0066] FIG. 17: Combinations of Anti-Ang-2 and Anti-TNF.alpha.
agents demonstrate efficacy in therapeutic treatment of arthritis
When administered in a therpeutic approach following the onset of
clinical disease, modest reductions in clinical signs of disease
progression (arthritic score) were observed with MEDI1/5 treatment,
while both doses of etanercept tested had no effect on disease
progression. A more dramatic inhibition of progression of disease
when MEDI1/5 (10 mg/kg) was administered in combination with the
higher dose of etanercept (4 mg/kg) (FIG. 17 (closed
circles=isotype control, closed diamond=MEDI1/5, open diamond=1
mg/kg etanercept, grey diamond=combination of MEDI1/5 with 1 mg/kg
etanercept, open square=4 mg/kg etanercept, grey square=combination
of MEDI1/5 with 4 mg/kg etanercept).
V. DETAILED DESCRIPTION
[0067] The inventors have found that certain modifications could be
made to a particular Ang-2 antibody, which renders the antibody
more stable under certain conditions. In particular, by altering
residue 49 of the light chain much less aggregation occurred. In
addition, when residue 37 of the heavy chain was changed, much less
aggregation occurred.
[0068] Accordingly, in one embodiment the invention is directed to
Ang-2 antibodies having one or more improved characteristics over
control antibody 3.19.3. Such characteristics include increased
stability, decreased aggregation and increased production
efficiency. In one embodiment, the antibodies of the invention
efficiently inhibit Ang-2 and/or Ang-1 signaling through the Tie2
receptor to modulate processes such as angiogenesis and tumor
growth.
[0069] Embodiments of the invention described herein relate to
monoclonal antibodies specific for Ang-2, which may be derived from
the antibody 3.19.3 and which exhibit increased stability and/or
production efficiencies. In some embodiments, the antibodies bind
to Ang-2 and inhibit the binding of Ang-2 to its receptor, Tie2.
Other embodiments of the invention include fully human anti-Ang-2
antibodies, and antibody preparations that are therapeutically
useful. Such anti-Ang-2 antibody preparations have desirable
therapeutic properties, including strong binding affinity for
Ang-2, the ability to neutralize Ang-2 in vitro, and the ability to
inhibit Ang-2-induced angiogenesis in vivo. Antibodies of the
invention comprise the ability to specifically bind Ang-2 and
inhibit tumor angiogenesis and reduce tumor growth. Mechanisms by
which this can be achieved can include and are not limited to
either inhibition of binding of Ang-2 to its receptor Tie2,
inhibition of Ang-2 induced Tie2 signaling, or increased clearance
of Ang-2, therein reducing the effective concentration of
Ang-2.
[0070] In other embodiments, the antibodies may bind to both Ang-2
and Ang-1 and/or modulate one or more functional activities of both
Ang-1 and Ang-2.
[0071] One aspect of the invention provides stabilized antibodies
which comprise a substitution of an amino acid at position 49 (as
compared to the light chain variable amino acid sequence of Ang-2
antibody 3.19.3, see SEQ ID No.1) as defined by the Kabat numbering
system (Kabat et al., Sequences of Proteins of Immunological
Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md.
(1991), vols. 1-3.). In one embodiment, the amino acid substitution
at position 49 may be any amino acid. In a specific embodiment, the
amino acid substitution at position 49 is selected from the group
consisting of Asp, Thr, Asn, and Ala.
[0072] In another embodiment, antibodies of the invention further
comprises a substitution of Val substitution at position 37 of the
heavy chain (as compared to the heavy chain variable amino acid
sequence of Ang-2 antibody 3.19.3, see, SEQ ID No. 2) as defined by
the EU numbering system ((Kabat et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, NIH Publication 91-3242,
Bethesda Md. (1991), vols. 1-3.).
[0073] In one embodiment, the antibodies of the invention exhibit
enhanced stability as compared to antibody 3.19.3. In another
embodiment, the antibodies of the invention exhibit enhanced
production yields as compared to 3.19.3.
[0074] In another embodiment, the antibodies of the invention may
comprise variable light chain acid sequences selected from the
group consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4),
MEDI3 (SEQ ID No.:5), MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID
No.:6). In another embodiment, the antibodies of the invention may
comprise the heavy chain variable sequence of MEDI5 (SEQ ID No.:7).
In another embodiment, the antibodies of the invention may comprise
variable light chain sequences selected from the group consisting
of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4), MEDI3 (SEQ ID
No.:5), MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID No.:6) and further
comprise the heavy chain variable sequence of MEDI5 (SEQ ID No.:7).
As used herein, an antibody of the invention comprising a light
chain and a heavy chain may be referred to as a MEDIX/MEDIY wherein
X represents the light chain sequence and Y represents the heavy
chain sequence.
[0075] In another embodiment, the antibodies of the invention may
comprise variable light chain acid sequences selected from the
group consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4),
MEDI3 (SEQ ID No.:5), MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID
No.:6), but having a different amino acid substitution at position
49. In another embodiment, the antibodies of the invention may
further comprise the heavy chain variable sequence of MEDI5 (SEQ ID
No.:7). In another embodiment, the antibodies of the invention may
comprise variable light chain sequences selected from the group
consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4), MEDI3
(SEQ ID No.:5), MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID No.:6), but
having a different amino acid substitution at position 49, and
further comprise the heavy chain variable sequence of MEDI5 (SEQ ID
No.:7).
Modulation of Unpaired Cysteine Residues:
[0076] Disulfide bond formation in proteins is a complex process,
which is determined by the redox potential of the environment and
specialized thiol-disulfide exchanging enzymes (Creighton, Methods
Enzymol. 107, 305-329, 1984; Houee-Levin, Methods Enzymol. 353,
35-44,2002). In general, cysteine residues in proteins (for
example, antibodies specific for Ang-2) are either engaged in
cysteine-cysteine disulfide bonds or sterically protected from the
disulfide bond formation when they are a part of folded protein
region. When a cysteine residue does not have a pair in protein
structure and is not sterically protected by folding, it can form a
disulfide bond with a free cysteine from solution in a process
known as disulfide shuffling. In another process known as disulfide
scrambling, free cysteines may also interfere with naturally
occurring disulfide bonds (such as those present in antibody
structures) and lead to low binding, low biological activity and/or
low stability.
Modulation of Glycosylation Sites:
[0077] It has been shown that glycosylation in the variable region
can have a negative effect on antibody binding affinity, likely due
to steric hindrance (Co, M. S., et al., Mol. Immunol. (1993)
30:1361-1367). The heterogeneity of the glycosylation process may
also lead to a number of antibody species with altered binding
properties. As such, it is desirable to remove or alter the
interfering glycosylation site to ensure a consistent antigen
binding profile. One method to remove potential or observed
glycosylation sites is site-directed mutagenesis to substitute at
least one potential glycosylation site (such as an asparagine,
threonine or serine amino acid) with another amino acid that cannot
serve as a glycosylation site. Accordingly, in one embodiment, the
antibodies of the invention comprise substituted amino acids that
do not serve as glycosylation sites. In one embodiment, the
glycosylation site to be modified occurs in the variable region. In
another embodiment, the glycosylation site to be modified occurs in
a complementary determining region (CDR) of an antibody. In another
embodiment, the glycosylation site to be modified is the 2.sup.nd
light chain CDR. In other embodiments, the sequences surrounding
the glycosylation site are modified. In another embodiment, the
glycosylation site to be modified occurs in the constant region. In
another embodiment, the antibodies of the invention comprise at
least one, at least two, at least three, at least four or more
modified glycosylation sites.
[0078] In some embodiments, antibodies of the invention comprise a
light chain which is engineered to remove at least one
O-glycosylation site. In some embodiments, antibodies of the
invention comprise a light chain selected from the group consisting
of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4), MEDI3 (SEQ ID
No.:5), and MEDI4 (SEQ ID No.:6) wherein said light chain further
comprises an amino acid substitution at Kabat position 59, wherein
said amino acid is not proline. In a specific embodiment,
antibodies of the invention comprise a light chain having the
sequence corresponding to MEDI6 (SEQ ID NO:8).
[0079] The amino acid sequences of selected antibody heavy and
light chains can be compared to germline heavy and light chain
amino acid sequences. In cases where certain framework residues of
the selected VL and/or VH chains differ from the germline
configuration (e.g., as a result of somatic mutation of the
immunoglobulin genes used to prepare the phage library), it may be
desirable to "backmutate" the altered framework residues of the
selected antibodies to the germline configuration (i.e., change the
framework amino acid sequences of the selected antibodies so that
they are the same as the germline framework amino acid sequences).
Such "backmutation" (or "germlining") of framework residues can be
accomplished by standard molecular biology methods for introducing
specific mutations (e.g., site-directed mutagenesis; PCR-mediated
mutagenesis, and the like). In one embodiment, the variable light
and/or heavy chain framework residues are backmutated. In another
embodiment, the variable heavy chain of an antibody of the
invention is backmutated. In another embodiment, the variable heavy
chain of an antibody of the invention comprises at least one, at
least two, at least three, at least four or more backmutations. In
a specific embodiment, the variable heavy chain of an antibody of
the invention comprises a backmutation of the glycine residue
occupying position 37. In another specific embodiment, the variable
heavy chain of an antibody of the invention comprises a
backmutation of position 37 corresponding to a glycine to valine
substitution.
Modulation of the Fc Region
[0080] The invention also provides antibodies with altered Fc
regions (also referred to herein as "variant Fc regions").
Accordingly, in one embodiment of the invention, antibodies of the
invention comprise a variant Fc region (i.e., Fc regions that have
been altered as discussed below). Antibodies of the invention
comprising a variant Fc region are also referred to here as "Fc
variant protein(s)."
[0081] In the description of variant Fc regions, it is understood
that the Fc regions of the antibodies of the invention comprise the
numbering scheme according to the EU index as in Kabat et al.
(1991, NIH Publication 91-3242, National Technical Information
Service, Springfield, Va.).
[0082] It is known that variants of the Fc region (e.g., amino acid
substitutions and/or additions and/or deletions) enhance or
diminish effector function (see Presta et al., 2002, Biochem Soc
Trans 30:487-490; U.S. Pat. Nos. 5,624,821, 5,885,573 and PCT
publication Nos. WO 00/42072, WO 99/58572 and WO 04/029207).
Accordingly, in one embodiment, the antibodies of the invention
comprise variant Fc regions. In one embodiment, the variant Fc
regions of antibodies exhibit a similar level of inducing effector
function as compared to the native Fc. In another embodiment, the
variant Fc region exhibits a higher induction of effector function
as compared to the native Fc. In another embodiment, the variant Fc
region exhibits lower induction of effector function as compared to
the native Fc. In another embodiment, the variant Fc region
exhibits higher induction of ADCC as compared to the native Fc. In
another embodiment, the variant Fc region exhibits lower induction
of ADCC as compared to the native Fc. In another embodiment, the
variant Fc region exhibits higher induction of CDC as compared to
the native Fc. In another embodiment, the variant Fc region
exhibits lower induction of CDC as compared to the native Fc.
Specific embodiments of variant Fc regions are detailed infra.
[0083] It is also known in the art that the glycosylation of the Fc
region can be modified to increase or decrease effector function
(see for examples, Umana et al, 1999, Nat. Biotechnol 17:176-180;
Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al,
2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol
Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No.
10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO
01/292246A 1; PCT WO 02/311140A1; PCT WO 02/30954A 1;
Potillegent.TM. technology (Biowa, Inc. Princeton, N.J.);
GlycoMAb.TM. glycosylation engineering technology (GLYCART
biotechnology AG, Zurich, Switzerland). Accordingly, in one
embodiment the Fc regions of antibodies of the invention comprise
altered glycosylation of amino acid residues. In another
embodiment, the altered glycosylation of the amino acid residues
results in lowered effector function. In another embodiment, the
altered glycosylation of the amino acid residues results in
increased effector function. In a specific embodiment, the Fc
region has reduced fucosylation. In another embodiment, the Fc
region is afucosylated (see for examples, U.S. Patent Application
Publication No. 2005/0226867).
[0084] Recent research suggests that the addition of sialic acid to
the oligosaccharides on IgG molecules enhances their
anti-inflammatory activity and alter their cytotoxicity (Keneko et
al., Science 313, 670-673 (2006), Scallon et al., Mol. Immuno. 2007
March; 44(7):1524-34). Thus, the efficacy of antibody therapeutics
may be optimized by selection of a glycoform that is best suited to
the intended application. The two oligosaccharide chains interposed
between the two CH2 domains of antibodies are involved in the
binding of the Fc region to its receptors. The studies referenced
above demonstrate that IgG molecules with increased sialylation
have anti-inflammatory properties whereas IgG molecules with
reduced sialylation have increased immunostimulatory properties.
Therefore, an antibody therapeutic can be "tailor-made" with an
appropriate sialylation profile for a particular application.
Methods for modulating the sialylation state of antibodies are
presented in WO2007/005786 entitled "Methods And Compositions With
Enhanced Therapeutic Activity", and WO2007/117505 entitled
"Polypeptides With Enhanced Anti-Inflammatory And Decreased
Cytotoxic Properties And Related Methods" each of which are
incorporated by reference in their entireties for all purposes.
[0085] In one embodiment, the Fc regions of antibodies of the
invention comprise an altered sialylation profile compared to a
reference unaltered Fc region. In one embodiment, the Fc regions of
antibodies of the invention comprise an increased sialylation
profile compared to a reference unaltered Fc region. In some
embodiments the Fc regions of antibodies of the invention comprise
an increase in sialylation of about 5%, about 10%, about 15%, about
20%, about 25%, about 30%, about 35%, about 40%, about 45%, about
50%, about 60%, about 65%, about 70%, about 80%, about 85%, about
90%, about 95%, about 100%, about 125%, about 150% or more as
compared to a reference unaltered Fc region. In some embodiments
the Fc regions of antibodies of the invention comprise an increase
in sialylation of about 2 fold, about 3 fold, about 4 fold, about 5
fold, about 10 fold, about 20 fold, about 50 fold or more as
compared to an unaltered reference Fc region.
[0086] In another embodiment, the Fc regions of antibodies of the
invention comprise a decreased sialylation profile compared to a
reference unaltered Fc region. In some embodiments, the Fc regions
of antibodies of the invention comprise a decrease in sialylation
of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%,
about 35%, about 40%, about 45%, about 50%, about 60%, about 65%,
about 70%, about 80%, about 85%, about 90%, about 95%, about 100%,
about 125%, about 150% or more as compared to a reference unaltered
Fc region. In some embodiments the Fc regions of antibodies of the
invention comprise a decrease in sialylation of about 2 fold, about
3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold,
about 50 fold or more as compared to an unaltered reference Fc
region.
[0087] It is also known in the art that the Fc region can be
modified to increase the half-lives of proteins. The increase in
half-life allows for the reduction in amount of drug given to a
patient as well as reducing the frequency of administration.
Accordingly, antibodies of the invention with increased half-lives
may be generated by modifying (for example, substituting, deleting,
or adding) amino acid residues identified as involved in the
interaction between the Fc and the FcRn receptor (see, for
examples, PCT publication Nos. 97/34631 and 02/060919 each of which
are incorporated by reference in their entireties). In addition,
the half-life of antibodies of the invention may be increase by
conjugation to PEG or Albumin by techniques widely utilized in the
art. In some embodiments the Fc regions of antibodies of the
invention comprise an increase in half-life of about 5%, about 10%,
about 15%, about 20%, about 25%, about 30%, about 35%, about 40%,
about 45%, about 50%, about 60%, about 65%, about 70%, about 80%,
about 85%, about 90%, about 95%, about 100%, about 125%, about 150%
or more as compared to a reference unaltered Fc region. In some
embodiments the Fc regions of antibodies of the invention comprise
an increase in half-life of about 2 fold, about 3 fold, about 4
fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold or
more as compared to an unaltered reference Fc region.
[0088] The present invention encompasses Fc variant proteins which
have altered binding properties for an Fc ligand (e.g., an Fc
receptor, C1q) relative to a comparable molecule (e.g., a protein
having the same amino acid sequence except having a wild type Fc
region). Examples of binding properties include but are not limited
to, binding specificity, equilibrium dissociation constant
(K.sub.D), dissociation and association rates (k.sub.off and
k.sub.on, respectively), binding affinity and/or avidity. It is
generally understood that a binding molecule (e.g., a Fc variant
protein such as an antibody) with a low K.sub.D may be more
desirable to a binding molecule with a high K.sub.D. However, in
some instances the value of the k.sub.on or k.sub.off may be more
relevant than the value of the K.sub.D. One skilled in the art can
determine which kinetic parameter is most important for a given
antibody application.
[0089] The affinities and binding properties of an Fc region for
its ligand may be determined by a variety of in vitro assay methods
(biochemical or immunological based assays) known in the art for
determining Fc-Fc.gamma.R interactions, i.e., specific binding of
an Fc region to an Fc.gamma.R including but not limited to,
equilibrium methods (e.g., enzyme-linked immunoabsorbent assay
(ELISA), or radioimmunoassay (RIA)), or kinetics (e.g.,
BIACORE.RTM. analysis), and other methods such as indirect binding
assays, competitive inhibition assays, fluorescence resonance
energy transfer (FRET), gel electrophoresis and chromatography
(e.g., gel filtration). These and other methods may utilize a label
on one or more of the components being examined and/or employ a
variety of detection methods including but not limited to
chromogenic, fluorescent, luminescent, or isotopic labels. A
detailed description of binding affinities and kinetics can be
found in Paul, W. E., ed., Fundamental Immunology, 4th Ed.,
Lippincott-Raven, Philadelphia (1999), which focuses on
antibody-immunogen interactions.
[0090] In one embodiment, the Fc variant protein has enhanced
binding to one or more Fc ligand relative to a comparable molecule.
In another embodiment, the Fc variant protein has an affinity for
an Fc ligand that is at least 2 fold, or at least 3 fold, or at
least 5 fold, or at least 7 fold, or a least 10 fold, or at least
20 fold, or at least 30 fold, or at least 40 fold, or at least 50
fold, or at least 60 fold, or at least 70 fold, or at least 80
fold, or at least 90 fold, or at least 100 fold, or at least 200
fold greater than that of a comparable molecule. In a specific
embodiment, the Fc variant protein has enhanced binding to an Fc
receptor. In another specific embodiment, the Fc variant protein
has enhanced binding to the Fc receptor Fc.gamma.RIIIA. In a
further specific embodiment, the Fc variant protein has enhanced
biding to the Fc receptor Fc.gamma.RIIB. In still another specific
embodiment, the Fc variant protein has enhanced binding to the Fc
receptor FcRn. In yet another specific embodiment, the Fc variant
protein has enhanced binding to C1q relative to a comparable
molecule.
[0091] The serum half-life of proteins comprising Fc regions may be
increased by increasing the binding affinity of the Fc region for
FcRn. In one embodiment, the Fc variant protein has enhanced serum
half life relative to comparable molecule.
[0092] The ability of any particular Fc variant protein to mediate
lysis of the target cell by ADCC can be assayed. To assess ADCC
activity an Fc variant protein of interest is added to target cells
in combination with immune effector cells, which may be activated
by the antigen antibody complexes resulting in cytolysis of the
target cell. Cytolysis is generally detected by the release of
label (e.g. radioactive substrates, fluorescent dyes or natural
intracellular proteins) from the lysed cells. Useful effector cells
for such assays include peripheral blood mononuclear cells (PBMC)
and Natural Killer (NK) cells. Specific examples of in vitro ADCC
assays are described in Wisecarver et al., 1985 79:277-282;
Bruggemann et al., 1987, Exp Med 166:1351-1361; Wilkinson et al.,
2001, J Immunol Methods 258:183-191; Patel et al., 1995 J Immunol
Methods 184:29-38. ADCC activity of the Fc variant protein of
interest may also be assessed in vivo, e.g., in an animal model
such as that disclosed in Clynes et al., 1998, Proc. Natl. Acad.
Sci. USA 95:652-656.
[0093] In one embodiment, an Fc variant protein has enhanced ADCC
activity relative to a comparable molecule. In a specific
embodiment, an Fc variant protein has ADCC activity that is at
least 2 fold, or at least 3 fold, or at least 5 fold or at least 10
fold or at least 50 fold or at least 100 fold greater than that of
a comparable molecule. In another specific embodiment, an Fc
variant protein has enhanced binding to the Fc receptor
Fc.gamma.RIIIA and has enhanced ADCC activity relative to a
comparable molecule. In other embodiments, the Fc variant protein
has both enhanced ADCC activity and enhanced scrum half life
relative to a comparable molecule.
[0094] In one embodiment, an Fc variant protein has reduced ADCC
activity relative to a comparable molecule. In a specific
embodiment, an Fc variant protein has ADCC activity that is at
least 2 fold, or at least 3 fold, or at least 5 fold or at least 10
fold or at least 50 fold or at least 100 fold lower than that of a
comparable molecule. In another specific embodiment, an Fc variant
protein has reduced binding to the Fc receptor Fc.gamma.RIIIA and
has reduced ADCC activity relative to a comparable molecule. In
other embodiments, the Fc variant protein has both reduced ADCC
activity and enhanced serum half life relative to a comparable
molecule.
[0095] In one embodiment, an Fc variant protein has enhanced CDC
activity relative to a comparable molecule. In a specific
embodiment, an Fc variant protein has CDC activity that is at least
2 fold, or at least 3 fold, or at least 5 fold or at least 10 fold
or at least 50 fold or at least 100 fold greater than that of a
comparable molecule. In other embodiments, the Fc variant protein
has both enhanced CDC activity and enhanced serum half life
relative to a comparable molecule. In one embodiment, the Fc
variant protein has reduced binding to one or more Fc ligand
relative to a comparable molecule. In another embodiment, the Fc
variant protein has an affinity for an Fc ligand that is at least 2
fold, or at least 3 fold, or at least 5 fold, or at least 7 fold,
or a least 10 fold, or at least 20 fold, or at least 30 fold, or at
least 40 fold, or at least 50 fold, or at least 60 fold, or at
least 70 fold, or at least 80 fold, or at least 90 fold, or at
least 100 fold, or at least 200 fold lower than that of a
comparable molecule. In a specific embodiment, the Fc variant
protein has reduced binding to an Fc receptor. In another specific
embodiment, the Fc variant protein has reduced binding to the Fc
receptor Fc.gamma.RIIIA. In a further specific embodiment, an Fc
variant described herein has an affinity for the Fc receptor
Fc.gamma.RIIIA that is at least about 5 fold lower than that of a
comparable molecule, wherein said Fc variant has an affinity for
the Fc receptor Fc.gamma.RIIB that is within about 2 fold of that
of a comparable molecule. In still another specific embodiment, the
Fc variant protein has reduced binding to the Fc receptor FcRn. In
yet another specific embodiment, the Fc variant protein has reduced
binding to C1q relative to a comparable molecule.
[0096] In one embodiment, the present invention provides Fc
variants, wherein the Fc region comprises a non naturally occurring
amino acid residue at one or more positions selected from the group
consisting of 234, 235, 236, 237, 238, 239, 240, 241, 243, 244,
245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267,
268, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316,
325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343,
370, 373, 378, 392, 416, 419, 421, 440 and 443 as numbered by the
EU index as set forth in Kabat. Optionally, the Fc region may
comprise a non naturally occurring amino acid residue at additional
and/or alternative positions known to one skilled in the art (see,
e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent
Publications WO 01/58957; WO 02/06919; WO 04/016750; WO 04/029207;
WO 04/035752; WO 04/074455; WO 04/099249; WO 04/063351; WO
05/070963; WO 05/040217, WO 05/092925 and WO 06/020114).
[0097] In a specific embodiment, the present invention provides an
Fc variant, wherein the Fc region comprises at least one non
naturally occurring amino acid residue selected from the group
consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V,
234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H,
235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T,
239H, 239Y, 240I, 240A, 240T, 240M, 241W, 241 L, 241Y, 241E, 241 R,
243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F,
252Y, 254T, 255L, 256E, 256M, 262I, 262A, 262T, 262E, 263I, 263A,
263T, 263M, 264L, 264I, 264W, 264T, 264R, 264F, 264M, 264Y, 264E,
265G, 265N, 265Q, 265Y, 265F, 265V, 265I, 265L, 265H, 265T, 266I,
266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E,
280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L,
296I, 296H, 269G, 297S, 297D, 297E, 298H, 298I, 298T, 298F, 299I,
299L, 299A, 299S, 299V, 299H, 299F, 299E, 305I, 313F, 316D, 325Q,
325L, 325I, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N,
327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328I, 328V, 328T,
328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y,
330V, 330I, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 331F, 331W,
331K, 331Q, 331E, 331S, 331V, 331I, 331C, 331Y, 331H, 331R, 331N,
331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H,
332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K,
440Y and 434W as numbered by the EU index as set forth in Kabat.
Optionally, the Fc region may comprise additional and/or
alternative non naturally occurring amino acid residues known to
one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821;
6,277,375; 6,737,056; PCT Patent Publications WO 01/58957; WO
02/06919; WO 04/016750; WO 04/029207; WO 04/035752 and WO
05/040217).
Antibody Affinity
[0098] In one embodiment of the invention there is provided an
antibody that binds to Angiopoietin-1 and prevents Angiopoietin-1
binding to Tie-2. Yet another embodiment of the invention is a
monoclonal antibody that binds to Angiopoietin-1 and/or
Angiopoietin-2 and inhibits Angiopoietin-1 and/or Angiopoietin-2
induced Tie-2 phosphorylation. In one embodiment, the antibody
binds Angiopoietin-1 and/or Angiopoietin-2 with a K.sub.d of less
than 1 nanomolar (nM). In other embodiments, the antibody binds
with a IQ less than 500 picomolar (pM). In other embodiments, the
antibody binds with a K.sub.d less than 100 picomolar (pM). In yet
other embodiments, the antibody binds with a K.sub.d less than 30
picomolar (pM). In further embodiments, the antibody binds with a
IQ of less than 20 .mu.M. In yet further embodiments, the antibody
binds with a IQ of less than 10 or 5 .mu.M.
[0099] Antibodies of the invention may have a high binding affinity
Ang-1 and/or Ang-2. For example, an antibody described herein may
have an association rate constant or k.sub.on rate (antibody
(Ab)+antigen->Ab-Ag) of at least 2.times.10.sup.5
M.sup.-1s.sup.-1, at least 5.times.10.sup.5 M.sup.-1s.sup.-1, at
least 10.sup.6 M.sup.-1s.sup.-1, at least 5.times.10.sup.6
M.sup.-1s.sup.-1, at least 10.sup.7 M.sup.-1s.sup.-1, at least
5.times.10.sup.7M.sup.-1s.sup.-1, or at least 10.sup.8
M.sup.-1s.sup.-1.
[0100] In another embodiment, an antibody may have a k.sub.off rate
(Ab-Ag->Ab+Ag) of less than 5.times.10.sup.-1 s.sup.-1, less
than 10.sup.-1 s.sup.-1, less than 5.times.10.sup.-2 s.sup.-1, less
than 10.sup.-2 s.sup.-1, less than 5.times.10.sup.-3 s.sup.-1, less
than 10.sup.-3 s.sup.-1, less than 5.times.10.sup.-4 s.sup.-1, or
less than 10.sup.-4 s.sup.-1. In a another embodiment, an antibody
of the invention has a k.sub.off of less than 5.times.10.sup.-5
s.sup.-1, less than 10.sup.-5 s.sup.-1, less than 5.times.10.sup.-6
s.sup.-1, less than 10.sup.-6 s.sup.-1, less than 5.times.10.sup.-7
s.sup.-1, less than 10.sup.-7 s.sup.-1, less than 5.times.10.sup.-8
s.sup.-1, less than 10.sup.-8 s.sup.-1, less than 5.times.10.sup.-9
s.sup.-1, less than 10.sup.-9 s.sup.-1, or less than 10.sup.-10
s.sup.-1.
[0101] In another embodiment, an antibody may have an affinity
constant or K.sub.a (k.sub.on/k.sub.off) of at least 10.sup.2
M.sup.-1, at least 5.times.10.sup.2 M.sup.-1, at least 10.sup.3
M.sup.-1, at least 5.times.10.sup.3 M.sup.-1, at least 10.sup.4
M.sup.-1, at least 5.times.10.sup.4 M.sup.-1, at least 10.sup.5
M.sup.-1, at least 5.times.10.sup.5 M.sup.-1, at least 10.sup.6
M.sup.-1, at least 5.times.10.sup.6 M.sup.-1, at least 10.sup.7
M.sup.-1, at least 5.times.10.sup.7 M.sup.-1, at least 10.sup.8
M.sup.-1, at least 5.times.10.sup.8 M.sup.-1, at least 10.sup.9
M.sup.-1, at least 5.times.10.sup.9 M.sup.-1, at least 10.sup.10
M.sup.-1, at least 5.times.10.sup.10 M.sup.-1, at least 10.sup.11
M.sup.-1, at least 5.times.10.sup.11 M.sup.-1, at least 10.sup.12
M.sup.-1 at least 5.times.10.sup.12 M.sup.-1, at least 10.sup.13
M.sup.-1, at least 5.times.10.sup.13 M.sup.-1, at least 10.sup.14
M.sup.-1, at least 5.times.10.sup.14 M.sup.-1, at least 10.sup.15
M.sup.-1, or at least 5.times.10.sup.15 M.sup.-1. In yet another
embodiment, an antibody may have a dissociation constant or IQ
(k.sub.off/k.sub.on) of less than 5.times.10.sup.-2 M, less than
10.sup.-2 M, less than 5.times.10.sup.-3 M, less than 10.sup.-3 M,
less than 5.times.10.sup.-4 M, less than 10.sup.-4 M, less than
5.times.10.sup.-5 M, less than 10.sup.-5 M, less than
5.times.10.sup.-6 M, less than 10.sup.-6 M, less than
5.times.10.sup.-7 M, less than 10.sup.-7 M, less than
5.times.10.sup.-8 M, less than 10.sup.-8 M, less than
5.times.10.sup.-9 M, less than 10.sup.-9 M, less than
5.times.10.sup.10 M, less than 10.sup.-10 M, less than
5.times.10.sup.-11 M, less than 10.sup.-11 M, less than
5.times.10.sup.-12 M, less than 10.sup.-12 M, less than
5.times.10.sup.-13 M, less than 10.sup.-13 M, less than
5.times.10.sup.-14 M, less than 10.sup.-14 M, less than
5.times.10.sup.-15 M, or less than 10.sup.15 M.
[0102] An antibody used in accordance with a method described
herein may have a dissociation constant (K.sub.d) of less than 3000
pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less
than 1000 pM, less than 750 pM, less than 500 pM, less than 250 pM,
less than 200 pM, less than 150 pM, less than 100 pM, less than 75
pM as assessed using a method described herein or known to one of
skill in the art (e.g., a BIAcore assay, ELISA) (Biacore
International AB, Uppsala, Sweden). In a specific embodiment, an
antibody used in accordance with a method described herein may have
a dissociation constant (K.sub.d) of between 25 to 3400 pM, 25 to
3000 pM, 25 to 2500 pM, 25 to 2000 pM, 25 to 1500 pM, 25 to 1000
pM, 25 to 750 pM, 25 to 500 pM, 25 to 250 pM, 25 to 100 pM, 25 to
75 pM, or 25 to 50 pM as assessed using a method described herein
or known to one of skill in the art (e.g., a BIAcore assay, ELISA).
In another embodiment, an antibody used in accordance with a method
described herein may have a dissociation constant (K.sub.d) of 500
pM, 100 pM, 75 pM or 50 pM as assessed using a method described
herein or known to one of skill in the art (e.g., a BIAcore assay,
ELISA).
[0103] One embodiment of the invention includes an antibody that
binds to and neutralizes Ang-2, but does not bind to Ang-1. In
another embodiment, the antibody binds to both Ang-2 and Ang-1, but
only neutralizes Ang-2. In another embodiment, the antibody binds
to both Ang-2 and Ang-1, and neutralizes binding of both Ang-1 and
Ang-2 to Tie2.
[0104] In one embodiment, antibodies of the invention
preferentially bind Ang-2 over Ang-1. In some embodiments,
antibodies of the invention bind Ang-2 over Ang-1 in a ratio of at
least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1,
at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least
15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1,
at least 250:1, at least 500:1, at least 1000:1 or at least
10,000:1 or higher.
[0105] In one embodiment, antibodies of the invention
preferentially bind Ang-1 over Ang-2. In some embodiments,
antibodies of the invention bind Ang-1 over Ang-2 in a ratio of at
least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1,
at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least
15:1, at least 20:1, at least 25:1, at least 50:1, at least 100:1,
at least 250:1, at least 500:1, at least 1000:1 or at least
10,000:1 or higher.
[0106] Embodiments of the invention also include isolated binding
fragments of anti-Ang-2 antibodies. In one embodiment, the binding
fragments are derived from fully human anti-Ang-2 antibodies.
Exemplary fragments include Fv, Fab' or other well know antibody
fragments, as described in more detail below. Embodiments of the
invention also include cells that express fully human antibodies
against Ang-2. Examples of cells include hybridomas, or
recombinantly created cells, such as Chinese hamster ovary (CHO)
cells, variants of CHO cells (for example DG44), 293 cells and NS0
cells that produce antibodies against Ang-2. Additional information
about variants of CHO cells can be found in Andersen and Reilly
(2004) Current Opinion in Biotechnology 15, 456-462 which is
incorporated herein in its entirety by reference.
Preparation of Antibodies
Nucleic Acids Encoding Antibodies of the Invention
[0107] The invention also encompasses isolated nucleic acid
molecules encoding antibodies of the invention. In another
embodiment, the antibody is derived from the fully human monoclonal
antibody 3.19.3. In one embodiment there is provided an antibody
which binds to the same epitope or epitopes as fully human
monoclonal antibody 3.19.3.
[0108] In one embodiment, the isolated nucleic acid encodes an
antibody variable light chain corresponding to an amino acid
sequence selected from the group consisting of MEDI1 (SEQ ID
No.:3), MEDI2 (SEQ ID No.:4), MEDI3 (SEQ ID No.:5), MEDI6 (SEQ ID
NO:8), and MEDI4 (SEQ ID No.:6). In another embodiment, the
isolated nucleic acid encodes an antibody further comprising a
variable heavy chain corresponding to the amino acid sequence MEDI5
(SEQ ID NO:7). In a specific embodiment, the nucleic acids of the
invention encode an antibody comprising a variable light chain
corresponding to an amino acid sequence selected from the group
consisting of MEDI1 (SEQ ID No.:3), MEDI2 (SEQ ID No.:4), MEDI3
(SEQ ID No.:5), MEDI6 (SEQ ID NO:8), and MEDI4 (SEQ ID No.:6); and
further comprises a variable heavy chain further comprising the
amino acid sequence MEDI5 (SEQ ID No.: 7).
Recombinant Expression Systems
[0109] Recombinant expression of an antibody of the invention
requires construction of an expression vector containing a
polynucleotide that encodes the antibody of the invention. Once a
polynucleotide encoding the antibody of the invention has been
obtained, the vector for the production of the antibody may be
produced by recombinant DNA technology using techniques well-known
in the art (e.g., U.S. Pat. No. 6,331,415, which is incorporated
herein by reference in its entirety). Thus, methods for preparing a
protein by expressing a polynucleotide containing an encoding
nucleotide sequence are described herein. The antibodies of the
invention can be produced in many different expression systems. In
one embodiment, the antibodies of the invention are produced and
secreted by mammalian cells. In another embodiment, the antibodies
of the invention are produced and secreted in human cells. In a
specific embodiment, the antibodies of the invention are produced
in cells of the 293F, CHO, or NS0 cell line.
[0110] Methods which are known to those skilled in the art can be
used to construct expression vectors containing protein coding
sequences and appropriate transcriptional and translational control
signals. These methods include, for example, in vitro recombinant
DNA techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule
operably linked to a promoter.
[0111] Once the expression vector is transferred to a host cell by
conventional techniques, the transfected cells are then cultured by
conventional techniques to produce an antibody. Thus, the invention
includes host cells containing a polynucleotide encoding a protein
of the invention operably linked to a heterologous promoter.
[0112] A variety of host-expression vector systems may be utilized
to express antibodies of the invention or portions thereof as
described in U.S. Pat. No. 5,807,715. For example, mammalian cells
such as Chinese hamster ovary cells (CHO), in conjunction with a
vector such as the major intermediate early gene promoter element
from human cytomegalovirus is an effective expression system for
antibodies (Foecking et al., Gene, 45:101 (1986); and Cockett et
al., Bio/Technology, 8:2 (1990)). In addition, a host cell strain
may be chosen which modulates the expression of inserted sequences,
or modifies and processes the gene product in the specific fashion
desired. Such modifications (e.g., glycosylation) and processing
(e.g., cleavage) of protein products may be important for the
function of the protein. Different host cells have characteristic
and specific mechanisms for the post-translational processing and
modification of proteins and gene products. Appropriate cell lines
or host systems can be chosen to ensure the correct modification
and processing of the protein of the invention. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, MDCK, 293, 293F, 293T, 3T3, W138, BT483, Hs578T, HTB2, BT2O
and T47D, NS0, CRL7O3O and HsS78Bst cells.
[0113] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
protein molecule being expressed. For example, when a large
quantity of such an antibody is to be produced, for the generation
of pharmaceutical compositions comprising an antibody of the
invention, vectors which direct the expression of high levels of
fusion protein products that are readily purified may be desirable.
Such vectors include, but are not limited to, the E. coli
expression vector pUR278 (Ruther et al., EMBO, 12:1791 (1983)), in
which the coding sequence may be ligated individually into the
vector in frame with the lac Z coding region so that a fusion
protein is produced; pIN vectors (Inouye & Inouye, 1985,
Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster,
1989, J. Biol. Chem., 24:5503-5509 (1989)); and the like. pGEX
vectors may also be used to express foreign polypeptides as fusion
proteins with glutathione-S-transferase (GST). In general, such
fusion proteins are soluble and can easily be purified from lysed
cells by adsorption and binding to glutathione-agarose affinity
matrix followed by elution in the presence of free glutathione. The
pGEX vectors are designed to introduce a thrombin and/or factor Xa
protease cleavage sites into the expressed polypeptide so that the
cloned target gene product can be released from the GST moiety.
[0114] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The protein
coding sequence may be cloned individually into non-essential
regions (for example, the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example, the polyhedrin
promoter).
[0115] In mammalian host cells, a number of virus based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the coding sequence of interest may be ligated
to an adenovirus transcription/translation control complex, e.g.,
the late promoter and tripartite leader sequence. This chimeric
gene may then be inserted in the adenovirus genome by in vitro or
in vivo recombination. Insertion into a non-essential region of the
viral genome (e.g., region E1 or E3) will result in a recombinant
virus that is viable and capable of expressing the antibody
molecule in infected hosts (e.g., see, Logan & Shenk, Proc.
Natl. Acad. Sci. USA, 81:355-359 (1984)). Specific initiation
signals may also be required for efficient translation of inserted
antibody coding sequences. These signals include the ATG initiation
codon and adjacent sequences. Furthermore, the initiation codon
should generally be in frame with the reading frame of the desired
coding sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see, e.g., Bittner et al., Methods in Enzymol.,
153:51-544 (1987)).
[0116] Stable expression can be used for long-term, high-yield
production of recombinant proteins. For example, cell lines which
stably express the protein molecule may be generated. Host cells
can be transformed with an appropriately engineered vector
comprising expression control elements (e.g., promoter, enhancer,
transcription terminators, polyadenylation sites, etc.), and a
selectable marker gene. Following the introduction of the foreign
DNA, cells may be allowed to grow for 1-2 days in an enriched
media, and then are switched to a selective media. The selectable
marker in the recombinant plasmid confers resistance to the
selection and allows cells that stably integrated the plasmid into
their chromosomes to grow and form foci which in turn can be cloned
and expanded into cell lines. Plasmids that encode an antibody of
the invention can be used to introduce the gene/cDNA into any cell
line suitable for production in culture.
[0117] A number of selection systems may be used, including, but
not limited to, the herpes simplex virus thymidine kinase (Wigler
et al., Cell, 11:223 (1977)), hypoxanthineguanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA, 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell, 22:8-17 (1980)) genes
can be employed in tk-, hgprt- or aprT-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA, 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA, 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991);
Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);
Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann.
Rev. Biochem. 62:191-217 (1993); May, TIB TECH 11(5):155-2 15
(1993)); and hygro, which confers resistance to hygromycin
(Santerre et al., Gene, 30:147 (1984)). Methods commonly known in
the art of recombinant DNA technology may be routinely applied to
select the desired recombinant clone, and such methods are
described, for example, in Ausubel et al. (eds.), Current Protocols
in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,
Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds.),
Current Protocols in Human Genetics, John Wiley & Sons, NY
(1994); Colberre-Garapin et al., 1981, J. Mol. Biol., 150:1, which
are incorporated by reference herein in their entireties.
[0118] Once an antibody of the invention has been produced by
recombinant expression, it may be purified by any method known in
the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigens Protein A or
Protein G, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins. Further, the proteins of the present
invention or fragments thereof may be fused to heterologous
polypeptide sequences described herein or otherwise known in the
art to facilitate purification.
Scalable Production of Antibodies
[0119] In an effort to obtain large quantities, antibodies of the
invention may be produced by a scalable process (hereinafter
referred to as "scalable process of the invention"). In some
embodiments, antibodies may be produced by a scalable process of
the invention in the research laboratory that may be scaled up to
produce the antibodies of the invention in analytical scale
bioreactors (for example, but not limited to 5 L, 10 L, 15 L, 30 L,
or 50 L bioreactors). In other embodiments, the antibodies may be
produced by a scalable process of the invention in the research
laboratory that may be scaled up to produce the antibodies of the
invention in production scale bioreactors (for example, but not
limited to 75 L, 100 L, 150 L, 300 L, or 500 L). In some
embodiments, the scalable process of the invention results in
little or no reduction in production efficiency as compared to the
production process performed in the research laboratory. In other
embodiments, the scalable process of the invention produces
antibodies at production efficiency of about 10 mg/L, about 20 m/L,
about 30 mg/L, about 50 mg/L, about 75 mg/L, about 100 mg/L, about
125 mg/L, about 150 mg/L, about 175 mg/L, about 200 mg/L, about 250
mg/L, or about 300 mg/L or higher.
[0120] In other embodiments, the scalable process of the invention
produces antibodies at production efficiency of at least about 10
mg/L, at least about 20 m/L, at least about 30 mg/L, at least about
50 mg/L, at least about 75 mg/L, at least about 100 mg/L, at least
about 125 mg/L, at least about 150 mg/L, at least about 175 mg/L,
at least about 200 mg/L, at least about 250 mg/L, or at least about
300 mg/L or higher.
[0121] In other embodiments, the scalable process of the invention
produces antibodies at production efficiency from about 10 mg/L to
about 300 mg/L, from about 10 mg/L to about 250 mg/L, from about 10
mg/L to about 200 mg/L, from about 10 mg/L to about 175 mg/L, from
about 10 mg/L to about 150 mg/L, from about 10 mg/L to about 100
mg/L, from about 20 mg/L to about 300 mg/L, from about 20 mg/L to
about 250 mg/L, from about 20 mg/L to about 200 mg/L, from 20 mg/L
to about 175 mg/L, from about 20 mg/L to about 150 mg/L, from about
20 mg/L to about 125 mg/L, from about 20 mg/L to about 100 mg/L,
from about 30 mg/L to about 300 mg/L, from about 30 mg/L to about
250 mg/L, from about 30 mg/L to about 200 mg/L, from about 30 mg/L
to about 175 mg/L, from about 30 mg/L to about 150 mg/L, from about
30 mg/L to about 125 mg/L, from about 30 mg/L to about 100 mg/L,
from about 50 mg/L to about 300 mg/L, from about 50 mg/L to about
250 mg/L, from about 50 mg/L to about 200 mg/L, from 50 mg/L to
about 175 mg/L, from about 50 mg/L to about 150 mg/L, from about 50
mg/L to about 125 mg/L, or from about 50 mg/L to about 100
mg/L.
[0122] In one embodiment, the antibodies of the invention exhibit
increased stability and/or enhanced production efficiency. In one
embodiment, the antibodies of the invention exhibit a production
efficiency at least 2 times, at least 3 times, at least 4 times, at
least 5 time, at least 6 times, at least 7 times, at least 8 times,
at least 10 times or that exhibited by antibody 3.19.3.
Antibody Purification and Isolation
[0123] When using recombinant techniques, the antibodies of the
invention can be produced intracellularly, in the periplasmic
space, or directly secreted into the medium. If the protein is
produced intracellularly, as a first step, the particulate debris,
either host cells or lysed fragments, is removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology,
10:163-167 (1992) describe a procedure for isolating antibodies
which are secreted into the periplasmic space of E. coli. Briefly,
cell paste is thawed in the presence of sodium acetate (pH 3.5),
EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.
Cell debris can be removed by centrifugation. Where the antibody is
secreted into the medium, supernatants from such expression systems
are generally first concentrated using a commercially available
protein concentration filter, for example, an Amicon or Millipore
Pellicon ultrafiltration unit. A protease inhibitor such as PMSF
may be included in any of the foregoing steps to inhibit
proteolysis and antibiotics may be included to prevent the growth
of adventitious contaminants.
[0124] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography,
hydrophobic interaction chromatography, ion exchange
chromatography, gel electrophoresis, dialysis, and/or affinity
chromatography either alone or in combination with other
purification steps. The suitability of protein A as an affinity
ligand depends on the species and isotype of any immunoglobulin Fc
that is present in the antibody. Protein A can be used to purify
antibodies that are based on human .gamma.1, .gamma.2, or .gamma.4
heavy chains (Lindmark et al., J. Immunol. Methods, 62:1-13
(1983)). Protein G is recommended for all mouse isotypes and for
human .gamma.3 (Cuss et al., EMBO J., 5:15671575 (1986)). The
matrix to which the affinity ligand is attached is most often
agarose, but other matrices are available. Mechanically stable
matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
protein of the invention comprises a CH3 domain, the Bakerbond ABX
resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification.
Other techniques for protein purification such as fractionation on
an ion-exchange column, ethanol precipitation, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin, SEPHAROSE
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered.
[0125] Following any preliminary purification step(s), the mixture
comprising the antibodies of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, and performed at low
salt concentrations (e.g., from about 0-0.25 M salt).
[0126] Recombinant protein isolation and purification can be
accomplished by many art-accepted techniques exploiting the
physical characteristics of the protein of interest, such as size,
charge, hydrophobicity, affinity, etc. In one embodiment, the
proteins of the invention are subjected to isolation/purification
methods known in the art such as size exclusion chromatography,
ion-exchange chromatography, and affinity chromatography. In
another embodiment, the proteins of the invention are purified
through protein A affinity chromatography. In another embodiment,
the proteins of the invention are purified through affinity
chromatography exploiting one or more binding specificities within
the protein.
[0127] To ensure the stability of the antibodies of the invention,
suitable assays have been developed. In one embodiment, the
stability of proteins of the invention is characterized by known
techniques in the art. In other embodiments, the stability of the
proteins of the invention can be assessed by aggregation and/or
fragmentation rate or profile. To determine the level of
aggregation or fragmentation, many techniques may be used. In one
embodiment, the aggregation and/or fragmentation profile may be
assessed by the use of analytical ultracentrifugation (AUC),
size-exclusion chromatography (SEC), high-performance
size-exclusion chromatography (HPSEC), melting temperature
(T.sub.m), polyacrylamide gel electrophoresis (PAGE), capillary gel
electrophoresis (CGE), light scattering (SLS), Fourier Transform
Infrared Spectroscopy (FTIR), circular dichroism (CD), urea-induced
protein unfolding techniques, intrinsic tryptophan fluorescence,
differential scanning calorimetry, or
1-anilino-8-naphthalenesulfonic acid (ANS) protein binding
techniques. In another embodiment, the stability of proteins of the
invention is characterized by polyacrylamide gel electrophoresis
(PAGE) analysis. In another embodiment, the stability of the
proteins of the invention is characterized by size exclusion
chromatography (SEC) profile analysis.
[0128] Another measure of stability is the relative resistance to
protease degradation exhibited by a protein. In one embodiment, the
stability of the proteins of the invention is characterized by a
protease resistance assay. In one embodiment, the protease utilized
in the protease resistance assay is a serine protease, threonine
protease, cysteine protease, aspartic acid protease,
metalloprotease, or a glutamic acid protease. In one embodiment,
the proteins of the invention are subjected to a protease
resistance assay in which the protease is trypsin, chymotrypsin,
cathepsin B, D, L, or G, pepsin, papain, elastase, HIV-1 protease,
chymosin, renin, plasmepsin, plasmin, carboxypeptidase E, caspase
1-10, or calpain. In another embodiment, proteins of the invention
exhibit a low level of protease degradation. In some embodiments,
the antibodies of the invention exhibit protease resistance in
which at least 70%, at least 75%, at least 80%, at least 85%, at
least 90%, at least 95% or more of the protein remains undigested
after incubation with the protease under standard conditions for
the protease selected.
[0129] The invention also provides methods of testing the binding
of antibodies of the invention. The binding specificities of an
antibody can be assessed by many different art accepted techniques
such as phage display and other ELISA based technologies. In one
embodiment, the binding specificities of the antibodies of the
invention may be tested by any well known technique in the art. In
another embodiment, the antibodies of the invention may be analyzed
by any of the techniques presented in the specification. In another
embodiment, the binding specificities for antibodies of the
invention may be tested by an ELISA based assay.
Methods of Monitoring the Stability and Aggregation of Antibody
Formulations
[0130] There are various methods available for assessing the
stability of protein formulations based on the physical and
chemical structures of the proteins as well as on their biological
activities. For example, to study denaturation of proteins, methods
such as charge-transfer absorption, thermal analysis, fluorescence
spectroscopy, circular dichroism, NMR, rCGE (reducing capillary gel
electrophoresis) and HPSEC (high performance size exclusion
chromatography), are available (See, for example, Wang et al.,
1988, J. of Parenteral Science & Technology
42(Suppl):S4-S26).
[0131] The rCGE and HPSEC are the most common and simplest methods
to assess the formation of protein aggregates, protein degradation,
and protein fragmentation. Accordingly, the stability of the liquid
formulations of the present invention may be assessed by these
methods.
[0132] The liquid formulations of the present invention comprise an
antibody of the invention and exhibit low to undetectable levels of
aggregation as measured by HPSEC or rCGE, that is, no more than 5%,
no more than 4%, no more than 3%, no more than 2%, no more than 1%,
or no more than 0.5% aggregate by weight protein, and low to
undetectable levels of fragmentation, that is, 80% or higher, 85%
or higher, 90% or higher, 95% or higher, 98% or higher, or 99% or
higher, or 99.5% or higher of the total peak area in the peak(s)
representing intact antibodies. Antibody formulations often
comprise antibodies at a concentration of about 1-100 mg/ml along
with an appropriate excipient. These antibody formulations may be
analyzed for aggregation levels at 1, 2, 3, 4, 5, 6, 7, 14, 21, 28,
35, or 45 days or more after formulation. Also, in stability
studies, antibody formulations are often incubated at 2-4.degree.
C., 10-15.degree. C., 22-27.degree. C., 30-37.degree. C., or
40-42.degree. C. to assess aggregation rates. In the case of
SDS-PAGE, the density or the radioactivity of each band stained or
labeled with radioisotope can be measured and the % density or %
radioactivity of the band representing non-degraded antibodies of
the invention can be obtained.
[0133] In one embodiment, the antibodies of the invention exhibit a
lowered aggregation rate than antibody 3.19.3. In one embodiment,
the antibodies of the invention exhibit an aggregation rate that is
at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, at least 90% or at
least 95% lower than the aggregation rate exhibited by antibody
3.19.3 assessed under similar experimental conditions. In another
embodiment, the antibodies of the invention exhibit an aggregation
rate of at least 25%, at least 20%, at least 15%, at least 10%, at
least 5%, at least 2%, at least 1%, or at least 0.5% as measured by
the experimental conditions outlined in Example 2.
[0134] The stability of the liquid formulations of the present
invention can be also assessed by any assays which measure the
biological activity of the antibodies in the formulation. The
biological activities of antibodies include, but are not limited
to, antigen-binding activity, complement-activation activity,
Fc-receptor binding activity, receptor/ligand neutralizing
activity, receptor agonism or antagonism and so forth.
Antigen-binding activity of the antibodies can be measured by any
method known to those skilled in the art, including but not limited
to ELISA, radioimmunoassay, Western blot, and the like (Also see
Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor
Laboratory Press, 2nd ed. 1988) (incorporated by reference herein
in its entirety). The purity of the liquid antibody formulations of
the invention may be measured by any method well-known to one of
skill in the art such as, e.g., HPSEC. The sterility of the liquid
antibody formulations may be assessed as follows: sterile
soybean-casein digest medium and fluid thioglycollate medium are
inoculated with a test liquid antibody formulation by filtering the
liquid formulation through a sterile filter having a nominal
porosity of 0.45 .mu.m. When using the Sterisure.TM. or
Steritest.TM. method, each filter device is aseptically filled with
approximately 100 ml of sterile soybean-casein digest medium or
fluid thioglycollate medium. When using the conventional method,
the challenged filter is aseptically transferred to 100 ml of
sterile soybean-casein digest medium or fluid thioglycollate
medium. The media are incubated at appropriate temperatures and
observed three times over a 14 day period for evidence of bacterial
or fungal growth.
Methods of Using Antibodies
[0135] In addition, embodiments of the invention include methods of
using these antibodies for treating diseases. Anti-Ang-2 antibodies
are useful for preventing Ang-2 mediated Tie2 signal transduction,
thereby inhibiting angiogenesis. The mechanism of action of this
inhibition may include inhibition of Ang-2/Ang-1 from binding to
the receptor Tie2; inhibition of Ang-2/Ang-1 induced Tie2
signaling; Ang-2/Ang1 mediated phosphorylation of Tie-2; or
enhanced clearance of Ang-2 therein lowering the effective
concentration of Ang-2 for binding to Tie-2. In another embodiment,
the antibodies of the invention may act through reducing
circulating Ang-2/Ang-1 levels.
[0136] Diseases that are treatable through this inhibition
mechanism include, but are not limited to, neoplastic diseases,
such as, melanoma, small cell lung cancer, non-small cell lung
cancer, glioma, hepatocellular (liver) carcinoma, glioblastoma, and
cancers and tumors of the thyroid, stomach, prostate, breast,
ovary, bladder, lung, uterus, kidney, colon, and pancreas, salivary
gland, and colorectal.
[0137] Other embodiments of the invention include diagnostic assays
for specifically determining the quantity of Ang-2 in a biological
sample. The assay kit can include anti-Ang-2 antibodies along with
the necessary labels for detecting such antibodies. These
diagnostic assays are useful to screen for angiogenesis-related
diseases including, but not limited to, neoplastic diseases, such
as, melanoma, small cell lung cancer, non-small cell lung cancer,
glioma, hepatocellular (liver) carcinoma, glioblastoma, and
carcinoma of the thyroid, stomach, prostate, breast, ovary,
bladder, lung, uterus, kidney, colon, and pancreas, salivary gland,
and colorectum.
[0138] According to another aspect of the invention there is
provided an antagonist of the biological activity of Angiopoietin-1
and Angiopoietin-2 wherein the antagonist binds to Angiopoietin-1
and Angiopoietin-2.
[0139] According to another aspect of the invention there is
provided an antagonist of the biological activity of Angiopoietin-1
and Angiopoietin-2 wherein the antagonist is not a compound.
[0140] In one embodiment there is provided an antagonist of the
biological activity of Angiopoietin-1 and Angiopoietin-2 wherein
the Angiopoietin-1 antagonist activity and the Angiopoietin-2
antagonist activity is comprised within one molecule. In an
alternative embodiment there is provided an antagonist wherein the
Angiopoietin-1 antagonist activity and the Angiopoietin-2
antagonist activity is comprised within more than one molecule.
[0141] In one embodiment there is provided an antagonist of the
biological activity of Angiopoietin-1 and Angiopoietin-2 wherein
the antagonist may bind to: [0142] I. the Tie-2 receptor; [0143]
II. Angiopoietin-1 and/or Angiopoietin-2; [0144] III. Tie-2
receptor-Angiopoietin-1 complex; or [0145] IV. Tie-2
receptor-Angiopoietin-2 complex, [0146] V. or any combination of
these.
[0147] In one embodiment the antagonist of the biological activity
of Angiopoietin-1 and Angiopoietin-2 may bind to Angiopoietin-1
and/or Angiopoietin-2 and/or Tie-2 and thereby prevent
Angiopoietin-1 and Angiopoietin-2 mediated Tie-2 signal
transduction, thereby inhibiting angiogenesis. The mechanism of
action of this inhibition may include; [0148] I. binding of the
antagonist to Angiopoietin-1 and inhibiting the binding of
Angiopoietin-1 to its receptor, Tie-2, and/or [0149] II. binding of
the antagonist to Angiopoietin-2 and inhibit the binding of
Angiopoietin-2 to its receptor, Tie-2, and/or [0150] III. enhancing
the clearance of Angiopoietin-1 and/or Angiopoietin-2 therein
lowering the effective concentration of Angiopoietin-1 and/or
Angiopoietin-2 available for binding to Tie-2, [0151] IV. or any
combination of these, sufficient to antagonize the biological
activity of Angiopoietin-1 and Angiopoietin-2.
[0152] Without wishing to be bound by theoretical considerations,
mechanisms by which antagonism of the biological activity of
Angiopoietin-1 and/or Angiopoietin-2 can be achieved include, but
are not limited to, inhibition of binding of Angiopoietin-1 and/or
Angiopoietin-2 to the receptor Tie-2, inhibition of Angiopoietin-1
and/or Angiopoietin-2 induced Tie-2 signaling, reduced
Angiopoietin-1 and/or Angiopoietin-2 mediated Tie-2 phosphorylation
or increased clearance of Angiopoietin-1 and/or Angiopoietin-2,
therein reducing the effective concentration of Angiopoietin-1
and/or Angiopoietin-2.
[0153] According to another aspect of the invention there is
provided a method of antagonizing the biological activity of
Angiopoietin-1 and Angiopoietin-2 comprising administering an
antagonist as described hereinabove. The method may include
selecting an animal in need of treatment for disease-related
angiogenesis, and administering to said animal a therapeutically
effective dose of an antagonist of the biological activity of
Angiopoietin-1 and Angiopoietin-2.
[0154] According to another aspect of the invention there is
provided a method of antagonizing the biological activity of
Angiopoietin-1 and Angiopoietin-2 comprising administering an
antibody as described hereinabove. The method may include selecting
a subject in need of treatment for disease-related angiogenesis,
and administering to said subject a therapeutically effective dose
of an antibody which antagonizes the biological activity of
Angiopoietin-1 and Angiopoietin-2.
[0155] According to another aspect there is provided a method of
treating disease-related angiogenesis in a mammal comprising
administering a therapeutically effective amount of an antagonist
of the biological activity of Angiopoietin-1 and Angiopoietin-2.
The method may include selecting a subject in need of treatment for
disease-related angiogenesis, and administering to said subject a
therapeutically effective dose of an antagonist of the biological
activity of Angiopoietin-1 and Angiopoietin-2.
[0156] According to another aspect there is provided a method of
treating disease-related angiogenesis in a subject comprising
administering a therapeutically effective amount of an antibody
which antagonizes the biological activity of Angiopoietin-1 and
Angiopoietin-2. The method may include selecting a subject in need
of treatment for disease-related angiogenesis, and administering to
said subject a therapeutically effective dose of an antibody which
antagonizes the biological activity of Angiopoietin-1 and
Angiopoietin-2. The antibody can be administered alone, or can be
administered in combination with additional antibodies or
chemotherapies, biological therapies/immunotherapies, radiation
therapies, hormonal therapies, or surgery.
[0157] According to another aspect there is provided a method of
treating cancer in a mammal comprising administering a
therapeutically effective amount of an antagonist of the biological
activity of Angiopoietin-1 and Angiopoietin-2. The method may
include selecting an animal in need of treatment for cancer, and
administering to said animal a therapeutically effective dose of an
antagonist which antagonizes the biological activity of
Angiopoietin-1 and Angiopoietin-2. The antagonist can be
administered alone, or can be administered in combination with
additional antibodies or chemotherapies, biological
therapies/immunotherapies, radiation therapies, hormonal therapies,
or surgery.
[0158] According to another aspect of the invention there is
provided the use of an antibody of the invention for the
manufacture of a medicament for the treatment of disease-related
angiogenesis.
[0159] According to another aspect of the invention there is
provided the use of an antibody which antagonizes the biological
activity of Angiopoietin-1 and Angiopoietin-2 for the manufacture
of a medicament for the treatment of disease-related
angiogenesis.
[0160] In one embodiment the present invention is particularly
suitable for use in antagonizing Angiopoietin-1 or Angiopoietin-2,
in patients with a tumor which is dependent alone, or in part, on a
Tie-2 receptor.
[0161] The invention also provides methods of using antibodies to
ameliorate, treat, or prevent cancer or symptoms thereof. In one
embodiment, methods of the invention are useful in the treatment of
cancers of the head, neck, eye, mouth, throat, esophagus, chest,
skin, bone, lung, colon, rectum, colorectal, stomach, spleen,
kidney, skeletal muscle, subcutaneous tissue, metastatic melanoma,
endometrial, prostate, breast, ovaries, testicles, thyroid, blood,
lymph nodes, kidney, liver, pancreas, brain, or central nervous
system. Examples of cancers that can be prevented, managed, treated
or ameliorated in accordance with the methods of the invention
include, but are not limited to, cancer of the head, neck, eye,
mouth, throat, esophagus, chest, bone, lung, colon, rectum,
stomach, prostate, breast, ovaries, kidney, liver, pancreas, and
brain. Additional cancers include, but are not limited to, the
following: leukemias such as but not limited to, acute leukemia,
acute lymphocytic leukemia, acute myelocytic leukemias such as
myeloblastic, promyelocytic, myelomonocytic, monocytic,
erythroleukemia leukemias and myelodysplastic syndrome, chronic
leukemias such as but not limited to, chronic myclocytic
(granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell
leukemia; polycythemia vera; lymphomas such as but not limited to
Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as
but not limited to smoldering multiple mycloma, nonsecretory
myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary
plasmacytoma and extramedullary plasmacytoma; Waldenstrom's
macroglobulinemia; monoclonal gammopathy of undetermined
significance; benign monoclonal gammopathy; heavy chain disease;
bone cancer and connective tissue sarcomas such as but not limited
to bone sarcoma, myeloma bone disease, multiple myeloma,
cholesteatoma-induced bone osteosarcoma, Paget's disease of bone,
osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell
tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma,
soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma,
Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma,
neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors
such as but not limited to, glioma, astrocytoma, brain stem glioma,
ependymoma, oligodendroglioma, non-glial tumor, acoustic neurinoma,
craniopharyngioma, medulloblastoma, meningioma, pineocytoma,
pineoblastoma, and primary brain lymphoma; breast cancer including
but not limited to adenocarcinoma, lobular (small cell) carcinoma,
intraductal carcinoma, medullary breast cancer, mucinous breast
cancer, tubular breast cancer, papillary breast cancer, Paget's
disease (including juvenile Paget's disease) and inflammatory
breast cancer; adrenal cancer such as but not limited to
pheochromocytom and adrenocortical carcinoma; thyroid cancer such
as but not limited to papillary or follicular thyroid cancer,
medullary thyroid cancer and anaplastic thyroid cancer; pancreatic
cancer such as but not limited to, insulinoma, gastrinoma,
glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or
islet cell tumor; pituitary cancers such as but limited to
Cushing's disease, prolactin-secreting tumor, acromegaly, and
diabetes insipius; eye cancers such as but not limited to ocular
melanoma such as iris melanoma, choroidal melanoma, and cilliary
body melanoma, and retinoblastoma; vaginal cancers such as squamous
cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as
squamous cell carcinoma, melanoma, adenocarcinoma, basal cell
carcinoma, sarcoma, and Paget's disease; cervical cancers such as
but not limited to, squamous cell carcinoma, and adenocarcinoma;
uterine cancers such as but not limited to endometrial carcinoma
and uterine sarcoma; ovarian cancers such as but not limited to,
ovarian epithelial carcinoma, borderline tumor, germ cell tumor,
and stromal tumor; esophageal cancers such as but not limited to,
squamous cancer, adenocarcinoma, adenoid cyctic carcinoma,
mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma,
melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small
cell) carcinoma; stomach cancers such as but not limited to,
adenocarcinoma, fungating (polypoid), ulcerating, superficial
spreading, diffusely spreading, malignant lymphoma, liposarcoma,
fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers;
liver cancers such as but not limited to hepatocellular carcinoma
and hepatoblastoma, gallbladder cancers such as adenocarcinoma;
cholangiocarcinomas such as but not limited to pappillary, nodular,
and diffuse; lung cancers such as non-small cell lung cancer,
squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma,
large-cell carcinoma and small-cell lung cancer; testicular cancers
such as but not limited to germinal tumor, seminoma, anaplastic,
classic (typical), spermatocytic, nonseminoma, embryonal carcinoma,
teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate
cancers such as but not limited to, adenocarcinoma, leiomyosarcoma,
and rhabdomyosarcoma; penal cancers; oral cancers such as but not
limited to squamous cell carcinoma; basal cancers; salivary gland
cancers such as but not limited to adenocarcinoma, mucoepidermoid
carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but
not limited to squamous cell cancer, and verrucous; skin cancers
such as but not limited to, basal cell carcinoma, squamous cell
carcinoma and melanoma, superficial spreading melanoma, nodular
melanoma, lentigo malignant melanoma, acral lentiginous melanoma;
kidney cancers such as but not limited to renal cell cancer,
adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell
cancer (renal pelvis and/or ureter); Wilms' tumor; bladder cancers
such as but not limited to transitional cell carcinoma, squamous
cell cancer, adenocarcinoma, carcinosarcoma. In addition, cancers
include myxosarcoma, osteogenic sarcoma, endotheliosarcoma,
lymphangioendotheliosarcoma, mesothelioma, synovioma,
hemangioblastoma, epithelial carcinoma, cystadenocarcinoma,
bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland
carcinoma, papillary carcinoma and papillary adenocarcinomas (for a
review of such disorders, see Fishman et al., 1985, Medicine, 2d
Ed., J. B. Lippincott Co., Philadelphia and Murphy et al., 1997,
Informed Decisions: The Complete Book of Cancer Diagnosis,
Treatment, and Recovery, Viking Penguin, Penguin Books U.S.A.,
inc., United States of America). It is also contemplated that
cancers caused by aberrations in apoptosis can also be treated by
the methods and compositions of the invention. Such cancers may
include, but not be limited to, follicular lymphomas, carcinomas
with p53 mutations, hormone dependent tumors of the breast,
prostate and ovary, and precancerous lesions such as familial
adenomatous polyposis, and myelodysplastic syndromes.
[0162] The invention also provides methods of using antibodies to
deplete a cell population. In one embodiment, methods of the
invention are useful in the depletion of the following cell types:
eosinophil, basophil, neutrophil, T cell, B cell, mast cell,
monocytes, endothelial cell and tumor cell. Tumor cells may be
cells derived from any of the cancer disorders described
herein.
[0163] The antibodies of the invention and compositions comprising
the same are useful for many purposes, for example, as therapeutics
against a wide range of chronic and acute diseases and disorders
including, but not limited to, autoimmune and/or inflammatory
disorders, which include Sjogren's syndrome, rheumatoid arthritis,
lupus psoriasis, atherosclerosis, diabetic and other retinopathies,
retrolental fibroplasia, age-related macular degeneration,
neovascular glaucoma, hemangiomas, thyroid hyperplasias (including
Grave's disease), corneal and other tissue transplantation, and
chronic inflammation, sepsis, rheumatoid arthritis, peritonitis,
Crohn's disease, reperfusion injury, septicemia, endotoxic shock,
cystic fibrosis, endocarditis, psoriasis, arthritis (e.g.,
psoriatic arthritis), anaphylactic shock, organ ischemia,
reperfusion injury, spinal cord injury and allograft rejection.
Other Examples of autoimmune and/or inflammatory disorders include,
but are not limited to, alopecia areata, ankylosing spondylitis,
antiphospholipid syndrome, autoimmune Addison's disease, autoimmune
diseases of the adrenal gland, autoimmune hemolytic anemia,
autoimmune hepatitis, autoimmune oophoritis and orchitis, Sjogren's
syndrome, psoriasis, atherosclerosis, diabetic and other
retinopathies, retrolental fibroplasia, age-related macular
degeneration, neovascular glaucoma, hemangiomas, thyroid
hyperplasias (including Grave's disease), corneal and other tissue
transplantation, and chronic inflammation, sepsis, rheumatoid
arthritis, peritonitis, Crohn's disease, reperfusion injury,
septicemia, endotoxic shock, cystic fibrosis, endocarditis,
psoriasis, arthritis (e.g., psoriatic arthritis), anaphylactic
shock, organ ischemia, reperfusion injury, spinal cord injury and
allograft rejection. autoimmune thrombocytopenia, Behcet's disease,
bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis,
chronic fatigue immune dysfunction syndrome (CFIDS), chronic
inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome,
cicatrical pemphigoid, CREST syndrome, cold agglutinin disease,
Crohn's disease, discoid lupus, essential mixed cryoglobulinemia,
fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease,
Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary
fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA
neuropathy, juvenile arthritis, lichen planus, lupus erythematosus,
Meniere's disease, mixed connective tissue disease, multiple
sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia
gravis, pemphigus vulgaris, pernicious anemia, polyarteritis
nodosa, polychrondritis, polyglandular syndromes, polymyalgia
rheumatica, polymyositis and dermatomyositis, primary
agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic
arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid
arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man
syndrome, systemic lupus erythematosus, lupus erythematosus,
takayasu arteritis, temporal artcristis/giant cell arteritis,
ulcerative colitis, uveitis, vasculitides such as dermatitis
herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.
Examples of inflammatory disorders include, but are not limited to,
asthma, encephilitis, inflammatory bowel disease, chronic
obstructive pulmonary disease (COPD), allergic disorders, septic
shock, pulmonary fibrosis, undifferentitated spondyloarthropathy,
undifferentiated arthropathy, arthritis, inflammatory osteolysis,
and chronic inflammation resulting from chronic viral or bacteria
infections.
[0164] In one embodiment disease-related angiogenesis may be bone
and joint disease: for example, but not limited to arthritis
associated with or including osteoarthritis/osteoarthrosis, both
primary and secondary to, for example, congenital hip dysplasia;
cervical and lumbar spondylitis, and low back and neck pain;
rheumatoid arthritis and Still's disease; seronegative
spondyloarthropathies including ankylosing spondylitis, psoriatic
arthritis, reactive arthritis and undifferentiated
spondarthropathy; septic arthritis and other infection-related
arthopathies and bone disorders such as tuberculosis, including
Potts' disease and Poncet's syndrome; acute and chronic
crystal-induced synovitis including urate gout, calcium
pyrophosphate deposition disease, and calcium apatite related
tendon, bursal and synovial inflammation; Behcet's disease; primary
and secondary Sjogren's syndrome; systemic sclerosis and limited
scleroderma; systemic lupus erythematosus, mixed connective tissue
disease, and undifferentiated connective tissue disease;
inflammatory myopathies including dermatomyositits and
polymyositis; polymalgia rheumatica; juvenile arthritis including
idiopathic inflammatory arthritides of whatever joint distribution
and associated syndromes, and rheumatic fever and its systemic
complications; vasculitides including giant cell arteritis,
Takayasu's arteritis, Churg-Strauss syndrome, polyarteritis nodosa,
microscopic polyarteritis, and vasculitides associated with viral
infection, hypersensitivity reactions, cryoglobulins, and
paraproteins; low back pain; Familial Mediterranean fever,
Muckle-Wells syndrome, and Familial Hibernian Fever, Kikuchi
disease; drug-induced arthalgias, tendonititides, and myopathies;
pain and connective tissue remodelling of musculoskeletal disorders
due to injury [for example sports injury] or disease: for example
arthitides (for example rheumatoid arthritis, osteoarthritis, gout
or crystal arthropathy), other joint disease (such as
intervertebral disc degeneration or temporomandibular joint
degeneration), bone remodelling disease (such as osteoporosis,
Paget's disease or osteonecrosis), polychondritits, scleroderma,
reactive arthritis, polymyalgia, mixed connective tissue disorder,
spondyloarthropathies or periodontal disease (such as
periodontitis).
[0165] In one embodiment disease-related angiogenesis may be
selected from one of the inflammatory arthritis group of diseases
including seronegative arthritis, seropositive arthritis, arthritis
related to other arthropathies, osteoarthritis or systemic lupus
erythematosus (SLE). In another embodiment disease-related
angiogenesis may be rheumatoid arthritis, seronegative
spondyloarthropathy, arthritis related to other arthropathies or
SLE. In one embodiment the seronegative spondyloarthropathy is
selected from ankylosing spondylitis, psoriatic arthritis, reactive
arthritis or inflammatory bowel disorder-related arthropathies. In
a specific embodiment disease-related angiogenesis is rheumatoid
arthritis. In another specific embodiment disease-related
angiogenesis is osteoarthritis.
[0166] In some embodiments, methods of the invention can be used to
reduce or inhibit disease-related angiogenesis. In some
embodiments, methods of the invention comprise a reduction or
inhibition of disease-related angiogenesis by at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% of the original
disease-related angiogenesis. In other embodiments, methods of the
invention comprise a reduction or inhibition of disease-related
angiogenesis by at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, or at least 95% of the disease-related angiogenesis prior to
treatment. In some embodiments, reduction in angiogenesis may be
measured by the methods presented herein in the Examples, or by
other methods known in the art. In specific embodiments, reduction
of angiogensis may be measured by staining of biopsy samples, or by
FITC-dextran accumulation in a tissue.
[0167] In some embodiments, methods of the invention can be used to
reduce or inhibit angiogenesis mediated by angiogenic factors. Such
factors include, but are not limited to FGF, FGF2, VEGF (and
various isoforms thereof), PDGF, TGF-.beta., endoglin, MCP-1, and
ephrins. In some embodiments, methods of the invention comprise a
reduction or inhibition of angiogenesis mediated by angiogenic
factors by at least 5%, at least 10%, at least 15%, at least 20%,
at least 25%, at least 30%, at least 35%, at least 40%, at least
45%, at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
or at least 95% of the level of angiogenesis mediated in the
absence of treatment. In a specific embodiment, methods of the
invention reduce angiogenesis induced by at least one or more
angiogenic factor selected from the group consisting of FGF, FGF2,
VEGF (and various isoforms thereof), PDGF, TGF-13, endoglin, MCP-1,
and ephrins.
[0168] In a specific embodiment, methods of the invention can be
used to reduce or inhibit FGF2-mediated angiogenesis. In some
embodiments, methods of the invention comprise a reduction or
inhibition of FGF2-mediated angiogenesis by at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, or at least 95% of the level of a
control FGF2-mediated angiogenesis sample.
[0169] In some embodiments, methods of the invention can be used to
reduce or inhibit symptoms associated with disease-related
angiogenesis. In some embodiments, methods of the invention
comprise a reduction or inhibition of symptoms associated with
disease-related angiogenesis by at least 5%, at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, or at least 95% of the original symptoms
associated with disease-related angiogenesis. In other embodiments,
methods of the invention comprise a reduction or inhibition of
disease-related angiogenesis by at least 5%, at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, or at least 95% of the symptoms associated with
disease-related angiogenesis prior to treatment. Such symptoms
include swelling, inflammation, fever, pain, edema, pleural
effusions, anemia, malaise, morning stiffness, lows of weight, poor
circulation, numbness in the limbs, and the like.
[0170] In other embodiments, methods of the invention can be used
to maintain disease-related angiogenesis. In some embodiments,
methods of the invention can be used to prevent disease-related
angiogenesis.
[0171] The compositions and methods of the invention can be used
with one or more conventional therapies that are used to prevent,
manage or treat the above diseases.
[0172] In one embodiment, the invention provides a method of
preventing development of cancer from a pre-cancerous state in an
animal. The pre-cancerous state may be dysplasia, hyperplasia or
cancer in situ (cancer in place).
[0173] In another embodiment, the invention provides a method of
treating, preventing, ameliorating or managing symptoms of cancer
in an animal. Symptoms of cancer may vary greatly depending on the
nature of the cancer and state of progression. Symptoms associated
with some cancer types may include; Bladder cancer: blood in the
urine, pain or burning upon urination; frequent urination; or
cloudy urine; Bone cancer: pain in the bone or swelling around the
affected site; fractures in bones; weakness, fatigue; weight loss;
repeated infections; nausea, vomiting, constipation, problems with
urination; weakness or numbness in the legs; bumps and bruises that
persist; Brain cancer: dizziness; drowsiness; abnormal eye
movements or changes in vision; weakness, loss of feeling in arms
or legs or difficulties in walking; fits or convulsions; changes in
personality, memory or speech; headaches that tend to be worse in
the morning and ease during the day, that may be accompanied by
nausea or vomiting; Breast cancer: a lump or thickening of the
breast; discharge from the nipple; change in the skin of the
breast; a feeling of heat; or enlarged lymph nodes under the arm;
Colorectal cancer: rectal bleeding (red blood in stools or black
stools); abdominal cramps; constipation alternating with diarrhea;
weight loss; loss of appetite; weakness; pallid complexion; Kidney
cancer: blood in urine; dull ache or pain in the back or side; lump
in kidney area, sometimes accompanied by high blood pressure or
abnormality in red blood cell count; Leukemia: weakness, paleness;
fever and flu-like symptoms; bruising and prolonged bleeding;
enlarged lymph nodes, spleen, liver; pain in bones and joints;
frequent infections; weight loss; night sweats; Lung cancer:
wheezing, persistent cough for months; blood-streaked sputum;
persistent ache in chest; congestion in lungs; enlarged lymph nodes
in the neck; Melanoma: change in mole or other bump on the skin,
including bleeding or change in size, shape, color, or texture;
Non-Hodgkin's lymphoma: painless swelling in the lymph nodes in the
neck, underarm, or groin; persistent fever; feeling of fatigue;
unexplained weight loss; itchy skin and rashes; small lumps in
skin; bone pain; swelling in the abdomen; liver or spleen
enlargement; Oral cancer: a lump in the mouth, ulceration of the
lip, tongue or inside of the mouth that does not heal within a
couple of weeks; dentures that no longer fit well; oral pain,
bleeding, foul breath, loose teeth, and changes in speech; Ovarian
cancer: abdominal swelling; in rare cases, abnormal vaginal
bleeding; digestive discomfort; Pancreatic cancer: upper abdominal
pain and unexplained weight loss; pain near the center of the back;
intolerance of fatty foods; yellowing of the skin; abdominal
masses; enlargement of liver and spleen; Prostate cancer: urination
difficulties due to blockage of the urethra; bladder retains urine,
creating frequent feelings of urgency to urinate, especially at
night; bladder not emptying completely; burning or painful
urination; bloody urine; tenderness over the bladder; and dull ache
in the pelvis or back; Stomach cancer: indigestion or heartburn;
discomfort or pain in the abdomen; nausea and vomiting; diarrhea or
constipation; bloating after meals; loss of appetite; weakness and
fatigue; bleeding--vomiting blood or blood in the stool; Uterine
cancer: abnormal vaginal bleeding, a watery bloody discharge in
postmenopausal women; a painful urination; pain during intercourse;
pain in pelvic area.
[0174] In another embodiment, the invention provides a method of
promoting tumor regression of a cancer. In one embodiment, the
method involves regression of the tumor by at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, at least 50%, at least 55%,
at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%, at least 90%, at least 95% of the original tumor
size. In a specific embodiment, the method comprises the
elimination of the tumor by inhibition of angiogenesis.
[0175] Cell proliferation rates may be assayed by many means known
in the art such as thymidine incorporation, DNA content, or cell
counts. In another embodiment, the invention provides a method of
inhibiting tumor cell proliferation. In one embodiment the method
comprises a reduction of tumor cell proliferation. In another
embodiment, the method comprises a reduction of tumor cell
proliferation by at least 5%, at least 10%, at least 15%, at least
20%, at least 25%, at least 30%, at least 35%, at least 40%, at
least 45%, at least 50%, at least 55%, at least 60%, at least 65%,
at least 70%, at least 75%, at least 80%, at least 85%, at least
90%, at least 95% of the original tumor cell proliferation
rate.
[0176] In another embodiment the invention provides a method of
depleting malignant tumor cells. In some embodiments, the method
comprises depleting tumor cells resident in the primary tumor. In
other embodiments, the method comprises depleting tumor cells in
circulation. In other embodiments, the method comprises depleting
tumor cells in a secondary site.
[0177] In another embodiment, the invention provides a method of
inhibiting angiogenesis of a cancer tumor. In some embodiments, the
method comprises inhibition of angiogenesis of a primary tumor
site. In other embodiments, the method comprises inhibition of
angiogenesis of a secondary tumor site.
[0178] The present invention provides methods of preventing,
treating, managing or ameliorating an inflammatory disorder or an
autoimmune disorder or one or more symptoms thereof in a subject,
said methods comprising administering to said subject an antibody
of the invention. In some embodiments, the present invention
provides methods of preventing, treating, managing or ameliorating
an inflammatory disorder or an autoimmune disorder associated with
inflammation or one or more symptoms thereof in a subject, said
methods comprising administering to said subject an antibody of the
invention and one or more TNF.alpha. antagonists. In further
embodiments, at least one of the TNF-.alpha. antagonists is a
soluble TNF-.alpha. receptor such as etanercept (ENBREL.TM.;
Immunex) or a fragment, derivative or analog thereof, or an
antibody that immunospecifically binds to TNF-.alpha. such as
infliximab (REMICADE.TM.; Centocor) or adalimumab (HUMIRA.TM.;
Abbott) a derivative, analog or antigen-binding fragment thereof.
In specific embodiments the methods of the invention are performed
prophylactically or therapeutically.
[0179] In some embodiments, the present invention provides methods
of preventing, treating, managing or ameliorating at least one
symptom associated with an inflammatory or autoimmune disorder.
Such symptoms may include anemia, swelling, inflammation, edema,
rash, swelling in the joints, bone synovial hyperplasia, synovitis,
synovial fibrosis, periostitis, or bone mineral density (loss). In
some embodiments, the present invention provides methods of
treating or managing at least one symptom in an individual
associated with an inflammatory or autoimmune disorder wherein said
method results in a reduction of at least 10%, at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, or more over the severity of at least
one symptom in an individual in the absence of treatment.
Combinations
[0180] The anti-angiogenic treatment defined herein may be applied
as a sole therapy or may involve, in addition to the compounds of
the invention, conventional surgery or radiotherapy or
chemotherapy. Such chemotherapy may include one or more of the
following categories of anti tumor agents:
[0181] (i) cytostatic agents such as antioestrogens (for example
tamoxifen, toremifene, raloxifene, droloxifene and iodoxyfene),
estrogen receptor down-regulators (for example fulvestrant),
antiandrogens (for example bicalutamide, flutamide, nilutamide and
cyproterone acetate), LHRH antagonists or LHRH agonists (for
example goserelin, leuprorelin and buserelin), progestogens (for
example megestrol acetate), aromatase inhibitors (for example as
anastrozole, letrozole, vorazole and exemestane) and inhibitors of
5' reductase such as finasteride;
[0182] (ii) agents which inhibit cancer cell invasion (for example
metalloproteinase inhibitors like marimastat and inhibitors of
urokinase plasminogen activator receptor function);
[0183] (iii) inhibitors of growth factor function, for example such
inhibitors include growth factor antibodies, growth factor receptor
antibodies (for example the anti-ErbB2 antibody trastuzumab
[Herceptin.TM.] and the anti-ErbB1 antibody cetuximab [C225]),
farnesyl transferase inhibitors, tyrosine kinase inhibitors and
serine/threonine kinase inhibitors, for example inhibitors of the
epidermal growth factor family (for example EGFR family tyrosine
kinase inhibitors such as N (3-chloro 4-fluorophenyl) 7-methoxy 6
(3-morpholinopropoxy)quinazolin 4-amine (gefitinib, AZD1839), N
(3-ethynylphenyl) 6,7 bis(2-methoxyethoxy)quinazolin 4-amine
(erlotinib, OSI 774) and 6-acrylamido N (3-chloro 4-fluorophenyl) 7
(3-morpholinopropoxy)quinazolin 4-amine (CI 1033)), for example
inhibitors of the platelet derived growth factor family and for
example inhibitors of the hepatocyte growth factor family;
[0184] (iv) antiangiogenic agents such as those which inhibit the
effects of vascular endothelial growth factor, (for example the
anti vascular endothelial cell growth factor antibody bevacizumab
(Avastin.RTM.), anti-vascular endothelial growth factor receptor
antibodies such anti-KDR antibodies and anti-flt1 antibodies,
compounds such as those disclosed in International Patent
Applications WO 97/22596, WO 97/30035, WO 97/3285, WO 98/13354,
WO00/47212 and WO01/32651) and compounds that work by other
mechanisms (for example linomide, inhibitors of integrin
.alpha.v.beta.3 function and angiostatin);
[0185] (v) vascular damaging agents such as Combretastatin A4 and
compounds disclosed in International Patent Applications WO
99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO
02/08213;
[0186] (vi) antisense therapies, for example those which are
directed to the targets listed above, such as ISIS 2503, an
anti-ras antisense;
[0187] (vii) gene therapy approaches, including for example
approaches to replace aberrant genes such as aberrant p53 or
aberrant BRCA1 or BRCA2, GDEPT (gene directed enzyme pro drug
therapy) approaches such as those using cytosine deaminase,
thymidine kinase or a bacterial nitroreductase enzyme and
approaches to increase patient tolerance to chemotherapy or
radiotherapy such as multi drug resistance gene therapy; and
[0188] (viii) immunotherapy approaches, including for example ex
vivo and in vivo approaches to increase the immunogenicity of
patient tumor cells, such as transfection with cytokines such as
interleukin-2, interleukin-4 or granulocyte macrophage colony
stimulating factor, approaches to decrease T cell anergy,
approaches using transfected immune cells such as cytokine
transfected dendritic cells, approaches using cytokine transfected
tumor cell lines and approaches using anti idiotypic
antibodies.
[0189] In one embodiment of the invention the anti-angiogenic
treatments of the invention are combined with agents which inhibit
the effects of vascular endothelial growth factor (VEGF), (for
example the anti-vascular endothelial cell growth factor antibody
bevacizumab (Avastin.RTM.), anti-vascular endothelial growth factor
receptor antibodies such anti-KDR antibodies and anti-flt1
antibodies, compounds such as those disclosed in International
Patent Applications WO 97/22596, WO 97/30035, WO 97/3285, WO
98/13354, WO00/47212 and WO01/32651) and compounds that work by
other mechanisms (for example linomide, inhibitors of integrin
.alpha.vb3 function and angiostatin); In another embodiment of the
invention the anti-angiogenic treatments of the invention are
combined agents which inhibit the tyrosine kinase activity of the
vascular endothelial growth factor receptor, KDR (for example
AZD2171 or AZD6474). Additional details on AZD2171 may be found in
Wedge et al (2005) Cancer Research. 65(10):4389-400. Additional
details on AZD6474 may be found in Ryan & Wedge (2005) British
Journal of Cancer. 92 Suppl 1:S6-13. Both publications are herein
incorporated by reference in their entireties. In another
embodiment of the invention the fully human antibodies 3.19.3,
3.3.2 or 5.88.3 are combined alone or in combination with
Avastin.RTM., AZD2171 or AZD6474.
[0190] Such conjoint treatment may be achieved by way of the
simultaneous, sequential or separate dosing of the individual
components of the treatment. Such combination products employ the
compounds of this invention, or pharmaceutically acceptable salts
thereof, within the dosage range described hereinbefore and the
other pharmaceutically active agent within its approved dosage
range.
Combinations of Ang2 Antagonists and Chemotherapy Agents
[0191] We have found that certain combinations of an antagonist of
the biological activity of Angiopoietin-2 (including, but not
limited to monoclonal antibody 3.19.3) and a chemotherapeutic agent
produces significantly better effects on tumors as compared to use
of the antagonist of the biological activity of Angiopoietin-2 or a
chemotherapeutic agent used alone.
[0192] Accordingly, embodiments of the present invention provide
methods of production of an anti-cancer effect in a patient, which
comprises administering to said patient a therapeutically effective
amount of an antagonist of the biological activity Angiopoietin-2,
and/or Tie-2, before, after or simultaneously with an effective
amount of a chemotherapeutic agent. In some embodiments, the method
comprises selecting a patient in need of an anti-cancer effect, and
administering to the patient a therapeutically effective dose of a
combination of an antagonist of the biological activity of
Angiopoietin-2, and/or Tie-2, and a chemotherapeutic agent.
[0193] In other embodiments, methods of the invention comprise the
production of an antiangiogenic and/or vascular permeability
reducing effect in a patient which comprises administering to said
patient an effective amount of an antagonist of the biological
activity of Angiopoietin-2, and/or Tie-2, before, after or
simultaneously with an effective amount of a chemotherapeutic
agent. In some embodiments, the method comprises selecting a
patient in need an antiangiogenic and/or vascular permeability
reducing effect, and administering to the patient a therapeutically
effective dose of a combination of an antagonist of the biological
activity of Angiopoietin-2, and/or Tie-2, and a chemotherapeutic
agent.
[0194] In other embodiments, the invention provides a method for
the treatment of disease-related angiogenesis in a patient which
comprises administering to said patient an effective amount of an
antagonist of the biological activity of Angiopoietin-2, and/or
Tie-2, before, after or simultaneously with an effective amount of
a chemotherapeutic agent. In some embodiments, the method comprises
selecting a patient in need of treatment of disease-related
angiogenesis, and administering to the patient a therapeutically
effective dose of a combination of an antagonist of the biological
activity of Angiopoietin-2, and/or Tie-2, and a chemotherapeutic
agent.
[0195] In other embodiments, the invention provides a method of
antagonizing the biological activity of Angiopoietin-2, and/or
Tie-2 in a patient, which comprises administering to said patient
in need thereof an effective amount of an antagonist of the
biological activity Angiopoietin-2, and/or Tie-2, before, after or
simultaneously with an effective amount of a chemotherapeutic
agent.
[0196] According to a further aspect of the present invention there
is provided a method of treatment comprising the administration of
an effective amount of an antagonist of the biological activity of
Angiopoietin-2, and/or Tie-2, or a pharmaceutically acceptable salt
thereof, optionally together with a pharmaceutically acceptable
excipient or carrier, and the simultaneous, sequential or separate
administration of an effective amount of a chemotherapeutic agent
or a pharmaceutically acceptable salt thereof, wherein the latter
may optionally be administered together with a pharmaceutically
acceptable excipient or carrier, to a patient in need of such
therapeutic treatment.
[0197] In one embodiment the antagonist of the biological activity
of Angiopoietin-2 is an antibody. In further embodiments, the
antagonist of Angiopoietin-2 is a monoclonal antibody. In yet
further embodiments, the antagonist of Angiopoietin-2 is a fully
human monoclonal antibody. In some embodiments the fully human
monoclonal antibody is selected from any one of: 3.31.2, or 5.16.3,
or 5.86.1, or 5.88.3, or 3.3.2, or 5.103.1, or 5.101.1, or 3.19.3,
or 5.28.1, or 5.78.3, MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, or
MEDI4/5. In further embodiments, the fully human monoclonal
antibody binds to the same epitope as any one of fully human
monoclonal antibody: 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2,
5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3 which are disclosed in
International Publication Number WO2006/068953 or AMG 386 (Amgen,
International Publication Number WO200330833).
[0198] In another embodiment the antagonist of the biological
activity of Angiopoietin-2 is a peptibody such as the peptibody
(AMG386) as disclosed in International Publication Number
WO2003057134.
[0199] In another embodiment the antagonist of the biological
activity of Tie-2 is an antibody. In further embodiments, the Tie-2
antibody is a monoclonal, humanized, or fully human antibody.
[0200] In one embodiment a chemotherapeutic agent comprises
alkylating agents (for example cisplatin, carboplatin, oxaliplatin,
cyclophosphamide, nitrogen mustard, melphalan, chlorambucil,
busulphan and nitrosoureas); antimetabolites (for example
antifolates such as fluoropyrimidines like 5-fluorouracil and
tegafur, raltitrexed, gemcitabine, capecitabine, methotrexate,
pemetrexed (Alimta), cytosine arabinoside and hydroxyurea, or, for
example, one of the antimetabolites disclosed in European Patent
Application No. 562734 such as
(2S)-2-{o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-ylmethy-
l)-N-(prop-2-ynyl)amino]benzamido}-4-(tetrazol-5-yl)butyric acid);
pharmaceutical combinations which comprise an alkylating agent and
an antimetabolite (for example Folfox (a combination of
fluorouracil (5FU), leucovorin and oxaliplatin)); antitumor
antibiotics (for example anthracyclines like adriamycin, bleomycin,
doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
dactinomycin and mithramycin); antimitotic agents (for example
vinca alkaloids like vincristine, vinblastine, vindesine and
vinorelbine and taxoids like taxol and taxotere); topoisomerase
inhibitors (for example epipodophyllotoxins like etoposide and
teniposide, irinotecan, amsacrine, topotecan and camptothecin); or
proteasome inhibitors (for example bortezomib). In one embodiment
there is provided a combination of the invention additionally
comprising Folfox.
[0201] In another embodiment a chemotherapeutic agent comprises
docetaxel, and other antimitotic agents (for example vinca
alkaloids like vincristine, vinblastine, vindesine and vinorelbine
and taxoids like taxol and taxotere); 5-fluorouracil, gemcitabine
and other antimetabolites (for example antifolates such as
fluoropyrimidines, tegafur, raltitrexed, capecitabine,
methotrexate, pemetrexed (Alimta), cytosine arabinoside and
hydroxyurea); irinotecan and other topoisomerase inhibitors (for
example etoposide topotecan, camptothecin teniposide, and
amsacrine); oxaliplatin and other alkylating or DNA binding agents
(for example cis-platin, and carboplatin). In one embodiment there
is provided a combination of the invention additionally comprising
Folfox.
[0202] In another embodiment a chemotherapeutic agent comprises Eg5
inhibitors, for example AZD4877.
Combinations of Ang2 Antagonists and CSF1/CSFR1 Antagonists
[0203] The present invention also provides pharmaceutical
combinations comprising an antagonist of the biological activity of
Angiopoietin-2, and/or Tie-2, and an antagonist of the biological
activity of CSF1R, and/or CSF1, and uses of such combinations.
[0204] According to one aspect of the invention there is provided a
pharmaceutical combination comprising an antagonist of the
biological activity of Angiopoietin-2, and/or Tie-2 and an
antagonist of the biological activity of CSF1R, and/or CSF1.
[0205] In one embodiment the antagonist of the biological activity
of Angiopoietin-2 is an antibody. In further embodiments, the
antagonist of Angiopoietin-2 is a monoclonal antibody. In yet
further embodiments, the antagonist of Angiopoietin-2 is a fully
human monoclonal antibody. In some embodiments the fully human
monoclonal antibody is selected from any one of; 3.31.2, or 5.16.3,
or 5.86.1, or 5.88.3, or 3.3.2, or 5.103.1, or 5.101.1, or 3.19.3,
or 5.28.1, or 5.78.3, MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, or
MEDI4/5. In further embodiments, the fully human monoclonal
antibody binds to the same epitope as any one of fully human
monoclonal antibody; 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2,
5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3 which are disclosed in
International Publication Number WO2006/068953 or AMG 386 (Amgen,
International Publication Number WO200330833).
[0206] In another embodiment there is provided a pharmaceutical
combination as described above, wherein the antagonist of the
biological activity of Tie-2 is an antibody. In one embodiment the
antagonist is a monoclonal antibody. In one embodiment the
antagonist is a fully human monoclonal antibody.
[0207] In another embodiment there is provided a pharmaceutical
combination as described above, wherein the antagonist of the
biological activity of CSF1R is an antibody. In one embodiment the
antagonist is a monoclonal antibody. In one embodiment the
antagonist is a fully human monoclonal antibody.
[0208] In another embodiment there is provided a pharmaceutical
combination as described above, wherein the antagonist of the
biological activity of CSF1 is an antibody. In one embodiment the
antagonist is a monoclonal antibody. In one embodiment the
antagonist is monoclonal antibody PD-360324 (Pfizer). In one
embodiment the antagonist is a fully human monoclonal antibody.
[0209] In another embodiment there is provided a pharmaceutical
combination as described above, wherein the antagonist of the
biological activity of CSF1R is a compound, or a pharmaceutically
acceptable salt thereof. In one embodiment the antagonist is a
tyrosine kinase inhibitor, or a pharmaceutically acceptable salt
thereof. In one embodiment the tyrosine kinase inhibitor, or a
pharmaceutically acceptable salt thereof, is selected from the
compounds disclosed in International Patent Application No.s
WO2004/004985, WO2007/119046, WO2008/056148 or WO2008/090353,
ABT-869 (Abbott), Sutent (Pfizer), KI-20227 (Kirin Brewery),
CYC-10268 (Cytopia), YM-359445 (Astellas Pharma), PLX-647 (Phenomix
Corp./Plexxikon), JNJ-27301937 (Johnson & Johnson), GW-2580
(GlaxoSmithKline) or any of the compounds disclosed in US
Provisional Application Numbers US05/0131022, US05/0113566,
International Patent Application Numbers WO2004/096795
WO2005/009967, WO2006/047277, WO2006/047504 or WO2003/093238.
[0210] In one embodiment the tyrosine kinase inhibitor, or a
pharmaceutically acceptable salt thereof, is selected from the
compounds disclosed in International Patent Application No.
WO2004/004985, WO2007/119046, WO 2008/090353, WO 2008/056148, WO
2007/119046, WO 2007/071955 each of which are incorporated by
reference in their entireties.
[0211] In another embodiment the antagonist of the biological
activity of CSF1R is selected from any one of: [0212]
2-chloro-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;
[0213]
2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-(trifluoromethyl)benzoyl]a-
mino}benzamide; [0214]
2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-fluoro-5-(trifluoromethyl)benzoyl-
]amino}-benzamide; [0215]
2-methyl-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;
[0216]
5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-pyridin--
3-ylbenzamide; [0217]
2-chloro-5-[(3-cyclopropylbenzoyl)amino]-N-pyridin-3-ylbenzamide;
[0218] 2-chloro-5-[(3-chlorobenzoyl)amino]-N-pyridin-3-ylbenzamide;
[0219]
5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;
[0220]
5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbe-
nzamide; [0221]
5-[(3-chlorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide; [0222]
5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-thia-
zol-5-yl)benzamide; [0223]
2-chloro-N-1,3-thiazol-5-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamid-
e; [0224]
2-chloro-5-[(3-chlorobenzoyl)amino]-N-1,3-thiazol-5-ylbenzamide;
[0225]
2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-1,3-thiazol-5-ylbenzamid-
e; [0226]
5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-1,3-thiaz-
ol-5-ylbenzamide; [0227]
2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)benzoyl]ami-
no}benzamide; [0228]
2-chloro-5-[(3-chlorobenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamid-
e; [0229]
2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-(2-methyl-1,3-thiazol--
5-yl)benzamide; [0230]
2-chloro-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)benzoyl]ami-
no}benzamide; [0231]
2-chloro-5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-N-(2-methyl-1,3-t-
hiazol-5-yl)benzamide; [0232]
5-[(5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methylbenzoyl)amino]-N--
methyl-1,3-thiazole-2-carboxamide; [0233]
5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-t-
hiazol-5-yl)benzamide; [0234]
5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl-
)benzamide; [0235]
5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-
-5-yl)benzamide; [0236]
5-[(3-chlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamid-
e; [0237]
5-[(3,4-dichlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol--
5-yl)benzamide; [0238]
5-[(3-cyclopropylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)ben-
zamide; [0239]
5-[(3,5-dimethylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benz-
amide; [0240]
2-methyl-5-[(3-methylbenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamid-
e; [0241]
2,6-dichloro-N-(4-methyl-3-{[(2-methyl-1,3-thiazol-5-yl)amino]ca-
rbonyl}phenyl)isonicotinamide; [0242]
2-methyl-5-{[(3-methylcyclohexyl)carbonyl]amino}-N-(2-methyl-1,3-thiazol--
5-yl)benzamide; [0243]
2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-(pentanoylamino)benzamide;
[0244]
2-methyl-5-[(4-methylhexanoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)-
benzamide; [0245]
4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinolin-
e-3-carboxamide; [0246]
4-[(2,3-dichlorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinolin-
e-3-carboxamide; [0247]
7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)-
quinoline-3-carboxamide; [0248]
4-[(3-chloro-2-fluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)qui-
noline-3-carboxamide; [0249]
7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)qui-
noline-3-carboxamide; [0250]
7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)qui-
noline-3-carboxamide; [0251]
4-[(2,4-difluorophenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-1--
yl)quinoline-3-carboxamide; [0252]
4-[(2-fluoro-4-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperaz-
in-1-yl)quinoline-3-carboxamide; [0253]
[(2-fluoro-5-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-
-1-yl)quinoline-3-carboxamide; [0254]
4-[(2-fluoro-4-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)-7-(2-met-
hoxyethoxy)quino line-3-carboxamide; [0255]
7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-methylpiperidin-4-yl)qui-
noline-3-carboxamide; [0256]
4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-methylpiperidin-4-yl)quinolin-
e-3-carboxamide; [0257]
4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-isopropylpiperidin-4-yl)quino-
line-3-carboxamide; [0258]
7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-
quinoline-3-carboxamide; [0259]
4-[(2-fluoro-4-methylphenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)qu-
inoline-3-carboxamide; [0260]
4-[(3-chloro-2-fluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)qu-
inoline-3-carboxamide; [0261]
4-[(2,4-difluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)quinoli-
ne-3-carboxamide; [0262]
4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methox-
yquinoline-3-carboxamide; [0263]
4-[(2,4-difluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxyquin-
oline-3-carboxamide; and [0264]
4-[(3-chloro-2-fluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methox-
yquinoline-3-carboxamide; [0265]
7-Ethoxy-4-[(2-fluoro-4-methyl-phenyl)amino]-6-(4-methylpiperazin-1-yl)ci-
nnoline-3-carboxamide; [0266]
4-(2-Fluoro-4-methylphenylamino)-7-methoxy-6-(4-methylpiperazin-1-yl)cinn-
oline-3-carboxamide; [0267]
4-[(2,4-Difluorophenyl)amino]-7-methoxy-6-(4-methylpiperazin-1-yl)cinnoli-
ne-3-carboxamide; [0268]
6-[(3R,5S)-3,5-Dimethylpiperazin-1-yl]-4-[(2-fluoro-4-methylphenyl)amino]-
-7-methoxycinnoline-3-carboxamide; [0269]
4-[(2-Fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazin-1-yl]-7-
-methoxycinnoline-3-carboxamide; [0270]
7-Ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazi-
n-1-yl]cinnoline-3-carboxamide; [0271]
4-[(3-Chloro-2-fluorophenyl)amino]-6-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-
-7-methoxycinnoline-3-carboxamide; [0272]
4-[(2-Fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methox-
ycinnoline-3-carboxamide hydrochloride; [0273]
4-[(2-Fluoro-4-methylphenyl)amino]-6-[1-(2-hydroxyethyl)piperidin-4-yl]-7-
-methoxycinnoline-3-carboxamide; and [0274]
4-[(2-Fluoro-4-methylphenyl)amino]-6-{4-[(2R)-2-hydroxypropanoyl]piperazi-
n-1-yl}-7-methoxycinnoline-3-carboxamide or a pharmaceutically
acceptable salt thereof.
[0275] In another embodiment there is provided a pharmaceutical
combination as described above, wherein the antagonist of the
biological activity of CSF1 is a compound, or a pharmaceutically
acceptable salt thereof.
[0276] In another embodiment there is provided a pharmaceutical
combination as described above, wherein the antagonist of the
biological activity of Angiopoietin-2 is a compound, or a
pharmaceutically acceptable salt thereof.
[0277] In another embodiment there is provided a pharmaceutical
combination as described above, wherein the antagonist of the
biological activity of Tie-2 is a compound, or a pharmaceutically
acceptable salt thereof. In one embodiment the antagonist is a
tyrosine kinase inhibitor, or a pharmaceutically acceptable salt
thereof. In one embodiment the tyrosine kinase inhibitor, or a
pharmaceutically acceptable salt thereof, is selected from any of
the compounds disclosed in International Patent Application Numbers
WO2004/013141, WO2004/058776, WO2005/060970 or WO2005/060969, or is
GW697465X (GSK), CP-547632 (Pfizer), CE-245677 (Pfizer) or CGI1631
(Cellular Genomics).
[0278] In another aspect of the present invention there is provided
a pharmaceutical combination of the present invention for use as a
medicament comprising an antagonist of the biological activity of
Angiopoietin-2, and/or Tie-2, and an antagonist of the biological
activity of CSF1R, and/or CSF1, for use simultaneously, serially or
separately.
[0279] In another aspect of the present invention there is provided
a method of antagonizing Angiopoietin-2 and/or Tie-2, and
antagonizing CSF1R, and/or CSF1, in a patient, which comprises
administering to the patient a therapeutically effective amount of
a pharmaceutical combination or another composition of the present
invention. In one embodiment the method additionally comprises
selecting a patient in need of inhibition of Angiopoietin-2 and/or
Tie-2, and inhibition of CSF1R, and/or CSF1, and administering to
the patient a therapeutically effective dose of a pharmaceutical
combination or another pharmaceutical composition as described
herein.
[0280] In one embodiment the present invention is particularly
suitable for use in antagonizing the biological activity of
Angiopoietin-2, and/or Tie-2, and the biological activity of CSF1R,
and/or CSF1, in patients with a tumor which is dependent alone, or
in part, on Angiopoietin-2, and/or Tie-2, and CSF1R, and/or
CSF1.
[0281] In one embodiment the method or use of the invention may be
administered with one or more of the following agents, by way of
the simultaneous, sequential or separate administration with of the
antagonist of the biological activity of Angiopoietin-2, and/or
Tie-2, an antagonist of cytokine function, (e.g. an agent which act
on cytokine signalling pathways such as a modulator of the SOCS
system), such as an alpha-, beta-, and/or gamma-interferon;
modulators of insulin-like growth factor type I (IGF-1), its
receptors and associated binding proteins; interleukins (IL) e.g.
one or more of IL-1 to 33, and/or an interleukin antagonist or
inhibitor such as anakinra; inhibitors of receptors of interleukin
family members or inhibitors of specific subunits of such
receptors; a tumor necrosis factor alpha (TNF-.alpha.) inhibitor
such as an anti-TNF monoclonal antibody (for example infliximab;
adalimumab, and/or CDP-870), and/or a TNF receptor antagonist e.g.
an immunoglobulin molecule (such as etanercept) and/or a
low-molecular-weight agent such as pentoxyfylline; a modulator of B
cells, e.g. a monoclonal antibody targeting B-lymphocytes (such as
CD20 (rituximab) or MRA-aIL16R) or T-lymphocytes (e.g. CTLA4-Ig,
HuMax I1-15 or Abatacept); a modulator that inhibits osteoclast
activity, for example an antibody to RANKL; a modulator of
chemokine or chemokine receptor function such as an antagonist of
CCR1, CCR2, CCR2A, CCR2B, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9,
CCR10 and CCR11 (for the C-X-C family); CXCR1, CXCR2, CXCR3, CXCR4
and CXCR5 and CXCR6 (for the C-X-C family) and CX.sub.3CR1 for the
C-X.sub.3-C family; antiangiogenic agents such as those which
inhibit the effects of vascular endothelial growth factor, [for
example the anti-vascular endothelial cell growth factor antibody
bevacizumab (Avastin.RTM.) and VEGF receptor tyrosine kinase
inhibitors such as
4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)qu-
inazoline (ZD6474; Example 2 within WO 01/32651),
4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)-
quinazoline (AZD2171; Example 240 within WO 00/47212), vatalanib
(PTK787; WO 98/35985) and SU11248 (sunitinib; WO 01/60814),
compounds such as those disclosed in International Patent
Applications WO97/22596, WO 97/30035, WO 97/32856, WO 98/13354,
WO00/47212 and WO01/32651 and compounds that work by other
mechanisms (for example linomide, inhibitors of integrin
.alpha.v.beta.3 function and angiostatin)] or colony stimulating
factor 1 (CSF1) or CSF1 receptor; an inhibitor of matrix
metalloproteases (MMPs), i.e., one or more of the stromelysins, the
collagenases, and the gelatinases, as well as aggrecanase;
especially collagenase-1 (MMP1), collagenase-2 (MMP8),
collagenase-3 (MMP13), stromelysin-1 (MMP3), stromelysin-2 (MMP10),
and/or stromelysin-3 (MMP11) and/or MMP9 and/or MMP12, e.g. an
agent such as doxycycline; a leukotriene biosynthesis inhibitor,
5-lipoxygenase (5-LO) inhibitor or 5-lipoxygenase activating
protein (FLAP) antagonist such as; zileuton; ABT-761; fenleuton;
tepoxalin; Abbott-79175; Abbott-85761;
N-(5-substituted)-thiophene-2-alkylsulfonamides;
2,6-di-tert-butylphenolhydrazones; methoxytetrahydropyrans such as
Zeneca ZD-2138; the compound SB-210661; a pyridinyl-substituted
2-cyanonaphthalene compound such as L-739,010; a 2-cyanoquinoline
compound such as L-746,530; indole and/or a quinoline compound such
as MK-591, MK-886, and/or BAY x 1005; a receptor antagonist for
leukotrienes (LT) B4, LTC4, LTD4, and LTE4, selected from the group
consisting of the phenothiazin-3-1s such as L-651,392; amidino
compounds such as CGS-25019c; benzoxalamines such as ontazolast;
benzenecarboximidamides such as BIIL 284/260; and compounds such as
zafirlukast, ablukast, montelukast, pranlukast, verlukast (MK-679),
RG-12525, Ro-245913, iralukast (CGP 45715A), and BAY x 7195; a
phosphodiesterase (PDE) inhibitor such as a methylxanthanine, e.g.
theophylline and/or aminophylline; and/or a selective PDE isoenzyme
inhibitor e.g. a PDE4 inhibitor and/or inhibitor of the isoform
PDE4D, and/or an inhibitor of PDE5; a histamine type 1 receptor
antagonist such as cetirizine, loratadine, desloratadine,
fexofenadine, acrivastine, terfenadine, astemizole, azelastine,
levocabastine, chlorpheniramine, promethazine, cyclizine, and/or
mizolastine (generally applied orally, topically or parenterally);
a proton pump inhibitor (such as omeprazole) or gastroprotective
histamine type 2 receptor antagonist; an antagonist of the
histamine type 4 receptor; an alpha-1/alpha-2 adrenoceptor agonist
vasoconstrictor sympathomimetic agent, such as propylhexedrine,
phenylephrine, phenylpropanolamine, ephedrine, pseudoephedrine,
naphazoline hydrochloride, oxymetazoline hydrochloride,
tetrahydrozoline hydrochloride, xylometazoline hydrochloride,
tramazoline hydrochloride, and ethylnorepinephrine hydrochloride;
an anticholinergic agent, e.g. a muscarinic receptor (M1, M2, and
M3) antagonist such as atropine, hyoscine, glycopyrrrolate,
ipratropium bromide, tiotropium bromide, oxitropium bromide,
pirenzepine, and telenzepine; a beta-adrenoceptor agonist
(including beta receptor subtypes 1-4) such as isoprenaline,
salbutamol, formoterol, salmeterol, terbutaline, orciprenaline,
bitolterol mesylate, and/or pirbuterol, e.g. a chiral enantiomer
thereof; a chromone, e.g. sodium cromoglycate and/or nedocromil
sodium; a glucocorticoid, such as flunisolide, triamcinolone
acetonide, beclomethasone dipropionate, budesonide, fluticasone
propionate, ciclesonide, and/or mometasone furoate; an agent that
modulates nuclear hormone receptors such as a PPAR; an
immunoglobulin (Ig) or Ig preparation or an antagonist or antibody
modulating Ig function such as anti-IgE (e.g. omalizumab); other
systemic or topically-applied anti-inflammatory agent, e.g.
thalidomide or a derivative thereof, a retinoid, dithranol, and/or
calcipotriol; pharmaceutical combinations of aminosalicylates and
sulfapyridine such as sulfasalazine, mesalazine, balsalazide, and
olsalazine; and immunomodulatory agents such as the thiopurines,
and corticosteroids such as budesonide; an antibacterial agent e.g.
a penicillin derivative, a tetracycline, a macrolide, a
beta-lactam, a fluoroquinolone, metronidazole, and/or an inhaled
aminoglycoside; and/or an antiviral agent e.g. acyclovir,
famciclovir, valaciclovir, ganciclovir, cidofovir; amantadine,
rimantadine; ribavirin; zanamavir and/or oseltamavir; a protease
inhibitor such as indinavir, nelfinavir, ritonavir, and/or
saquinavir; a nucleoside reverse transcriptase inhibitor such as
didanosine, lamivudine, stavudine, zalcitabine, zidovudine; a
non-nucleoside reverse transcriptase inhibitor such as nevirapine,
efavirenz; a cardiovascular agent such as a calcium channel
blocker, beta-adrenoceptor blocker, angiotensin-converting enzyme
(ACE) inhibitor, angiotensin-2 receptor antagonist; lipid lowering
agent such as a statin, and/or fibrate; a modulator of blood cell
morphology such as pentoxyfylline; a thrombolytic, and/or an
anticoagulant e.g. a platelet aggregation inhibitor; a CNS agent
such as an antidepressant (such as sertraline), anti-Parkinsonian
drug (such as deprenyl, L-dopa, ropinirole, pramipexole, MAOB
inhibitor such as selegine and rasagiline, comP inhibitor such as
tasmar, A-2 inhibitor, dopamine reuptake inhibitor, NMDA
antagonist, nicotine agonist, dopamine agonist and/or inhibitor of
neuronal nitric oxide synthase), and an anti-Alzheimer's drug such
as donepezil, rivastigmine, tacrine, COX-2 inhibitor,
propentofylline or metrifonate; an agent for the treatment of acute
and chronic pain, e.g. a centrally or peripherally-acting analgesic
such as an opioid analogue or derivative, carbamazepine, phenyloin,
sodium valproate, amitryptiline or other antidepressant agent,
paracetamol, or non-steroidal anti-inflammatory agent; a
parenterally or topically-applied (including inhaled) local
anaesthetic agent such as lignocaine or an analogue thereof; an
anti-osteoporosis agent e.g. a hormonal agent such as raloxifene,
or a biphosphonate such as alendronate; (i) a tryptase inhibitor;
(ii) a platelet activating factor (PAF) antagonist; (iii) an
interleukin converting enzyme (ICE) inhibitor; (iv) an IMPDH
inhibitor; (v) an adhesion molecule inhibitors including VLA-4
antagonist; (vi) a cathepsin; (vii) a kinase inhibitor e.g. an
inhibitor of tyrosine kinases (such as Btk, Itk, Jak3 MAP examples
of inhibitors might include Gefitinib, Imatinib mesylate), a
serine/threonine kinase (e.g. an inhibitor of MAP kinase such as
p38, JNK, protein kinases A, B and C and IKK), or a kinase involved
in cell cycle regulation (e.g. a cylin dependent kinase); (viii) a
glucose-6 phosphate dehydrogenase inhibitor; (ix) a kinin-B.sub1.-
and/or B.sub2.-receptor antagonist; (x) an anti-gout agent, e.g.,
colchicine; (xi) a xanthine oxidase inhibitor, e.g., allopurinol;
(xii) a uricosuric agent, e.g., probenecid, sulfinpyrazone, and/or
benzbromarone; (xiii) a growth hormone secretagogue; (xiv)
transforming growth factor (TGF.beta.); (xv) platelet-derived
growth factor (PDGF); (xvi) fibroblast growth factor, e.g., basic
fibroblast growth factor (bFGF); (xvii) granulocyte macrophage
colony stimulating factor (GM-CSF); (xviii) capsaicin cream; (xix)
a tachykinin NK.sub1. and/or NK.sub3. receptor antagonist such
NKP-608C, SB-233412 (talnetant), and/or D-4418; (xx) an elastase
inhibitor e.g. UT-77 and/or ZD-0892; (xxi) a TNF-alpha converting
enzyme inhibitor (TACE); (xxii) induced nitric oxide synthase
(iNOS) inhibitor or (xxiii) a chemoattractant receptor-homologous
molecule expressed on TH2 cells, (such as a CRTH2 antagonist)
(xxiv) an inhibitor of a P38 (xxv) agent modulating the function of
Toll-like receptors (TLR) and (xxvi) an agent modulating the
activity of purinergic receptors such as P2.times.7; (xxvii) an
inhibitor of transcription factor activation such as NFkB, API,
and/or STATS; Non-steroidal anti-inflammatory agents (hereinafter
NSAIDs) including non-selective cyclo-oxygenase (COX)-1/COX-2
inhibitors whether applied topically or systemically (such as
piroxicam, diclofenac, propionic acids such as naproxen,
flurbiprofen, fenoprofen, ketoprofen and ibuprofen, fenamates such
as mefenamic acid, indomethacin, sulindac, azapropazone,
pyrazolones such as phenylbutazone, salicylates such as aspirin);
selective COX-2 inhibitors (such as meloxicam, celecoxib,
rofecoxib, valdecoxib, lumarocoxib, parecoxib and etoricoxib);
cyclo-oxygenase inhibiting nitric oxide donors (CINODs);
glucocorticosteroids (whether administered by topical, oral,
intramuscular, intravenous, or intra-articular routes);
methotrexate, leflunomide; hydroxychloroquine, d-penicillamine,
auranofin or other parenteral or oral gold preparations;
analgesics; diacerein; intra-articular therapies such as hyaluronic
acid derivatives; and nutritional supplements such as
glucosamine.
[0282] In some embodiments, the method or use of the invention may
comprise administration of an agent that antagonizes TNF-.alpha..
Any TNF-.alpha. antagonist well-known to one of skill in the art
can be used in the compositions and methods of the invention.
Non-limiting examples of TNF-.alpha. antagonists include proteins,
polypeptides, peptides, fusion proteins, antibodies (e.g., human,
humanized, chimeric, monoclonal, polyclonal, Fvs, ScFvs, Fab
fragments, F(ab).sub.2 fragments, and antigen-binding fragments
thereof) such as antibodies that immunospecifically bind to
TNF-.alpha., nucleic acid molecules (e.g., antisense molecules or
triple helices), organic molecules, inorganic molecules, and small
molecules that blocks, reduces, inhibits or neutralizes the
function, activity and/or expression of TNF-.alpha.. In various
embodiments, a TNF-.alpha. antagonist reduces the function,
activity and/or expression of TNF-.alpha. by at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95% or at least 99% relative to a
control such as phosphate buffered saline (PBS).
[0283] Examples of antibodies that immunospecifically bind to
TNF-.alpha. include, but are not limited to, infliximab
(REMICADE.TM.; Centocor), adalimumab (HUMIRA.TM.; Abbott
Laboratories), D2E7 (Abbott Laboratories/Knoll Pharmaceuticals Co.,
Mt. Olive, N.J.), CDP571 which is also known as HUMICADE.TM. and
CDP-870 (both of Celltech/Pharmacia, Slough, U.K.), and TN3-19.12
(Williams et al., 1994, Proc. Natl. Acad. Sci. USA 91: 2762-2766;
Thorbecke et al., 1992, Proc. Natl. Acad. Sci. USA 89:7375-7379).
The present invention also encompasses the use of antibodies that
immunospecifically bind to TNF-.alpha. disclosed in the following
U.S. patents in the compositions and methods of the invention:
5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,334,380; 5,360,716;
5,426,181; 5,436,154; 5,610,279; 5,644,034; 5,656,272; 5,658,746;
5,698,195; 5,736,138; 5,741,488; 5,808,029; 5,919,452; 5,958,412;
5,959,087; 5,968,741; 5,994,510; 6,036,978; 6,114,517; and
6,171,787; each of which are herein incorporated by reference in
their entirety. Examples of soluble TNF-.alpha. receptors include,
but are not limited to, sTNF-R1 (Amgen), etanercept (ENBREL.TM.;
Immunex) and its rat homolog RENBREL.TM., soluble inhibitors of
TNF-.alpha. derived from TNFrI, TNFrII (Kohno et al., 1990, Proc.
Natl. Acad. Sci. USA 87:8331-8335), and TNF-.alpha. Inh (Seckinger
et al, 1990, Proc. Natl. Acad. Sci. USA 87:5188-5192).
[0284] In one embodiment, a TNF-.alpha. antagonist used in the
compositions and methods of the invention is a soluble TNF-.alpha.
receptor. In a specific embodiment, a TNF-.alpha. antagonist used
in the compositions and methods of the invention is etanercept
(ENBREL.TM.; Immunex) or a fragment, derivative or analog thereof.
In another embodiment, a TNF-.alpha. antagonist used in the
compositions and methods of the invention is an antibody that
immunospecifically binds to TNF-.alpha.. In a specific embodiment,
a TNF-.alpha. antagonist used in the compositions and methods of
the invention is infliximab (REMICADE.TM.; Centocor) a derivative,
analog or antigen-binding fragment thereof. In another specific
embodiment, a TNF-.alpha. antagonist used in the compositions and
methods of the invention is adalimumab (HUMIRA.TM.; Abbott
Laboratories) a derivative, analog or antigen-binding fragment
thereof.
[0285] Other TNF-.alpha. antagonists encompassed by the invention
include, but are not limited to, IL-10, which is known to block
TNF-.alpha. production via interferon .gamma.-activated macrophages
(Oswald et al. 1992, Proc. Natl. Acad. Sci. USA 89:8676-8680),
TNFR-IgG (Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA
88:10535-10539), the murine product TBP-1 (Serono/Yeda), the
vaccine CytoTAb (Protherics), antisense molecule104838 (ISIS), the
peptide RDP-58 (SangStat), thalidomide (Celgene), CDC-801
(Celgene), DPC-333 (Dupont), VX-745 (Vertex), AGIX-4207
(AtheroGenics), ITF-2357 (Italfarmaco), NPI-13021-31 (Nereus),
SCIO-469 (Scios), TACE targeter (Immunix/AHP), CLX-120500 (Calyx),
Thiazolopyrim (Dynavax), auranofin (Ridaura) (SmithKline Beecham
Pharmaceuticals), quinacrine (mepacrine dichlorohydrate), tenidap
(Enablex), Melanin (Large Scale Biological), and anti-p38 MAPK
agents by Uriach.
[0286] Nucleic acid molecules encoding proteins, polypeptides, or
peptides with TNF-.alpha. antagonist activity or proteins,
polypeptides, or peptides with TNF-.alpha. antagonist activity can
be administered to a subject with an inflammatory or autoimmune
disease in accordance with the methods of the invention. Further,
nucleic acid molecules encoding derivatives, analogs, fragments or
variants of proteins, polypeptides, or peptides with TNF-.alpha.
antagonist activity, or derivatives, analogs, fragments or variants
of proteins, polypeptides, or peptides with TNF-.alpha. antagonist
activity can be administered to a subject with an inflammatory or
autoimmune disease in accordance with the methods of the invention.
Such derivatives, analogs, variants and fragments retain the
TNF-.alpha. antagonist activity of the full-length wild-type
protein, polypeptide, or peptide.
[0287] Proteins, polypeptides, or peptides that can be used as
TNF-.alpha. antagonists can be produced by any technique well-known
in the art or described herein. Proteins, polypeptides or peptides
with TNF-.alpha. antagonist activity can be engineered so as to
increase the in vivo half-life of such proteins, polypeptides, or
peptides utilizing techniques well-known in the art or described
herein. Preferably, agents that are commercially available and
known to function as TNF-.alpha. antagonists are used in the
compositions and methods of the invention. The TNF-.alpha.
antagonist activity of an agent can be determined in vitro and/or
in vivo by any technique well-known to one skilled in the art.
[0288] In one embodiment the method or use of the invention may
comprise administration of an antibody that is an antagonist of the
biological activity of Angiopoietin-2, and/or Tie-2 as an
immuno-conjugate with any one of the agents listed above.
Pharmaceutical Compositions
[0289] In another aspect, the present invention provides a
composition, for example, but not limited to, a pharmaceutical
composition, containing one or more antibodies of the present
invention, formulated together with a pharmaceutically acceptable
carrier. Such compositions may include one or a combination of, for
example, but not limited to two or more different antibodies of the
invention. For example, a pharmaceutical composition of the
invention may comprise a combination of antibodies that bind to
different epitopes on the target antigen or that have complementary
activities.
[0290] Pharmaceutical compositions of the invention also can be
administered in combination therapy, such as, combined with other
agents. For example, the combination therapy can include an
antibody of the present invention combined with at least one other
therapy wherein the therapy may be surgery, immunotherapy,
chemotherapy, radiation treatment, or drug therapy.
[0291] The pharmaceutical compounds of the invention may include
one or more pharmaceutically acceptable salts. Examples of such
salts include acid addition salts and base addition salts. Acid
addition salts include those derived from nontoxic inorganic acids,
such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,
hydroiodic, phosphorous and the like, as well as from nontoxic
organic acids such as aliphatic mono- and dicarboxylic acids,
phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic
acids, aliphatic and aromatic sulfonic acids and the like. Base
addition salts include those derived from alkaline earth metals,
such as sodium, potassium, magnesium, calcium and the like, as well
as from nontoxic organic amines, such as
N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,
choline, diethanolamine, ethylenediamine, procaine and the
like.
[0292] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: (1) water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and (3) metal
chelating agents, such as citric acid, ethylenediamine tetraacetic
acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the
like.
[0293] Examples of suitable aqueous and non-aqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0294] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures and by the inclusion of various
antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as aluminum monostearate and gelatin.
[0295] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, it will be
suitable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent that
delays absorption, for example, monostearate salts and gelatin.
[0296] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions,
selected methods of preparation are vacuum drying and freeze-drying
(lyophilization) that yield a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0297] In one embodiment the compositions of the invention are
pyrogen-free formulations which are substantially free of
endotoxins and/or related pyrogenic substances. Endotoxins include
toxins that are confined inside a microorganism and are released
when the microorganisms are broken down or die. Pyrogenic
substances also include fever-inducing, thermostable substances
(glycoproteins) from the outer membrane of bacteria and other
microorganisms. Both of these substances can cause fever,
hypotension and shock if administered to humans. Due to the
potential harmful effects, it is advantageous to remove even low
amounts of endotoxins from intravenously administered
pharmaceutical drug solutions. The Food & Drug Administration
("FDA") has set an upper limit of 5 endotoxin units (EU) per dose
per kilogram body weight in a single one hour period for
intravenous drug applications (The United States Pharmacopeial
Convention, Pharmacopeial Forum 26 (1):223 (2000)). When
therapeutic proteins are administered in amounts of several hundred
or thousand milligrams per kilogram body weight it is advantageous
to remove even trace amounts of endotoxin. In one embodiment,
endotoxin and pyrogen levels in the composition are less then 10
EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1
EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg. In
another embodiment, endotoxin and pyrogen levels in the composition
are less then about 10 EU/mg, or less then about 5 EU/mg, or less
then about 1 EU/mg, or less then about 0.1 EU/mg, or less then
about 0.01 EU/mg, or less then about 0.001 EU/mg.
[0298] In one embodiment, the invention comprises administering a
composition wherein said administration is oral, parenteral,
intramuscular, intranasal, vaginal, rectal, lingual, sublingual,
buccal, intrabuccal, intravenous, cutaneous, subcutaneous or
transdermal.
[0299] In another embodiment the invention further comprises
administering a composition in combination with other therapies,
such as surgery, chemotherapy, hormonal therapy, biological
therapy, immunotherapy or radiation therapy.
[0300] In another embodiment, the invention comprises administering
a composition comprising an antibody of the invention in
combination with an antagonist of the biological activity of CSF1R,
and/or CSF1, simultaneously, serially or separately.
[0301] In another embodiment, the invention comprises administering
a composition comprising an antibody of the invention in
combination with an antagonist of the biological activity of VEGF,
and/or VEGFR, simultaneously, serially or separately.
[0302] In another embodiment, the invention comprises administering
a composition comprising an antibody of the invention in
combination with an antagonist of the biological activity of
TNF-.alpha., simultaneously, serially or separately.
Dosing/Administration
[0303] To prepare pharmaceutical or sterile compositions including
an antibody of the invention, the antibody is mixed with a
pharmaceutically acceptable carrier or excipient. Formulations of
therapeutic and diagnostic agents can be prepared by mixing with
physiologically acceptable carriers, excipients, or stabilizers in
the form of, e.g., lyophilized powders, slurries, aqueous
solutions, lotions, or suspensions (see, e.g., Hardman, et al.
(2001) Goodman and Gilman's The Pharmacological Basis of
Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000)
Remington: The Science and Practice of Pharmacy, Lippincott,
Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993)
Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker,
NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:
Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)
Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY;
Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel
Dekker, Inc., New York, N.Y.).
[0304] Selecting an administration regimen for a therapeutic
depends on several factors, including the serum or tissue turnover
rate of the entity, the level of symptoms, the immunogenicity of
the entity, and the accessibility of the target cells in the
biological matrix. In certain embodiments, an administration
regimen maximizes the amount of therapeutic delivered to the
patient consistent with an acceptable level of side effects.
Accordingly, the amount of biologic delivered depends in part on
the particular entity and the severity of the condition being
treated. Guidance in selecting appropriate doses of antibodies,
cytokines, and small molecules are available (see, e.g.,
Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd,
Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies,
Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (ed.)
(1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune
Diseases, Marcel Dekker, New York, N.Y.; Baert, et al. (2003) New
Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med.
341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792;
Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh,
et al. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (2000)
New Engl. J. Med. 343:1594-1602).
[0305] Determination of the appropriate dose is made by the
clinician, e.g., using parameters or factors known or suspected in
the art to affect treatment or predicted to affect treatment.
Generally, the dose begins with an amount somewhat less than the
optimum dose and it is increased by small increments thereafter
until the desired or optimum effect is achieved relative to any
negative side effects. Important diagnostic measures include those
of symptoms of, e.g., the inflammation or level of inflammatory
cytokines produced.
[0306] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0307] Compositions comprising antibodies of the invention can be
provided by continuous infusion, or by doses at intervals of, e.g.,
one day, one week, or 1-7 times per week. Doses may be provided
intravenously, subcutaneously, topically, orally, nasally,
rectally, intramuscular, intracerebrally, or by inhalation. A
specific dose protocol is one involving the maximal dose or dose
frequency that avoids significant undesirable side effects. A total
weekly dose may be at least 0.05 .mu.g/kg body weight, at least 0.2
.mu.g/kg, at least 0.5 .mu.g/kg, at least 1 .mu.g/kg, at least 10
.mu.g/kg, at least 100 .mu.g/kg, at least 0.2 mg/kg, at least 1.0
mg/kg, at least 2.0 mg/kg, at least 10 mg/kg, at least 25 mg/kg, or
at least 50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J. Med.
349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698;
Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456;
Portielji, et al. (20003) Cancer Immunol. Immunother. 52:133-144).
The dose may be at least 15 .mu.g, at least 20 .mu.g, at least 25
.mu.g, at least 30 .mu.g, at least 35 .mu.g, at least 40 .mu.g, at
least 45 .mu.g, at least 50 .mu.g, at least 55 .mu.g, at least 60
.mu.g, at least 65 .mu.g, at least 70 .mu.g, at least 75 .mu.g, at
least 80 .mu.g, at least 85 .mu.g, at least 90 .mu.g, at least 95
.mu.g, or at least 100 .mu.g. The doses administered to a subject
may number at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, or
more.
[0308] For antibodies of the invention, the dosage administered to
a patient may be 0.0001 mg/kg to 100 mg/kg of the patient's body
weight. The dosage may be between 0.0001 mg/kg and 20 mg/kg, 0.0001
mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg,
0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and
0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001
to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to
0.10 mg/kg of the patient's body weight.
[0309] The dosage of the antibodies of the invention may be
calculated using the patient's weight in kilograms (kg) multiplied
by the dose to be administered in mg/kg. The dosage of the
antibodies of the invention may be 150 .mu.g/kg or less, 125
.mu.g/kg or less, 100 .mu.g/kg or less, 95 .mu.g/kg or less, 90
.mu.g/kg or less, 85 .mu.g/kg or less, 80 .mu.g/kg or less, 75
.mu.g/kg or less, 70 .mu.g/kg or less, 65 .mu.g/kg or less, 60
.mu.g/kg or less, 55 .mu.g/kg or less, 50 .mu.g/kg or less, 45
.mu.g/kg or less, 40 .mu.g/kg or less, 35 .mu.g/kg or less, 30
.mu.g/kg or less, 25 .mu.g/kg or less, 20 .mu.g/kg or less, 15
.mu.g/kg or less, 10 .mu.g/kg or less, 5 .mu.g/kg or less, 2.5
.mu.g/kg or less, 2 .mu.g/kg or less, 1.5 .mu.g/kg or less, 1
.mu.g/kg or less, 0.5 mg/kg or less, or 0.5 .mu.g/kg or less of a
patient's body weight.
[0310] Unit dose of the antibodies of the invention may be 0.1 mg
to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg
to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to
20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 to 8 mg,
0.25 mg to 7 m g, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg,
1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to
7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.
[0311] The dosage of the antibodies of the invention may achieve a
serum titer of at least 0.1 .mu.g/ml, at least 0.5 .mu.g/ml, at
least 1 .mu.g/ml, at least 2 .mu.g/ml, at least 5 .mu.g/ml, at
least 6 .mu.g/ml, at least 10 .mu.g/ml, at least 15 .mu.g/ml, at
least 20 .mu.g/ml, at least 25 .mu.g/ml, at least 50 .mu.g/ml, at
least 100 .mu.g/ml, at least 125 .mu.g/ml, at least 150 .mu.g/ml,
at least 175 .mu.g/ml, at least 200 .mu.g/ml, at least 225
.mu.g/ml, at least 250 .mu.g/ml, at least 275 .mu.g/ml, at least
300 .mu.g/ml, at least 325 .mu.g/ml, at least 350 .mu.g/ml, at
least 375 .mu.g/ml, or at least 400 .mu.g/ml in a subject.
Alternatively, the dosage of the antibodies of the invention may
achieve a scrum titer of at least 0.1 .mu.g/ml, at least 0.5
.mu.g/ml, at least 1 .mu.g/ml, at least, 2 .mu.g/ml, at least 5
.mu.g/ml, at least 6 .mu.g/ml, at least 10 .mu.g/ml, at least 15
.mu.g/ml, at least 20 .mu.g/ml, at least 25 .mu.g/ml, at least 50
.mu.g/ml, at least 100 .mu.g/ml, at least 125 .mu.g/ml, at least
150 .mu.g/ml, at least 175 .mu.g/ml, at least 200 .mu.g/ml, at
least 225 .mu.g/ml, at least 250 .mu.g/ml, at least 275 .mu.g/ml,
at least 300 .mu.g/ml, at least 325 .mu.g/ml, at least 350
.mu.g/ml, at least 375 .mu.g/ml, or at least 400 .mu.g/ml in the
subject.
[0312] Doses of antibodies of the invention may be repeated and the
administrations may be separated by at least 1 day, 2 days, 3 days,
5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3
months, or at least 6 months.
[0313] An effective amount for a particular patient may vary
depending on factors such as the condition being treated, the
overall health of the patient, the method route and dose of
administration and the severity of side affects (see, e.g.,
Maynard, et al. (1996) A Handbook of SOPs for Good Clinical
Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good
Laboratory and Good Clinical Practice, Urch Publ., London, UK).
[0314] The route of administration may be by, e.g., topical or
cutaneous application, injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intracerebrospinal, intralesional, or by sustained
release systems or an implant (see, e.g., Sidman et al. (1983)
Biopolymers 22:547-556; Langer, et al. (1981) J. Biomed. Mater.
Res. 15:167-277; Langer (1982) Chem. Tech. 12:98-105; Epstein, et
al. (1985) Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al.
(1980) Proc. Natl. Acad. Sci. USA 77:4030-4034; U.S. Pat. Nos.
6,350,466 and 6,316,024).
[0315] Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lidocaine to ease
pain at the site of the injection. In addition, pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent. See, e.g.,
U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272,
5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication
Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO
99/66903, each of which is incorporated herein by reference their
entirety. In one embodiment, an antibody, combination therapy, or a
composition of the invention is administered using Alkermes AIR.TM.
pulmonary drug delivery technology (Alkermes, Inc., Cambridge,
Mass.).
[0316] A composition of the present invention may also be
administered via one or more routes of administration using one or
more of a variety of methods known in the art. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results.
Selected routes of administration for antibodies of the invention
include intravenous, intramuscular, intradermal, intraperitoneal,
subcutaneous, spinal or other parenteral routes of administration,
for example by injection or infusion. Parenteral administration may
represent modes of administration other than enteral and topical
administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion. Alternatively, a
composition of the invention can be administered via a
non-parenteral route, such as a topical, epidermal or mucosal route
of administration, for example, intranasally, orally, vaginally,
rectally, sublingually or topically.
[0317] If the antibodies of the invention are administered in a
controlled release or sustained release system, a pump may be used
to achieve controlled or sustained release (see Langer, supra;
Sefton, 1987, CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al.,
1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med.
321:574). Polymeric materials can be used to achieve controlled or
sustained release of the therapies of the invention (see e.g.,
Medical Applications of Controlled Release, Langer and Wise (eds.),
CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,
Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.
5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S.
Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO
99/15154; and PCT Publication No. WO 99/20253. Examples of polymers
used in sustained release formulations include, but are not limited
to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),
poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one
embodiment, the polymer used in a sustained release formulation is
inert, free of leachable impurities, stable on storage, sterile,
and biodegradable. A controlled or sustained release system can be
placed in proximity of the prophylactic or therapeutic target, thus
requiring only a fraction of the systemic dose (see, e.g., Goodson,
in Medical Applications of Controlled Release, supra, vol. 2, pp.
115-138 (1984)).
[0318] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more antibodies of the invention.
See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO 91/05548,
PCT publication WO 96/20698, Ning et al., 1996, "Intratumoral
Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a
Sustained-Release Gel," Radiotherapy & Oncology 39:179-189,
Song et al., 1995, "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
& Technology 50:372-397, Cleek et al., 1997, "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854, and Lam et al., 1997, "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Int'l. Symp. Control Rel. Bioact. Mater. 24:759-760, each of
which is incorporated herein by reference in their entirety.
[0319] If the antibody of the invention is administered topically,
it can be formulated in the form of an ointment, cream, transdermal
patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or
other form well-known to one of skill in the art. See, e.g.,
Remington's Pharmaceutical Sciences and Introduction to
Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa.
(1995). For non-sprayable topical dosage forms, viscous to
semi-solid or solid forms comprising a carrier or one or more
excipients compatible with topical application and having a dynamic
viscosity, in some instances, greater than water are typically
employed. Suitable formulations include, without limitation,
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, and the like, which are, if desired, sterilized
or mixed with auxiliary agents (e.g., preservatives, stabilizers,
wetting agents, buffers, or salts) for influencing various
properties, such as, for example, osmotic pressure. Other suitable
topical dosage forms include sprayable aerosol preparations wherein
the active ingredient, in some instances, in combination with a
solid or liquid inert carrier, is packaged in a mixture with a
pressurized volatile (e.g., a gaseous propellant, such as freon) or
in a squeeze bottle. Moisturizers or humectants can also be added
to pharmaceutical compositions and dosage forms if desired.
Examples of such additional ingredients are well-known in the
art.
[0320] If the compositions comprising antibodies are administered
intranasally, it can be formulated in an aerosol form, spray, mist
or in the form of drops. In particular, prophylactic or therapeutic
agents for use according to the present invention can be
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebuliser, with the use of a suitable
propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0321] Methods for co-administration or treatment with a second
therapeutic agent, e.g., a cytokine, steroid, chemotherapeutic
agent, antibiotic, or radiation, are well known in the art (see,
e.g., Hardman, et al. (eds.) (2001) Goodman and Gilman's The
Pharmacological Basis of Therapeutics, 10.sup.th ed., McGraw-Hill,
New York, N.Y.; Poole and Peterson (eds.) (2001)
Pharmacotherapeutics for Advanced Practice: A Practical Approach,
Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo
(eds.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott,
Williams & Wilkins, Phila., Pa.). An effective amount of
therapeutic may decrease the symptoms by at least 10%; by at least
20%; at least about 30%; at least 40%, or at least 50%.
[0322] Additional therapies (e.g., prophylactic or therapeutic
agents), which can be administered in combination with the
antibodies of the invention may be administered less than 5 minutes
apart, less than 30 minutes apart, 1 hour apart, at about 1 hour
apart, at about 1 to about 2 hours apart, at about 2 hours to about
3 hours apart, at about 3 hours to about 4 hours apart, at about 4
hours to about 5 hours apart, at about 5 hours to about 6 hours
apart, at about 6 hours to about 7 hours apart, at about 7 hours to
about 8 hours apart, at about 8 hours to about 9 hours apart, at
about 9 hours to about 10 hours apart, at about 10 hours to about
11 hours apart, at about 11 hours to about 12 hours apart, at about
12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to
36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours
apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72
hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to
120 hours apart from the antibodies of the invention. The two or
more therapies may be administered within one same patient
visit.
[0323] The antibodies of the invention and the other therapies may
be cyclically administered. Cycling therapy involves the
administration of a first therapy (e.g., a first prophylactic or
therapeutic agent) for a period of time, followed by the
administration of a second therapy (e.g., a second prophylactic or
therapeutic agent) for a period of time, optionally, followed by
the administration of a third therapy (e.g., prophylactic or
therapeutic agent) for a period of time and so forth, and repeating
this sequential administration, i.e., the cycle in order to reduce
the development of resistance to one of the therapies, to avoid or
reduce the side effects of one of the therapies, and/or to improve
the efficacy of the therapies.
[0324] In certain embodiments, the antibodies of the invention can
be formulated to ensure proper distribution in vivo. For example,
the blood-brain barrier (BBB) excludes many highly hydrophilic
compounds. To ensure that the therapeutic compounds of the
invention cross the BBB (if desired), they can be formulated, for
example, in liposomes. For methods of manufacturing liposomes, see,
e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The
liposomes may comprise one or more moieties which are selectively
transported into specific cells or organs, thus enhance targeted
drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol.
29:685). Exemplary targeting moieties include folate or biotin
(see, e.g., U.S. Pat. No. 5,416,016 to Low et al.); mannosides
(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038);
antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140; M.
Owais et al. (1995) Antimicrob. Agents Chemother. 39:180);
surfactant protein A receptor (Briscoe et al. (1995) Am. J.
Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBS Lett.
346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods
4:273.
[0325] The invention provides protocols for the administration of
pharmaceutical composition comprising antibodies of the invention
alone or in combination with other therapies to a subject in need
thereof. The therapies (e.g., prophylactic or therapeutic agents)
of the combination therapies of the present invention can be
administered concomitantly or sequentially to a subject. The
therapy (e.g., prophylactic or therapeutic agents) of the
combination therapies of the present invention can also be
cyclically administered. Cycling therapy involves the
administration of a first therapy (e.g., a first prophylactic or
therapeutic agent) for a period of time, followed by the
administration of a second therapy (e.g., a second prophylactic or
therapeutic agent) for a period of time and repeating this
sequential administration, i.e., the cycle, in order to reduce the
development of resistance to one of the therapies (e.g., agents) to
avoid or reduce the side effects of one of the therapies (e.g.,
agents), and/or to improve, the efficacy of the therapies.
[0326] The therapies (e.g., prophylactic or therapeutic agents) of
the combination therapies of the invention can be administered to a
subject concurrently. The term "concurrently" is not limited to the
administration of therapies (e.g., prophylactic or therapeutic
agents) at exactly the same time, but rather it is meant that a
pharmaceutical composition comprising antibodies of the invention
are administered to a subject in a sequence and within a time
interval such that the antibodies of the invention can act together
with the other therapy(ies) to provide an increased benefit than if
they were administered otherwise. For example, each therapy may be
administered to a subject at the same time or sequentially in any
order at different points in time; however, if not administered at
the same time, they should be administered sufficiently close in
time so as to provide the desired therapeutic or prophylactic
effect. Each therapy can be administered to a subject separately,
in any appropriate form and by any suitable route. In various
embodiments, the therapies (e.g., prophylactic or therapeutic
agents) are administered to a subject less than 15 minutes, less
than 30 minutes, less than 1 hour apart, at about 1 hour apart, at
about 1 hour to about 2 hours apart, at about 2 hours to about 3
hours apart, at about 3 hours to about 4 hours apart, at about 4
hours to about 5 hours apart, at about 5 hours to about 6 hours
apart, at about 6 hours to about 7 hours apart, at about 7 hours to
about 8 hours apart, at about 8 hours to about 9 hours apart, at
about 9 hours to about 10 hours apart, at about 10 hours to about
11 hours apart, at about 11 hours to about 12 hours apart, 24 hours
apart, 48 hours apart, 72 hours apart, or 1 week apart. In other
embodiments, two or more therapies (e.g., prophylactic or
therapeutic agents) are administered to a within the same patient
visit.
[0327] The prophylactic or therapeutic agents of the combination
therapies can be administered to a subject in the same
pharmaceutical composition. Alternatively, the prophylactic or
therapeutic agents of the combination therapies can be administered
concurrently to a subject in separate pharmaceutical compositions.
The prophylactic or therapeutic agents may be administered to a
subject by the same or different routes of administration.
[0328] Another embodiment of the invention includes an assay kit
for detecting Angiopoietin-1 and/or Angiopoietin-2 in mammalian
tissues, cells, or body fluids to screen for angiogenesis-related
diseases. The kit includes an antibody that binds to Angiopoietin-1
and/or Angiopoietin-1 and a means for indicating the reaction of
the antibody with Angiopoietin-1 and/or Angiopoietin-2, if present.
The antibody may be a monoclonal antibody. In one embodiment, the
antibody that binds Angiopoietin-2 is labeled. In another
embodiment the antibody is an unlabeled primary antibody and the
kit further includes a means for detecting the primary antibody. In
one embodiment, the means includes a labeled second antibody that
is an anti-immunoglobulin. In some embodiments, the antibody is
labeled with a marker selected from the group consisting of a
fluorochrome, an enzyme, a radionuclide and a radio-opaque
material.
INCORPORATION BY REFERENCE
[0329] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated herein by reference in their entirety. In addition,
the following U.S. provisional patent applications 61/023,958 filed
Jan. 28, 2008, 61/100,063 filed Sep. 25, 2008, and 61/142,778 filed
Jan. 6, 2009 are hereby incorporated by reference herein in their
entireties for all purposes.
EQUIVALENTS
[0330] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The foregoing description and Examples detail certain
embodiments of the invention and describes the best mode
contemplated by the inventors. It will be appreciated, however,
that no matter how detailed the foregoing may appear in text, the
invention may be practiced in many ways and the invention should be
construed in accordance with the appended claims and any
equivalents thereof.
SPECIFIC EMBODIMENTS
[0331] 1. An isolated antibody that binds to Ang-2, wherein said
antibody comprises a variable light chain, said light chain
comprising a sequence selected from the group consisting of SEQ ID
No:3 (MEDI1); SEQ ID No:4 (MEDI2); SEQ ID No:5 (MEDI3); SEQ ID No:6
(MEDI4); and SEQ ID No:8 (MEDI6). [0332] 2. The antibody of
embodiment 1, wherein said antibody is an IgG1 or an IgG2 isotype
antibody. [0333] 3. The antibody of embodiment 1 or 2, wherein said
antibody further comprises a variable heavy chain region comprising
SEQ ID No:7 (MEDI5). [0334] 4. The antibody of any of embodiments
1-3, wherein said antibody, when produced, exhibits a production
efficiency in a mammalian host cell equal to or greater than 2
times the production efficiency of the Ang-2 antibody 3.19.3.
[0335] 5. The antibody of embodiment 4, wherein said production
efficiency is equal to or greater than 3 times the production
efficiency of the Ang-2 antibody 3.19.3. [0336] 6. The antibody of
embodiment 4, wherein said production efficiency is equal to or
greater than 5 times the production efficiency of the Ang-2
antibody 3.19.3. [0337] 7. A nucleic acid encoding the antibody of
any of embodiments 1-6. [0338] 8. A vector comprising the nucleic
acid of embodiment 7 [0339] 9. A host cell comprising the vector of
embodiment 8. [0340] 10. A pharmaceutical composition comprising
the antibody of any of embodiments 1-6 and an excipient. [0341] 11.
A method of preventing, treating, or managing cancer in an animal
in need thereof, said method comprising administering to said
animal a dose of an effective amount of the composition of
embodiment 10. [0342] 12. A method of preventing metastasis of
cancer in an animal in need thereof, said method comprising
administering to said animal a dose of an effective amount of the
composition of embodiment 10. [0343] 13. A method of preventing
recurrences of cancer in an animal in need thereof, said method
comprising administering to said animal a dose of an effective
amount of the composition of embodiment 10. [0344] 14. A method of
preventing advancement of cancer in an animal in need thereof, said
method comprising administering to said animal a dose of an
effective amount of the composition of embodiment 10. [0345] 15. A
method of preventing development of cancer from a pre-cancerous
state in an animal in need thereof, said method comprising
administering to said animal an effective amount of the composition
of embodiment 10. [0346] 16. A method of preventing symptoms of
cancer in an animal in need thereof, said method comprising
administering to said animal a dose of an effective amount of the
composition of embodiment 10. [0347] 17. A method of promoting
tumor regression of a cancer in an animal in need thereof, said
method comprising administering to said animal a dose of an
effective amount of the composition of embodiment 10. [0348] 18. A
method of inhibiting tumor cell proliferation in an animal in need
thereof, said method comprising administering to said animal a dose
of an effective amount of the composition of embodiment 10. [0349]
19. A method of depleting malignant tumor cells in an animal in
need thereof, said method comprising administering to said animal a
dose of an effective amount of the composition of embodiment 10.
[0350] 20. A method of inhibiting angiogenesis of a cancer tumor in
an animal in need thereof, said method comprising administering to
said animal a dose of an effective amount of the composition of
embodiment 10. [0351] 21. The method of any of embodiments 11-20,
wherein said method comprises an additional dosing to said animal
of one or more other cancer therapies. [0352] 22. The method of
embodiment 21, wherein said one or more other cancer therapies are
chemotherapies, biological therapies/immunotherapies, radiation
therapies, hormonal therapies, or surgery. [0353] 23. The method of
any of embodiments 11-22, wherein said method further comprises the
administration of another therapeutic agent that is not a cancer
therapeutic agent. [0354] 24. The method of embodiment 23, wherein
said therapeutic agent is an anti-emetic agent, anti-fungal agent,
anti-parasitic agent, anti-inflammatory agent, immunomodulatory
agent, anti-viral agent, or antibiotic. [0355] 25. The method of
any of embodiments 21-24, wherein said chemotherapy is selected
from the group consisting of 5-Fluorouracil, carboplatin, and
paclitaxel. [0356] 26. The method of any of embodiments 21-25,
wherein said immunotherapy is the administration of bevacizumab or
an antibody that competes for the same epitope as bevacizumab.
[0357] 27. The method of any of embodiments 11-26, wherein said
cancer or tumor is selected from the group consisting of melanoma,
colon, colorectal, lung, small cell lung carcinoma, non-small cell
lung carcinoma, breast, rectum, stomach, glioma, prostate, ovary,
testes, thyroid, blood, kidney, liver, hepatocellular carcinoma
pancreas, brain, neck, glioblastoma, endometrial cancer, and
central nervous system cancer. [0358] 28. The method of any of
embodiments 11-27, wherein said animal has been previously treated
by administration of one or more cancer therapies but not by
administration of the composition of embodiment 10. [0359] 29. The
method of any embodiments 11-27, wherein said animal has been
previously treated with chemotherapy alone, or in combination with
one or more radiation therapies, biological/immunotherapies,
hormonal therapies or surgery. [0360] 30. The method of any of
embodiments 11-27, wherein said animal has been previously treated
with radiation therapy alone, or in combination with one or more
chemotherapies, biological therapies/immunotherapies, hormonal
therapies or surgery. [0361] 31. The method of any of embodiments
11-27, wherein said animal has been previously treated with
biological therapies/immunotherapies alone, or in combination with
one or more chemotherapies, radiation therapy, hormonal therapies
or surgery. [0362] 32. The method of any of embodiments 11-27,
wherein said animal has been previously treated with hormonal
therapies alone, or in combination with one or more chemotherapies,
radiation therapy, biological therapies/immunotherapies or surgery.
[0363] 33. The method of any of embodiments 11-27, wherein said
animal has been previously treated with surgery alone, or in
combination with one or more chemotherapies, radiation therapy,
hormonal therapies or biological therapies/immunotherapies. [0364]
34. The method of any of embodiments 11-33, wherein said cancer is
refractory to chemotherapy or radiation therapy. [0365] 35. The
method of any of embodiments 11-34, wherein said administration is
intravenously, subcutaneously, intratumorally, intramuscularly,
parenterally, or orally. [0366] 36. The method of any of
embodiments 21-35, wherein said composition and cancer therapy are
administered by the same mode of administration. [0367] 37. The
method of any of embodiments 21-35, wherein said composition and
cancer therapy are administered by a different mode of
administration. [0368] 38. The method of any of embodiments 21-35,
wherein said composition and cancer therapy are administered in the
same dosage form. [0369] 39. The method of any of embodiments
21-35, wherein said composition and cancer therapy are administered
in different dosage forms. [0370] 40. The method of any of
embodiments 21-39, wherein said cancer therapy is selected from the
group consisting of radiation therapies, biological
therapies/immunotherapies, hormonal therapies and surgery. [0371]
41. A method of preventing, treating, or managing disease-related
angiogenesis in an animal in need thereof, said method comprising
administering to said animal a dose of an effective amount of the
composition of embodiment 10. [0372] 42. The method of embodiment
41, wherein the disease-related angiogenesis is associated with
seronegative arthritis, seropositive arthritis, arthritis related
to other arthropathies, osteoarthritis or SLE. [0373] 43. The
method of embodiment 42, wherein the seropositive arthritis is
rheumatoid arthritis. [0374] 44. A method of preventing recurrences
of disease-related angiogenesis in an animal in need thereof, said
method comprising administering to said animal a dose of an
effective amount of the composition of embodiment 10. [0375] 45. A
method of preventing advancement of disease-related angiogenesis in
an animal in need thereof, said method comprising administering to
said animal a dose of an effective amount of the composition of
embodiment 10. [0376] 46. A method of treating rheumatoid arthritis
in an animal in need thereof, said method comprising administering
to said animal an effective amount of the composition of embodiment
10. [0377] 47. A method of preventing symptoms of disease-related
angiogenesis in an animal in need thereof, said method comprising
administering to said animal a dose of an effective amount of the
composition of embodiment 10. [0378] 48. The method of any of
embodiments 41-47, wherein said method comprises an additional
dosing to said animal of one or more other anti-inflammatory
therapies. [0379] 49. The method of embodiment 48, wherein said one
or more other anti-inflammatory therapies are chemotherapies,
biological therapies/immunotherapies, radiation therapies, hormonal
therapies, or surgery. [0380] 50. The method of embodiment 49,
wherein said biological therapy/immunotherapy is a TNF-.alpha.
antagonist. [0381] 51. The method of embodiment 50, wherein said
TNF-.alpha. is selected from etanercept (ENBREL.RTM.), adalimumab
(HUMIRA.RTM.), and infliximab (REMICADE.RTM.). [0382] 52. The
method of any of embodiments 41-51, wherein said method further
comprises the administration of another therapeutic agent that is
not an anti-inflammatory therapeutic agent. [0383] 53. The method
of embodiment 52, wherein said therapeutic agent is an anti-emetic
agent, anti-fungal agent, anti-parasitic agent, anti-cancer agent,
immunomodulatory agent, anti-viral agent, or antibiotic. [0384] 54.
The method of any of embodiments 41-53, wherein said animal has
been previously treated by administration of one or more
anti-inflammatory therapies but not by administration of the
composition of embodiment 10. [0385] 55. The method of any of
embodiments 41-53, wherein said animal has been previously treated
with chemotherapy alone, or in combination with one or more
radiation therapies, biological/immunotherapies, hormonal therapies
or surgery. [0386] 56. The method of any of embodiments 41-53,
wherein said animal has been previously treated with radiation
therapy alone, or in combination with one or more chemotherapies,
biological therapies/immunotherapies, hormonal therapies or
surgery. [0387] 57. The method of any of embodiments 41-53, wherein
said animal has been previously treated with biological
therapies/immunotherapies alone, or in combination with one or more
chemotherapies, radiation therapy, hormonal therapies or surgery.
[0388] 58. The method of any of embodiments 41-53, wherein said
animal has been previously treated with hormonal therapies alone,
or in combination with one or more chemotherapies, radiation
therapy, biological therapies/immunotherapies or surgery. [0389]
59. The method of any of embodiments 41-53, wherein said animal has
been previously treated with surgery alone, or in combination with
one or more chemotherapies, radiation therapy, hormonal therapies
or biological therapies/immunotherapies. [0390] 60. A method of
reducing endothelial cell proliferation in an animal, said method
comprising administration of a dose of an effective amount of the
composition of embodiment 10. [0391] 61. A method of inhibiting
Ang-2 and/or Ang-1 binding to Tie-2 in an animal, said method
comprising administration of a dose of an effective amount of the
composition of embodiment 10. [0392] 62. A method of inhibiting
Tie-2 phosphorylation in an animal, said method comprising
administration of a dose of an effective amount of the composition
of embodiment 10. [0393] 63. A method of reducing levels of
circulating Ang-2 and/or Ang-1 polypeptide in an animal, said
method comprising administration of a dose of an effective amount
of the composition of embodiment 10. [0394] 64. A pharmaceutical
composition comprising a combination of i) an antagonist of the
biological activity of Angiopoietin-2 and/or Tie-2, and ii) an
antagonist of the biological activity of CSF1R, and/or CSF1. [0395]
65. The composition according to embodiment 64, wherein the
antagonist of Angiopoietin-2 is an antibody. [0396] 66. The
composition according to embodiment 65, wherein the antagonist of
Angiopoietin-2 is a fully human monoclonal antibody. [0397] 67. The
composition according to embodiments 65 or 66, wherein the antibody
binds to the same epitope as any one of fully human monoclonal
antibodies selected from the group consisting of 3.31.2, 5.16.3,
5.86.1, 5.88.3, 3.3.2, 5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3,
MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, and MEDI4/5. [0398] 68. The
composition according to embodiment 65, wherein the antibody is a
fully human monoclonal antibody selected from the group consisting
of: 33.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2, 5.103.1, 5.101.1,
3.19.3, 5.28.1, 5.78.3, MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, and
MEDI4/5. [0399] 69. The composition according to any of embodiments
64-68, wherein the antagonist of the biological activity of CSF1R
is a tyrosine kinase inhibitor. [0400] 70. The composition
according to embodiment 69, wherein the antagonist of the
biological activity of CSF1R is selected from any one of: [0401]
2-chloro-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;
[0402]
2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-(trifluoromethyl)benzoyl]a-
mino}benzamide; [0403]
2-chloro-N-(5-fluoropyridin-3-yl)-5-{[3-fluoro-5-(trifluoromethyl)benzoyl-
]amino}-benzamide; [0404]
2-methyl-N-pyridin-3-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamide;
[0405]
5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-pyridin--
3-ylbenzamide; [0406]
2-chloro-5-[(3-cyclopropylbenzoyl)amino]-N-pyridin-3-ylbenzamide;
[0407] 2-chloro-5-[(3-chlorobenzoyl)amino]-N-pyridin-3-ylbenzamide;
[0408]
5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide;
[0409]
5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbe-
nzamide; [0410]
5-[(3-chlorobenzoyl)amino]-2-methyl-N-pyridin-3-ylbenzamide; [0411]
5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-thia-
zol-5-yl)benzamide; [0412]
2-chloro-N-1,3-thiazol-5-yl-5-{[3-(trifluoromethyl)benzoyl]amino}benzamid-
e; [0413]
2-chloro-5-[(3-chlorobenzoyl)amino]-N-1,3-thiazol-5-ylbenzamide;
[0414]
2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-1,3-thiazol-5-ylbenzamid-
e; [0415]
5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methyl-N-1,3-thiaz-
ol-5-ylbenzamide; [0416]
2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)benzoyl]ami-
no}benzamide; [0417]
2-chloro-5-[(3-chlorobenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamid-
e;
[0418]
2-chloro-5-[(3,5-dimethylbenzoyl)amino]-N-(2-methyl-1,3-thiazol-5--
yl)benzamide; [0419]
2-chloro-N-(2-methyl-1,3-thiazol-5-yl)-5-{[3-(trifluoromethyl)benzoyl]ami-
no}benzamide; [0420]
2-chloro-5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-N-(2-methyl-1,3-t-
hiazol-5-yl)benzamide; [0421]
5-[(5-{[3-(1-cyano-1-methylethyl)benzoyl]amino}-2-methylbenzoyl)amino]-N--
methyl-1,3-thiazole-2-carboxamide; [0422]
5-{[3-fluoro-5-(trifluoromethyl)benzoyl]amino}-2-methyl-N-(2-methyl-1,3-t-
hiazol-5-yl)benzamide; [0423]
5-[(3-chloro-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl-
)benzamide; [0424]
5-[(3-cyclopropyl-5-fluorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-
-5-yl)benzamide; [0425]
5-[(3-chlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benzamid-
e; [0426]
5-[3,4-dichlorobenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-
-yl)benzamide; [0427]
5-[(3-cyclopropylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)ben-
zamide; [0428]
5-[(3,5-dimethylbenzoyl)amino]-2-methyl-N-(2-methyl-1,3-thiazol-5-yl)benz-
amide; [0429]
2-methyl-5-[(3-methylbenzoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)benzamid-
e; [0430]
2,6-dichloro-N-(4-methyl-3-{[(2-methyl-1,3-thiazol-5-yl)amino]ca-
rbonyl}phenyl)isonicotinamide; [0431]
2-methyl-5-{[(3-methylcyclohexyl)carbonyl]amino}-N-(2-methyl-1,3-thiazol--
5-yl)benzamide; [0432]
2-methyl-N-(2-methyl-1,3-thiazol-5-yl)-5-(pentanoylamino)benzamide;
[0433]
2-methyl-5-[(4-methylhexanoyl)amino]-N-(2-methyl-1,3-thiazol-5-yl)-
benzamide; [0434]
4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinolin-
e-3-carboxamide; [0435]
4-[(2,3-dichlorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinolin-
e-3-carboxamide; [0436]
7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)-
quinoline-3-carboxamide; [0437]
4-[(3-chloro-2-fluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)qui-
noline-3-carboxamide; [0438]
7-ethoxy-4-[(2-fluoro-5-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)qui-
noline-3-carboxamide; [0439]
7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(4-methylpiperazin-1-yl)qui-
noline-3-carboxamide; [0440]
4-[(2,4-difluorophenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperazin-1--
yl)quinoline-3-carboxamide; [0441]
4-[(2-fluoro-4-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperaz-
in-1-yl)quinoline-3-carboxamide; [0442]
4-[(2-fluoro-5-methylphenyl)amino]-7-(2-methoxyethoxy)-6-(4-methylpiperaz-
in-1-yl)quinoline-3-carboxamide; [0443]
4-[(2-fluoro-4-methylphenyl)amino]-6-(4-isopropylpiperazin-1-yl)-7-(2-met-
hoxyethoxy)quinoline-3-carboxamide; [0444]
7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-methylpiperidin-4-yl)qui-
noline-3-carboxamide; [0445]
4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-methylpiperidin-4-yl)quinolin-
e-3-carboxamide; [0446]
4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(1-isopropylpiperidin-4-yl)quino-
line-3-carboxamide; [0447]
7-ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-
quinoline-3-carboxamide; [0448]
4-[(2-fluoro-4-methylphenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)qu-
inoline-3-carboxamide; [0449]
4-[(3-chloro-2-fluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)qu-
inoline-3-carboxamide; [0450]
4-[(2,4-difluorophenyl)amino]-7-methoxy-6-(1-methylpiperidin-4-yl)quinoli-
ne-3-carboxamide; [0451]
4-[(2-fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methox-
yquinoline-3-carboxamide; [0452]
4-[(2,4-difluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methoxyquin-
oline-3-carboxamide; and [0453]
4-[(3-chloro-2-fluorophenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methox-
yquinoline-3-carboxamide; [0454]
7-Ethoxy-4-[(2-fluoro-4-methyl-phenyl)amino]-6-(4-methylpiperazin-1-yl)ci-
nnoline-3-carboxamide; [0455]
4-(2-Fluoro-4-methylphenylamino)-7-methoxy-6-(4-methylpiperazin-1-yl)cinn-
oline-3-carboxamide; [0456]
4-[(2,4-Difluorophenyl)amino]-7-methoxy-6-(4-methylpiperazin-1-yl)cinnoli-
ne-3-carboxamide; [0457]
6-[(3R,5S)-3,5-Dimethylpiperazin-1-yl]-4-[(2-fluoro-4-methylphenyl)amino]-
-7-methoxycinnoline-3-carboxamide; [0458]
4-[(2-Fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazin-1-yl]-7-
-methoxycinnoline-3-carboxamide; [0459]
7-Ethoxy-4-[(2-fluoro-4-methylphenyl)amino]-6-[4-(2-hydroxyethyl)piperazi-
n-1-yl]cinnoline-3-carboxamide; [0460]
4-[(3-Chloro-2-fluorophenyl)amino]-6-[(3R,5S)-3,5-dimethylpiperazin-1-yl]-
-7-methoxycinnoline-3-carboxamide; [0461]
4-[(2-Fluoro-4-methylphenyl)amino]-6-(1-isopropylpiperidin-4-yl)-7-methox-
ycinnoline-3-carboxamide hydrochloride; [0462]
4-[(2-Fluoro-4-methylphenyl)amino]-6-[1-(2-hydroxyethyl)piperidin-4-yl]-7-
-methoxycinnoline-3-carboxamide; and [0463]
4-[(2-Fluoro-4-methylphenyl)amino]-6-{4-[(2R)-2-hydroxypropanoyl]piperazi-
n-1-yl}-7-methoxycinnoline-3-carboxamide; or a pharmaceutically
acceptable salt thereof. [0464] 71. A pharmaceutical composition
comprising a combination according to any one of embodiments 62 to
70, in association with a pharmaceutically-acceptable excipient or
carrier. [0465] 72. A pharmaceutical composition according to
embodiment 71, for use in the treatment of disease-related
angiogenesis or inflammation. [0466] 73. A pharmaceutical
composition according to embodiment 71, for use in the treatment of
cancer. [0467] 74. A method of treating disease-related
angiogenesis or inflammation in an animal in need thereof with a
combination according to any one of embodiments 62-73. [0468] 75. A
method of treating cancer in an animal in need thereof with a
combination according to any one of embodiments 62-73. [0469] 76. A
composition comprising an antagonist of the biological activity of
Angiopoietin-2, and/or Tie-2; and a chemotherapeutic agent. [0470]
77. The composition according to embodiment 76, wherein the
antagonist of Angiopoietin-2 is an antibody. [0471] 78. The
composition according to embodiment 77, wherein the antagonist of
Angiopoietin-2 is a fully human monoclonal antibody. [0472] 79. The
composition according to any one of embodiments 77 or 78, wherein
the antibody binds to the same epitope as an antibody selected from
the group consisting of 3.31.2, 5.16.3, 5.86.1, 5.88.3, 3.3.2,
5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3, MEDI1/5, MEDI2/5,
MEDI3/5, MEDI6/5, and MEDI4/5. [0473] 80. The composition according
to embodiment 78, wherein the antibody is a fully human monoclonal
antibody selected from the group consisting of 3.31.2, 5.16.3,
5.86.1, 5.88.3, 3.3.2, 5.103.1, 5.101.1, 3.19.3, 5.28.1, 5.78.3,
MEDI1/5, MEDI2/5, MEDI3/5, MEDI6/5, and MEDI4/5. [0474] 81. The
composition according to any of embodiments 76-80, wherein the
chemotherapeutic agent is selected from the group consisting of
docetaxel, AZD4877, vincristine, vinblastine, vindesine and
vinorelbine, taxol, taxotere, 5-fluorouracil, gemcitabine,
fluoropyrimidines tegafur, raltitrexed, capecitabine, methotrexate,
pemetrexed, cytosine arabinoside, hydroxyurea; irinotecan,
etoposide topotecan, camptothecin teniposide, amsacrine,
oxaliplatin, cisplatin oxaliplatin, 5-fluorouracil, irinotecan,
gemcitabine and carboplatin. [0475] 82. The composition according
to any of embodiments 76 to 81 in association with a
pharmaceutically acceptable excipient or carrier. [0476] 83. A
method of antagonizing the biological activity of Angiopoietin-2,
and/or Tie-2 comprising administering the composition according to
any of embodiments 76 to 82. [0477] 84. A method of producing an
anti-cancer effect in a patient comprising administering a
therapeutically effective amount of a composition of any one of
embodiments 76 to 82. [0478] 85. A method of reducing tumor growth
in an animal comprising administering a therapeutically effective
amount of a composition of any one of embodiments 76 to 82.
VI. SEQUENCES
TABLE-US-00001 [0479] 3.19.3 light chain SEQ ID No.: 1 ##STR00001##
The boxed residue in this sequence represents an unpaired cysteine
(C49) that may be changed to any other amino acid. Examples of such
changes are highlighted in the light chain sequences below. 3.19.3
heavy chain SEQ ID No.: 2 ##STR00002## The boxed residue in this
sequence represents an example of a residue that may be
"backmutated " to another residue. One example of such a
"backmutation" is represented in the MEDI5 heavy chain sequence.
MEDI1 light chain SEQ ID No.: 3 ##STR00003## MEDI2 light chain SEQ
ID No.: 4 ##STR00004## MEDI3 light chain SEQ ID No.: 5 ##STR00005##
MEDI4 light chain SEQ ID No.: 6 ##STR00006## MEDI5 heavy chain SEQ
ID No.: 7 ##STR00007## MEDI6 light chain SEQ ID No.: 8
##STR00008##
VII. EXAMPLES
Example 1
Potency of Modified Ang-2 Antibodies
[0480] In this example, Ang-2 specific antibodies comprising the
light and heavy chains of the Ang-2 antibody 3.19.3 have been
modified such that amino acid substitutions for cysteine at
position 49 of the light chain were introduced. The potency of the
resultant antibodies was measured in an Ang-2:Tie-2 potency assay.
The results are presented in Table 1 below.
TABLE-US-00002 TABLE 1 Potency of modified Ang-2 antibodies
Antibody IC50 3.19.3 (WT) 0.06 C49A (MEDI4) SEQ ID No.: 6 0.03 C49D
(MEDI3) SEQ ID No.: 5 0.07 C49E 0.06 C49F 0.06 C49G 0.09 C49H 0.98
C49I 0.04 C49K 103.40 C49L 0.05 C49M 0.05 C49N (MEDI2) SEQ ID No.:
4 0.04 C49P 0.07 C49Q 0.12 C49R 5.48 C49S 0.14 C49T (MEDI1) SEQ ID
No.: 3 0.04 C49V 0.08 C49W 1.02 C49Y 0.73 Control Ab 0.05
[0481] Results:
[0482] In an Ang-2:Tie-2 potency assay, various modifications of
position 49 resulted in antibodies with similar potency. Exemplary
antibodies include but are not limited to C49A, C49T, C49N, and
C49D. Some of the modifications that included bulky, hydrophobic
substitutions, such as C49W resulted in lowered potency
demonstrated by an increased IC50 value. Other modifications,
including various charged residues such as C49K and C49H also
resulted in lowered potency demonstrated by an increased IC50
value.
Example 2
Improved Antibody Production Efficiency
[0483] In this example, Ang-2 specific antibodies comprising the
light and heavy chains of the Ang-2 antibody 3.19.3 have been
modified such that amino acid substitutions for cysteine at
position 49 of the light chain were introduced. The relative
expression of the resulting antibodies was measured. In addition,
position 37 of the heavy chain of several antibodies was also
modified to introduce a Val residue (MEDI5).
[0484] Materials and Methods: Vectors encoding the heavy and light
chains of 3.19.3 (SEQ ID Nos. 2 and 1, respectively), as well as,
heavy and light chains set forth as SEQ ID Nos. 7 and 3,
respectively (in both an IgG1 and IgG2 format) were expressed in
293F cells with the following protocol: Viable cells (>95%) were
diluted in Freestyle293.RTM. media (Invitrogen) at a cell density
of 1.0.times.10.sup.6 cells/min. The various DNA preparations were
diluted in 293Fectin.RTM. (Invitrogen) and added to cells as per
the manufacturers directions. On Day 6 of the transformation, the
expressed antibodies were harvested by collecting the culture
supernatant. Antibody levels were measured by Protein A binding
prior to purification.
[0485] Results: The production efficiencies of the "MEDI1/5 IgG1"
(having the heavy and light chains set forth as SEQ ID Nos. 7 and 3
in an IgG format), and "MEDI1/5 IgG2" (having the heavy and light
chains set forth as SEQ ID Nos. 7 and 3 in an IgG format)
antibodies were increased as compared to the production efficiency
of the 3.19.3 antibody. The results are summarized in Table 2.
TABLE-US-00003 TABLE 2 Improved production efficiency of Ang-2
antibodies Antibody 3.19.3 MEDI1/5 IgG1 MEDI1/5 IgG2 Yield 10 mg/L
140 mg/L 260 mg/L Recovery 0.2 g 1.4 g 2.7 g
[0486] These results demonstrate that the substitution at position
49 of cysteine for threonine in the variable light chain coupled
with the substitution at position 37 of glycine to valine in the
light chain of an Ang-2 specific antibody leads to greatly improved
production efficiencies (i.e. yield and/or recovery).
Example 3
Increased Stability of Ang-2 Antibodies
[0487] In an attempt to assess the increased stability of the
cysteine substituted Ang-2 antibodies, a stability study was
performed. The WT 3.19.3 antibody, as well as, MEDI1/5 in an IgG1
and IgG2 format were concentrated to 10 mg/ml in 10 mM histidine pH
6.0. Samples of the aforementioned formulations were incubated at
either 25.degree. C. or 40.degree. C. for two weeks. As a measure
of stability, the rate of aggregation (% aggregation/mth) was
calculated after the two week time point. The results are presented
in Table 3.
TABLE-US-00004 TABLE 3 Stability of Ang-2 antibodies Sample %
Agg/mth @ 40.degree. C. % Agg/mth @ 25.degree. C. WT (3.19.3) 42.6
2.97 MEDI1/5 IgG1 0.21 0.21 MEDI1/5 IgG2 0.82 0.61
[0488] As demonstrated in Table 2, the MEDI1/IgG1 and IgG2
antibodies exhibit an enhanced stability as compared to the WT
(3.19.3) antibody. Also, as demonstrated in FIG. 2, much of the
heterogeneity demonstrated by the wild type antibody (3.19.3) was
abolished in the MEDI1/5 antibodies in an IgG1 or IgG2 formats. The
chromatograph in FIG. 2 demonstrates that replacement of C49 in the
wild type antibody reduces or eliminates the multiple antibody
species present in samples.
Example 4
Increased Stability of Ang-2 Antibodies
[0489] In an attempt to assess the increased stability of the
cysteine substituted Ang-2 antibodies, a Differential Scanning
calorimetry (DSC) analysis of WT (3.19.3) and MEDI1/5 IgG1 and IgG2
antibodies was performed. In this example, formulations of WT,
MEDI1/5IgG1, and MEDI1/5 IgG2 antibodies were prepared at 1 g/L
antibody in 10 mM histidine, pH 6.0 and subjected to DSC analysis.
The results are presented in FIG. 1. As presented, the WT antibody
exhibits a melting temperature of about 61.degree. C. The MEDI1/5
IgG1 antibody exhibits a higher melting temperature of about
76.degree. C. The MEDI1/5 IgG2 also exhibits a higher melting
temperature of about 76.degree. C., however it subsequently falls
out of solution, which was observed in two independent trials
(trial 1 and 2).
[0490] These results suggest that MEDI1/5 antibodies exhibit
increased stability over the WT (3.19.3) antibody as measured by
melting temperature.
Example 5
Binding Profiles of Ang-2 Antibodies
[0491] In this example the Ang-2 antibody, 3.19.3, and a modified
3.19.3 antibody comprising a C49T (MEDI1/5) substitution were
analyzed for Ang-2 binding in a competitive ELISA format assay.
[0492] Materials and Methods:
[0493] Competitive Tie-2 Fc/Ang2 ELISA: Maxisorp ELISA plates
(Nunc, Rochester, N.Y.) were coated with 100 .mu.l of 4 .mu.g/ml
Tie-2 Fc (R & D Systems, Minneapolis, Minn.) in 0.1M carbonate
buffer pH 9.4 (Pierce, Rockford, Ill.) and incubated overnight at
4.degree. C. The following day, plates were blocked for 1 hour at
room temperature with 200 .mu.l of phosphate buffered saline (PBS)
(Invitrogen, Carlsbad, Calif.) containing 0.5% bovine serum albumin
(BSA) (Sigma, St. Louis, Mo.) and 0.1% Tween-20 (Sigma, St. Louis,
Mo.). Plates were washed 3 times with wash buffer (PBS containing
0.05% Tween-20). 50 .mu.l of 11-point serial tertiary dilutions (30
.mu.g/ml high concentration) of 3.19.3 or modified anti-Ang2
antibody were plated, with PBS as a negative control. For antibody
capture, 50 .mu.l of 200 ng/ml Biotin Ang2 (R & D Systems,
Minneapolis, Minn.) were added and plates were incubated for 2
hours at room temperature. Plates were washed 3 times with wash
buffer. Following the wash, 100 ul of 1:5000 streptavidin HRP
(Pierce, Rockford, Ill.) dilution in wash buffer were added and
incubated for 1 hour at room temperature. Plates were washed 3
times with wash buffer. Plates were developed by adding 100 .mu.l
of TMB peroxidase substrate (KPL, Gaithersburg, Md.) for 5 minutes.
The reaction was stopped by adding 100 .mu.l/well of 1M phosphoric
acid. Optical densities were measured at 450 nm with a microplate
reader (Molecular Devices, Sunnyvale, Calif.).
[0494] Results: As presented in FIG. 3, the ELISA results
demonstrate that the MEDI1/5 antibody in either an IgG1 or an IgG2
format exhibit a very similar binding profile for Ang-2 as compared
to the 3.19.3 antibody as measured by competition for Tie-2 in an
ELISA format.
Example 6
Combination Studies
[0495] The in vivo efficacy of 3.19.3 in combination with small
molecule CSF1R tyrosine kinase inhibitors has been evaluated.
[0496] Study 6.1 Determination of the Therapeutic Efficacy of mAb
3.19.3 in Combination with the CSF1R Antagonist AZD6495 in a MCF7
Breast Cancer Xenograft Model
[0497] The anti-tumor activity of the anti-Angiopoietin-2
monoclonal antibody 3.19.3 in combination with the small molecule
CSF1R tyrosine kinase inhibitor AZD6495
(4-[(2,4-difluorophenyl)amino]-7-ethoxy-6-(4-methylpiperazin-1-yl)quinoli-
ne-3-carboxamide) was evaluated in a xenograft model of human
breast carcinoma using the MCF7 cell line (FIG. 4a).
[0498] Materials and Methods: Breast carcinoma MCF7 cells were
cultured in flasks as routine until the cells reach sub-confluence.
Immunodeficient 7-10 week old male NCr-nude mice were
subcutaneously implanted with 8.times.10.sup.6 MCF7 cells suspended
1:1 in Matrigel in the right flank. 17-B-Estradiol pellets (0.72
mg/pellet) were also implanted as standard procedure to support the
growth of this ER positive (estrogen requiring) cell line.
[0499] The mice were then randomized into cohorts containing 15
mice once the tumors reached approximately 100 mm.sup.3. The mice
were treated by intraperitoneally (IP) injection with mAb 3.19.3
(10 mg/kg) twice per week for 3 weeks thereafter, or by oral
administration of CSF1R inhibitor A (AZD6495) at (30 mg/kg)
following bid dosing for 18 days. For all experiments, 0.5% HPMC
was used as an oral vehicle only control. The body weights of each
animal, and the dimensions of each tumor were measured twice per
week. The volume of the tumor was calculated as:
Volume=Length.times.(Width).times.0.5 cm.sup.3, or by bilateral
Vernier caliper measurement and, taking length to be the longest
diameter across the tumor and width the corresponding
perpendicular, calculated using the formula
(7c/6).times.(length.times.width).times.(length.times.width).
Growth inhibition from the start of treatment was assessed by
comparison of the differences in tumor volume between control and
treated groups.
The summary study design was as follows:
TABLE-US-00005 TABLE 4 Study design Group Treatment # Mice Schedule
Route A Veh (0.5% HPMC) 15 bid x 18 d p.o. B mAb3.19.3, 10 mg/kg 15
q3.5 d x 3 wk i.p. C AZD6495, 30 mg/kg 15 bid x 18 d p.o. D
mAb3.19.3, 10 mg/kg + 15 q3.5 d x 3 wk i.p. AZD6495, 30 mg/kg bid x
18 d p.o.
[0500] As illustrated in FIG. 4a, 3.19.3 and AZD6495 delayed the
growth of the MCF7 tumors as single agents. However the combination
of 3.19.3 and AZD6495 had a greater effect than the single agents
alone as illustrated in FIG. 4a. The % tumor growth inhibition
achieved is as follows:
[0501] 3.19.3 (10 mg/kg 2.times.wk)=62% inhibition; (p<0.04)
[0502] AZD6495 (30 mg/kg bid)=32% inhibition; (p<0.42)
[0503] Combination 3.19.3+AZD6495=81% inhibition (p<0.01)
[0504] No additional toxicity was observed with the combinations as
compared to single-agent treatment alone as determined by changes
in body weights (FIG. 4b). Changes in macrophage populations are
being measured via F4/80 staining and Fluorescence Activated Cell
Sorting and the tumor tissue analyzed via CD31+ vessel staining
density to examine any effects on the tumor associated vasculature.
CD31 staining density can be measured by threshold and by manual
grid counting methods. The combination of 3.19.3 with CSF1R
tyrosine kinase inhibitors is expected to produce a significantly
greater effect on both tumor associated macrophage populations and
CD31 staining blood vessels.
[0505] These results demonstrate that combination treatment with
the anti-Ang2 antibody 3.19.3 and the small molecule CSF1R
antagonist, AZD6495, leads to improvements in efficacy without
additive toxicity in a pre-clinical model of breast cancer.
[0506] Study 6.2. Determination of the Therapeutic Efficacy of mAb
3.19.3 in Combination with the CSF1R Antagonist AZD6495 in a
MDA-MB-231 Orthotopic Breast Cancer Xenograft Model
[0507] The anti-tumor activity of the anti-Angiopoietin-2
monoclonal antibody 3.19.3 in combination with the small molecule
CSF1R tyrosine kinase inhibitor AZD6495 was evaluated in a
xenograft model of human breast carcinoma using the MDA-MB-231 cell
line (FIG. 5a).
[0508] Breast adenocarcinoma MDA-MB-231 cells were cultured in
flasks as routine until the cells reach sub-confluence.
Immunodeficient 7-10 week old female nude mice were orthotopically
implanted with 8.times.10.sup.6 MDA-MB-231 cells suspended 1:1 in
Matrigel into the mammary fat pad. The mice were then randomized
into cohorts containing 10 mice once the tumors reached
approximately 100 mm.sup.3. The mice were treated by
intraperitoneally (IP) injection with mAb 3.19.3 (10 mg/kg) twice
per week for 3 weeks thereafter, or by oral administration of
AZD6495 (30 mg/kg) following bid dosing for 18 days. For all
experiments, 0.5% HPMC was used as an oral vehicle only control.
The body weights of each animal, and the dimensions of each tumor
were measured twice per week. The volume of the tumor was
calculated as: Volume=Length.times.(Width).times.0.5 cm.sup.3, or
by bilateral Vernier caliper measurement and, taking length to be
the longest diameter across the tumor and width the corresponding
perpendicular, calculated using the formula
(.pi./6).times.(length.times.width).times. (length.times.width).
Growth inhibition from the start of treatment was assessed by
comparison of the differences in tumor volume between control and
treated groups.
[0509] The summary study design was as follows;
TABLE-US-00006 TABLE 5 Study design Group Treatment # Mice Schedule
Route A Veh (0.5% HPMC) 10 bid x 18 d p.o. B mAb3.19.3, 10 mg/kg 10
q3.5 d x 3 wk i.p. C AZD6495, 30 mg/kg 10 bid x 18 d p.o. D
mAb3.19.3, 10 mg/kg + 10 q3.5 d x 3 wk i.p. AZD6495, 30 mg/kg bid x
18 d p.o.
[0510] As illustrated in FIG. 5a, 3.19.3 and AZD6495 delayed the
growth of the MDA-MS-231 tumors as single agents. However the
combination of 3.19.3 and AZD6495 had a greater effect than the
single agents alone as illustrated in FIG. 5a. The % tumor growth
inhibition achieved is as follows:
[0511] 3.19.3 (10 mg/kg 2.times.wk)=17% inhibition; (p<0.20)
[0512] AZD6495 (30 mg/kg bid)=25% inhibition; (p<0.04)
[0513] Combination 3.19.3+AZD6495=52% inhibition (p<0.09)
[0514] No additional toxicity was observed with the combinations as
compared to single-agent treatment alone as determined by changes
in body weights (FIG. 5b). Changes in macrophage populations are
being measured via F4/80 staining and Fluorescence Activated Cell
Sorting and the tumor tissue analyzed via CD31+ vessel staining
density to examine any effects on the tumor associated vasculature.
CD31 staining density can be measured by threshold and by manual
grid counting methods. The combination of 3.19.3 with CSF1R
tyrosine kinase inhibitors is expected to produce a significantly
greater effect on both tumor associated macrophage populations and
CD31 staining blood vessels.
[0515] These results demonstrate that combination treatment with
the anti-Ang2 antibody 3.19.3 and the small molecule CSF1R
antagonist AZD6495 leads to improvements in efficacy without
additive toxicity in a pre-clinical model of breast cancer.
Example 7
Combination Studies
[0516] The activity of the monoclonal antibody 3.19.3 was evaluated
in combination studies with chemotherapeutic agents to determine
the in vivo efficacy and tolerability in human tumor xenograft
models.
[0517] Study 7.1. Determination of the Therapeutic Efficacy of
Monoclonal Antibody 3.19.3 in Combination with 5-Fluorouracil in
LoVo Xenograft Tumors
[0518] The anti-tumor activity of 3.19.3 was evaluated in
combination with 5-Fluoruracil (5FU) in the LoVo xenograft model of
colorectal cancer. LoVo cells were cultured in flasks as routine
until the cells reached sub-confluence. Cell suspensions containing
approximately 3.times.10E6 cells were injected subcutaneously into
the flank of female Swiss nude mice. When the tumor volume reached
200 mm.sup.3, the mice were randomized in treatment groups of 8-10
mice and the treatments were initiated. 3.19.3 (10 mg/kg) in saline
was injected intraperitoneally, twice per week for 2 weeks and
5-Fluorouracil (100 mg/kg) was administered by intraperitoneal
administration following a weekly schedule. The dimensions of each
tumor and body weights were measured at least twice per week. The
volume of the tumor was calculated as:
Volume=Length.times.(Width).times.0.5 (cm.sup.3). As illustrated in
FIG. 6a, 3.19.3 and 5FU delayed the growth of the LoVo tumors as
single agent. However the combination of 3.19.3 and 5FU had a
greater effect than the single agents alone as illustrated in FIG.
6a. The % tumor growth inhibition achieved is as follows:
[0519] 3.19.3 (10 mg/kg 2.times.wk)=59% inhibition; (p<0.09)
[0520] 5 FU (100 mg/kg/week)=62% inhibition; (p<0.02)
[0521] Combination 3.19.3+5FU=85% inhibition (p<0.007)
[0522] No additional toxicity was observed with the combinations as
compared to single-agent treatment alone as determined by changes
in body weights (FIG. 6b). These results demonstrate that
combination treatment with the anti-Ang2 antibody 3.19.3 and
5-fluorouracil leads to improvements in efficacy without additive
toxicity in a pre-clinical model of colon cancer, providing the
basis for further clinical investigation of this combination.
[0523] Study 7.2. Determination of the Therapeutic Efficacy of
Monoclonal Antibody 3.19.3 in Combination with Irinotecan in HT-29
Xenograft Tumors
[0524] The anti-tumor activity of 3.19.3 was evaluated in
combination with Irinotecan in the HT-29 xenograft model of
colorectal cancer. HT-29 cells were cultured in flasks as routine
until the cells reached sub-confluence. Cell suspensions containing
approximately 3.times.10E6 cells were injected subcutaneously into
the flank of female Swiss nude mice. When the tumor volume reached
200 mm.sup.3, the mice were randomized in treatment groups of 8-10
mice and the treatments were initiated. 3.19.3 (10 mg/kg) in saline
was injected intraperitoneally, twice per week for 2 weeks.
Irinotecan (35 mg/kg) was administered by intravenous
administration following a weekly schedule. The dimensions of each
tumor and body weights were measured at least twice per week. The
volume of the tumor was calculated as:
Volume=Length.times.(Width).times.0.5 (cm.sup.3). As illustrated in
FIG. 7a, 3.19.3 and Irinotecan delayed the growth of the HT29
tumors as single agents. However the combination of 3.19.3 and
Irinotecan had a greater effect than the single agents alone as
illustrated in FIG. 7a. The % tumor growth inhibition achieved is
as follows:
[0525] 3.19.3 (10 mg/kg 2.times.wk)=44% inhibition;
(p<0.005)
[0526] Irinotecan (35 mg/kg/week)=56% inhibition; (p<0.006)
[0527] Combination 3.19.3+Irinotecan=71% inhibition
(p<0.0001)
[0528] No additional toxicity was observed with the combinations as
compared to single-agent treatment alone as determined by changes
in body weights (FIG. 7b). These results demonstrate that
combination treatment with anti-Ang2 antibody 3.19.3 and Irinotecan
leads to improvements in efficacy without additive toxicity in a
pre-clinical model of colon cancer, and providing the basis for
further clinical investigation of this combination.
[0529] Study 7.3. Determination of the Therapeutic Efficacy of
Monoclonal Antibody 3.19.3 in Combination with Gemcitabine in
Colo205 Xenograft Tumors
[0530] The anti-tumor activity of 3.19.3 was evaluated in
combination with Gemcitabine in the Colo205 xenograft model of
colorectal cancer. Colo205 cells were cultured in flasks as routine
until the cells reached sub-confluence. Cell suspensions containing
approximately 3.times.10E6 cells were injected subcutaneously into
the flank of female Swiss nude mice. When the tumor volume reached
200 mm.sup.3, the mice were randomized in treatment groups of 8-10
mice and the treatments were initiated. 3.19.3 (10 mg/kg) in saline
was injected intraperitoneally, twice per week for 2 weeks.
Gemcitabine (50 mg/kg) was administered by intravenous
administration following a q3d schedule. The dimensions of each
tumor and body weights were measured at least twice per week. The
volume of the tumor was calculated as:
Volume=Length.times.(Width).times.0.5 (cm.sup.3). As illustrated in
FIG. 8a, 3.19.3 delayed the growth of the Colo205 tumors as single
agent however the Colo205 tumors were fairly refractory to
Gemcitabine treatment resulting in a modest 8% tumor growth delay.
However the combination of 3.19.3 and Gemcitabine had a greater
effect than the single agents alone as illustrated in FIG. 8a. The
% tumor growth inhibition achieved is as follows:
[0531] 3.19.3 (10 mg/kg 2.times.wk)=74% inhibition;
(p<0.001)
[0532] Gemcitabine (50 mg/kg q3d.times.2)=8% inhibition;
(p<0.2)
[0533] Combination 3.19.3+Gemcitabine=88% inhibition
(p<0.0003)
[0534] No additional toxicity was observed with the combinations as
compared to single-agent treatment alone as determined by changes
in body weights (FIG. 8b). These results demonstrate that
combination treatment with anti-Ang2 antibody 3.19.3 and
Gemcitabine leads to improvements in efficacy without additive
toxicity in a pre-clinical model of colon cancer, and providing the
basis for further clinical investigation of this combination.
[0535] Study 7.4. Determination of the Therapeutic Efficacy of
Monoclonal Antibody 3.19.3 in Combination with Docetaxel in Calu6
Xenograft Tumors
[0536] The anti-tumor activity of 3.19.3 was evaluated in
combination with Docetaxel in the Calu6 xenograft model of lung
cancer. Calu6 cells were cultured in flasks as routine until the
cells reached sub-confluence. Cell suspensions containing
approximately 3.times.10E6 cells were injected subcutaneously into
the flank of female Swiss nude mice. When the tumor volume reached
200 mm.sup.3, the mice were randomized in treatment groups of 8-10
mice and the treatments were initiated. 3.19.3 (10 mg/kg) in saline
was injected intraperitoneally, twice per week for 2 weeks.
Docetaxel (15 mg/kg) was administered by intravenous administration
following a weekly schedule. The dimensions of each tumor and body
weights were measured at least twice per week. The volume of the
tumor was calculated as: Volume=Length.times.(Width).times.0.5
(cm.sup.3). As illustrated in FIG. 9a, 3.19.3 and Docetaxel delayed
the growth of the Calu6 tumors with the single agents. However the
combination of 3.19.3 and Docetaxel had a greater effect than the
single agents alone as illustrated in FIG. 9a. The % tumor growth
inhibition achieved is as follows:
[0537] 3.19.3 (10 mg/kg 2.times.wk)=20% inhibition; (p<0.12)
[0538] Docetaxel (15 mg/kg/week)=43% inhibition; (p<0.0007)
[0539] Combination 3.19.3+Docetaxel=71% inhibition
(p<0.0001)
[0540] No additional toxicity was observed with the combinations as
compared to single-agent treatment alone as determined by changes
in body weights (FIG. 9b). These results demonstrate that
combination treatment with anti-Ang2 antibody 3.19.3 and Docetaxel
leads to improvements in efficacy without additive toxicity in a
pre-clinical model of lung cancer, and providing the basis for
further clinical investigation of this combination.
[0541] Study 7.5. Determination of the Therapeutic Efficacy of
Monoclonal Antibody 3.19.3 in Combination with Oxaliplatin in H460
Xenograft Tumors
[0542] The anti-tumor activity of 3.19.3 was evaluated in
combination with Oxaliplatin in the H460 xenograft model of lung
cancer. H460 cells were cultured in flasks as routine until the
cells reached sub-confluence. Cell suspensions containing
approximately 3.times.10E6 cells were injected subcutaneously into
the flank of female Swiss nude mice. When the tumor volume reached
200 mm.sup.3, the mice were randomized in treatment groups of 8-10
mice and the treatments were initiated. 3.19.3 (10 mg/kg) in saline
was injected intraperitoneally, twice per week for 2 weeks.
Oxaliplatin (5 mg/kg) was administered by intraperitoneal
administration following a weekly schedule. The dimensions of each
tumor and body weights were measured at least twice per week. The
volume of the tumor was calculated as:
Volume=Length.times.(Width).times.0.5 (cm.sup.3). As illustrated in
FIG. 10a, 3.19.3 and Oxaliplatin delayed the growth of the H460
tumors with the single agents. However the combination of 3.19.3
and Oxaliplatin had a greater effect than the single agents alone
as illustrated in FIG. 10a. The % tumor growth inhibition achieved
is as follows:
[0543] 3.19.3 (10 mg/kg 2.times.wk)=67% inhibition;
(p<0.001)
[0544] Oxaliplatin (5 mg/kg/week)=35% inhibition; (p<0.01)
[0545] Combination 3.19.3+Oxaliplatin=75% inhibition
(p<0.0001)
[0546] No additional toxicity was observed with the combinations as
compared to single-agent treatment alone as determined by changes
in body weights (FIG. 10b). These results demonstrate that
combination treatment with anti-Ang2 antibody 3.19.3 and
Oxaliplatin leads to improvements in efficacy without additive
toxicity in a pre-clinical model of lung cancer, and providing the
basis for further clinical investigation of this combination.
[0547] Study 7.6. Determination of the Therapeutic Efficacy of
Monoclonal Antibody 3.19.3 in Combination with the Mitotic Eg5
Inhibitor AZD4877 in H460 Xenograft Tumors
[0548] The anti-tumor activity of 3.19.3 was evaluated in
combination with AZD4877 in the H460 xenograft model of lung
cancer. H460 cells were cultured in flasks as routine until the
cells reached sub-confluence. Cell suspensions containing
approximately 3.times.10E6 cells were injected subcutaneously into
the right flank of male NCr nu/nu mice. When the tumor volume
reached 200 mm.sup.3, the mice were randomized in treatment groups
of 8-10 mice and the treatments were initiated. 3.19.3 (10 mg/kg)
in saline was injected intraperitoneally, twice per week for 2
weeks. AZD4877 (10 mg/kg) was administered by intraperitoneal
administration following a q4d schedule. The dimensions of each
tumor and body weights were measured at least twice per week. The
volume of the tumor was calculated as:
Volume=Length.times.(Width).times.0.5 (cm.sup.3). As illustrated in
FIG. 11a, 3.19.3 and AZD4877 delayed the growth of the H460 tumors
with the single agents. However the combination of 3.19.3 and
AZD4877 had a greater effect than the single agents alone as
illustrated in FIG. 11a. The % tumor growth inhibition achieved is
as follows:
[0549] 3.19.3 (10 mg/kg 2.times.wk)=64% inhibition;
(p<0.001)
[0550] AZD4877 (10 mg/kg q4d.times.2)=50% inhibition;
(p<0.001)
[0551] Combination 3.19.3+AZD4877=78% inhibition (p<0.0001)
[0552] No additional toxicity was observed with the combinations as
compared to single-agent treatment alone as determined by changes
in body weights (FIG. 11b). These results demonstrate that
combination treatment with anti-Ang2 antibody 3.19.3 and AZD4877
leads to improvements in efficacy without additive toxicity in a
pre-clinical model of lung cancer, and providing the basis for
further clinical investigation of this combination.
[0553] The studies of the anti Ang-2 antibody, 3.19.3 in
combination with chemotherapeutic agents included Docetaxel,
5-fluorouracil, Irinotecan, Oxaliplatin, or Gemcitabine which
demonstrated at least additive activity with no increases in
toxicity with the combinations as indicated by body weights. These
results demonstrate that combination treatment with monoclonal
antibody 3.19.3 and chemotherapy leads to improvements in efficacy
without additive toxicity in pre-clinical models of cancer.
[0554] The results of the monoclonal antibody 3.19.3 xenograft
combination studies with VEGF inhibitors and chemotherapeutic
agents are summarized in below:
TABLE-US-00007 TABLE 6 Summary of 3.19.3 with VEGF inhibitors and
chemotherapeutic agents: Xenograft Chemotherapeutic Agent %
Inhibition Significance (T-test) Lovo 5-flurouracil (100 mg/kg) 62%
p < 0.02 3.19.3 (10 mg/kg) 59% p < 0.09 Combination 85% p
< 0.007 HT-29 Irinotecan (35 mg/kg) 56% p < 0.006 3.19.3 (10
mg/kg) 44% p < 0.0005 Combination 71% p < 0.0001 Colo205
Gemcitabine (50 mg/kg) 8% p < 0.2 3.19.3 (10 mg/kg) 74% p <
0.001 Combination 88% p < 0.0003 Calu6 Docetaxel (15 mg/kg) 43%
p < 0.0007 3.19.3 (10 mg/kg) 20% p < 0.12 Combination 71% p
< 0.0001 H460 Oxaliplatin (5 mg/kg) 35% p < 0.01 3.19.3 (10
mg/kg) 67% p < 0.001 Combination 75% p < 0.0001
Example 8
Effect of Therapeutic Administration of the Ang-2 Antibody, 3.19.3
on Disease Progression in the DBA/1 Murine Model of Collagen
Induced Arthritis In Vivo
[0555] Preparation of rat collagen type II emulsion: Bovine
Collagen Type II (MD Biosciences, Cat # IMBII; Lot 090205) was
stored at 4.degree. C. in the dark until use. Prior to immunisation
of animals, bovine collagen was dissolved in 0.01 M acetic acid at
a stock solution of 2 mg/mL and stored overnight in the dark at
4.degree. C. On the day of immunisation, collagen was emulsified
with equal volume of Freund's Complete Adjuvant (FCA [Difco, Cat
#231131; Lot 850262/R1]) to give a solution at 1 mg/mL.
[0556] Induction of arthritis: On day 0, male DBA/1 mice (6-8 weeks
old, Harlan Sprague Dawley, UK) were lightly anaesthetised with
3.5% isoflurane and immunized intra-dermal just above the root of
the tail with 100 .mu.g rat collagen type II emulsified in FCA (1
mg/mL; 0.1 mL/mouse).
[0557] Staphylococcal Enterotoxin B (SEB) booster: On day 21, all
mice were anaesthetised as before and given a booster injection of
30 .mu.g SEB (600 .mu.g/ml in water for injections [Toxin
Technology, Cat # BT202; Lot 70903] emulsified in an equal volume
of Freund's Incomplete Adjuvant [Sigma, Cat # F5506; Lot 112K8930]
to give a final concentration of 300 .mu.g/ml). 50 .mu.l.times.2
(equivalent to 30 .mu.g SEB) is injected intra-dermal adjacent to
the immunisation site.
[0558] Assessment of arthritis: Clinical observations regarding
welfare of the animals were carried out daily at time of dosing.
Observations for clinical signs of disease were carried out daily
from day 20 post immunisation, whereby the animals were removed
from their micro-environment, and scored using the scoring system
outlined below.
TABLE-US-00008 TABLE 7 Scoring system for hind and forepaws
Clinical score Description 0 Normal 1 Erythema and slight swelling
of one of more toes. 2 Erythema and obvious swelling of two or more
toes, or mild swelling of the ankle or wrist without toe
involvement. 3 Erythema and obvious swelling to some toes and ankle
or fore paw and wrist. 4 Erythema and severe swelling of ankle and
digits.
[0559] Dosing:
[0560] Animals were randomly assigned to treatment groups, as
outlined below.
TABLE-US-00009 TABLE 8 Treatment groups Group Treatment Number of
animals 1 PBS Vehicle i.p 15 2 3.19.3 10 mg/kg i.p 15 3 Human IgG
control antibody i.p 15 4 Prednisolone 3 mg/kg p.o 10
[0561] Animals in treatment group 2 were therapeutically dosed
intraperitonealy (i.p.) with 3.19.3 10 mL/kg every 3 days for 14
days from disease onset (defined as a clinical score of 2 in one or
more paw). Purified human IgG (hIgG) was used as a negative isotype
control. Animals in treatment group 4 were therapeutically dosed
per oral (p.o.) with Prednisolone 3 mg/kg daily for 14 days from
disease onset (defined as a clinical score of 2 in one or more
paw).
[0562] Termination: Animals were terminated 14 days post disease
onset by exposure to rising concentration of carbon dioxide. Mouse
paws were excised post mortem, fixed in 10% buffered formalin and
decalcified. Decalcified paws were routinely processed and then
embedded in paraffin blocks. Serial sections (10 .mu.m) were cut
and stained with hematoxylin and eosin for histologic analysis.
[0563] Data analysis: Area under the curve (AUC) for clinical
disease progression was calculated for each animal from disease
onset. Unless otherwise stated, statistical analysis was by one-way
ANOVA with Dunnett's post-hoc comparison to vehicle controls.
P<0.05 was considered to be statistically significant throughout
the study.
[0564] Results: Significant reductions in both clinical signs of
disease progression (arthritic score) and histological assessment
of synovitis and joint destruction were observed with 3.19.3 at a
dose of 10 mg/kg (One-Way ANOVA with Dunnett's post-hoc comparison
to vehicle control).
TABLE-US-00010 TABLE 9 Therapeutic effects of 3.19.3 on CIA disease
progression Treatment group Area Under Curve (AUC) % Inhibition PBS
Vehicle i.p 100.9 .+-. 10.9 -- 3.19.3 10 mg/kg i.p 36.0 .+-. 8.7
64.3% .+-. 8.6% hIgG isotype control i.p 92.4 .+-. 11.2 8.4% .+-.
11.1% Prednisolone 3 mg/kg p.o 24.5 .+-. 9.6 76.1% .+-. 9.6%
Table 9: Effect of therapeutic administration of 3.19.3 (10 mg/kg
i.p every 3 days) on clinical disease progression throughout the
time course of collagen-induced arthritis as measured by Area Under
Curve [AUC] (values represent means.+-.standard error of the mean,
n=15 for 3.19.3, PBS vehicle and hIgG treatment groups, n=10 for
Prednisolone control group)
[0565] Histolopathological evaluation of CIA model treatment
groups: Results showed unequivocal evidence of an anti-arthritic
effect following administration of 3.19.3--which morphologically
was most evident upon synovial hyperplasia and fibrosis. There were
no atypical cell forms in this study and no atypical presentation
of bone. There is good correlation between clinical score and
histological measurements in this study.
[0566] PBS Vehicle treatment group: Marked arthropathy was noted in
the majority of animals treated with PBS vehicle. The pathology
presentation was a widespread--essentially mononuclear
cell--synovitis invading the tibio-talus space and spreading
towards the superficial facet of the calcaneum. There was extensive
lytic destruction of bone--notably the talus and navicular
bones--from both contact synovitis and from pannus expansion into
the bone marrow cavities and stromal cavities. Fibrosis of the
synovium was common, with occasional fibrinoid deposits, with
variable degrees of synovial hyperplasia.
[0567] 3.19.3 10 mg/kg treatment group: The 3.19.3 treatment group
showed a histologically significant reduction in the incidence and
severity of all arthropathy lesions, although it was most marked by
reduction of synovial hyperplasia and fibrosis; together with a
reduction in severity of both articular space and bone marrow
localized pannus.
[0568] Human IgG isotype control treatment group: Histologically,
there were no significant differences between this group and the
PBS vehicle group.
[0569] Prednisolone 3 mg/kg treatment group: This group showed a
marked reduction in the incidence and severity of all arthropathy
lesions.
[0570] Summary: This study demonstrates that neutralization of
angiopoietin-2 has been shown to be efficacious in ameliorating
collagen-induced arthritis in male dba/1 mice (FIG. 12a) with no
significant change in mean body weight observed between each
treatment group throughout the period from disease onset,
suggesting that 3.19.3 therapy was well tolerated (FIG. 12b). This
study assessed efficacy of 3.19.3 at a dose of 10 mg/kg, and
demonstrated reductions in both clinical signs of disease
progression (arthritic score) and histological assessment of
synovitis and joint destruction.
Example 9
Anti-Ang-2 Antibodies Inhibit Retinal Vascularization
[0571] The effect of anti-Ang-2 antibodies on retinal
vascularization MEDI1/5 antibodies was studied by comparing retinal
samples from treated mice versus control treated mice.
[0572] Methods: CD1 pups are either left untreated or dosed
intraperitoneally with MEDI1/5 (1 mg/kg or 10 mg/kg) at p1, p3 and
p5 (p1 being day of birth). At p10, pups were anesthetized with
isoflurane and then perfused with 12.5 mg/ml FITC-dextran (Vector
Labs). A small slit was made in the cornea and the entire eye was
removed from the optic cup before placing into 10% neutral buffered
formalin. After 1 hour fixation in formalin, eyes were briefly
rinsed in PBS and then placed in a dish of PBS for dissection.
Retinas were carefully dissected and cut into a clover leaf
formation prior to mounting onto a glass slide with Vectashield
(Vector Labs). Images of flatmounts were examined and acquired
using fluorescence microscopy (Nikon) with attached digital camera
system.
[0573] Results: Retina vasculature in untreated pups (FIG. 13a) and
those treated with 0.3 mg/kg of MEDI1/5 (FIG. 13b) extend to the
outer edge of retina, indicated with white arrow. Upon treatment
with increasing dose of MEDI1/5, we see a dose response in the
level of inhibition of advancing retina vessels (FIGS. 13c and
13d). The outer rim of retina is demarcated with dotted line and at
both doses the hyloid vessels (white arrow) reach the outer rim of
the retina but the retina vessels (white arrowheads) in 1 mg/kg
MEDI1/5 (FIG. 13c) approach closer to the periphery of the retina
in comparison to the 10 mg/kg MEDI1/5 (FIG. 13d) treated group.
These results demonstrate a dose-dependent inhibition of retinal
angiogenesis by the treatment of MEDI1/5 anti-Ang-2 antibodies.
Example 10
Anti-Ang-2 Antibodies Inhibit FGF-Mediated Angiogenesis
[0574] The anti-Ang-2 antibody, MEDI1/5 was evaluated for
anti-angiogenic effects in a FGF2 (basic FGF)-induced Matrigel.TM.
plug assay. Recombinant murine FGF2 (rmFGF basic; R&D Systems)
was pre-mixed with Matrigel.TM. (reduced growth factor, phenol
red-free; Trevigen) at 1 .mu.g/ml. Each 5-6 week old female athymic
mouse was subcutaneously implanted with 500 .mu.l of
FGF2/Matrigel.TM. mixture. Antibody MEDI1/5 was administered 10
minutes prior to FGF2/Matrigel.TM. implantation and continued every
three days given intraperitoneal at 1, 10, and 20 mg/kg for a total
of 3 doses. The extent of angiogenesis was assessed after 10 days
by measuring dextran functioning vessels. Mice were intervenously
injected with 100 .mu.l FITC-Dextran (250,000 MW; Sigma) at 25
mg/ml in saline. Twenty minutes post FITC-Dextran injection, mice
are humanely euthanized and plugs dissected out. Plugs are then
placed in lysing matrix tubes A (MP Biomedicals) containing 1 ml of
PBS and homogenized on FastPrep machine (MP Biomedicals) for 60
seconds at 6.0M/S. Samples were then centrifuged at 10,000 rpm for
5 minutes and supernatant collected. 200 .mu.A of each sample (in
duplicates) was then placed into a white, clear bottom 96 well
plate and FITC output was read on EnVision instrument (Perkin
Elmer).
[0575] For histology purposes, plugs were harvested and placed into
10% neutral-buffered formalin, processed and then paraffin
embedded. The paraffin embedded tissues were then sectioned and
stained with hematoxylin and eosin.
[0576] Results: Approximately 0.78 .mu.g of FITC-dextran could be
detected in plugs induced with 1 .mu.g/ml of FGF2 (FIG. 14a). On
the other hand, when these FGF2-treated plugs were exposed to 3
doses of MEDI1/5, ranging from 1, 10, and 20 mg/kg, the amount of
FITC-dextran present in plugs harvested ranged from 0.05-3 .mu.g.
This significant reduction in FITC-dextran in MEDI1/5 treated
animals suggests that MEDI1/5 inhibits FGF2-induced angiogenesis in
the Matrigel.TM. plugs. Plugs when stained with hematoxylin and
eosin (FIG. 14b) reveal that there are fewer vessels in
FGF2+MEDI1/5 plugs compared to FGF2 plugs alone, thus further
providing evidence that the anti Ang-2 antibody MEDI1/5 inhibits
FGF-mediated angiogenesis.
Example 11
Anti-Ang-2 Antibodies Inhibit Disease Progression in a Mouse Model
of Arthritis
[0577] In this Example, the Anti-Ang-2 antibody 3.19.3 was used in
a collagen-induced arthritis (CIA) mouse model in an attempt to
demonstrate therapeutic efficacy.
[0578] Materials and Methods: To study the effect of treatment on
clinical disease progression in the collagen-induced arthritis
(CIA) disease model collagen-induced arthritis was induced in male
DBA/1 mice and animals dosed therapeutically with test
treatments.
[0579] Preparation of rat collagen type II emulsion: Bovine
Collagen Type II (MD Biosciences) was stored at 4.degree. C. in the
dark until use. Prior to immunization of animals, bovine collagen
was dissolved in 0.01 M acetic acid at a stock solution of 2 mg/mL
and stored overnight in the dark at 4.degree. C. On the day of
immunization, collagen was emulsified with equal volume of Freund's
Complete Adjuvant (FCA (Difco)) to give a solution at 1 mg/mL.
[0580] Induction of arthritis: On day 0, male DBA/1 mice (6-8 weeks
old, Harlan Sprague Dawley, UK) were lightly anaesthetized with
3.5% isoflurane and immunized intra-dermal just above the root of
the tail with 100 .mu.g rat collagen type II emulsified in FCA (1
mg/mL; 0.1 mL/mouse).
[0581] Staphylococcal Enterotoxin B (SEB) booster: On day 21, all
mice were anaesthetised as before and given a booster injection of
30 .mu.g SEB (600 .mu.g/ml in water for injections (Toxin
Technology) emulsified in an equal volume of Freund's Incomplete
Adjuvant (Sigma) to give a final concentration of 300 .mu.g/ml). 50
.mu.l.times.2 (equivalent to 30 .mu.g SEB) was injected intradermal
adjacent to the immunization site.
[0582] Assessment of arthritis: Clinical observations regarding
welfare of the animals were carried out daily at time of dosing.
Observations for clinical signs of disease were carried out daily
from day 20 post immunization, whereby the animals were removed
from their micro-environment, and scored using the scoring system
outlined below.
TABLE-US-00011 TABLE 10 Scoring system for hind and forepaws Score
Description 0 Normal 1 Erythema and slight swelling of one of more
toes 2 Erythema and obvious swelling of two or more toes, or mild
swelling of the ankle or wrist without toe involvement 3 Erythema
and obvious swelling to some toes and ankle or fore paw and wrist 4
Erythema and severe swelling of ankle and digits
[0583] Dosing:
[0584] Animals were randomly assigned to treatment groups, as
outlined in Table 11 below.
TABLE-US-00012 TABLE 11 Treatment groups Group Treatment No. of
animals 1 PBS Vehicle i.p. 20 2 0.1 mg/kg Antibody 3.19.3 20 3 1
mg/kg 3.19.3 i.p. 20 4 10 mg/kg 3.19.3 i.p. 20 5 Human IgG control
i.p. 20 6 Prednisolone 3 mg/kg p.o. 10
[0585] Animals in treatment groups 1-5 were therapeutically dosed
intraperitoneally (i.p.) with 10 mL/kg every 3 days for 14 days
from disease onset (defined as a clinical score of 2 in one or more
paws). Purified human IgG (hIgG) was used as a negative isotype
control. Animals in treatment group 6 were therapeutically dosed
orally (p.o.) with Prednisolone 3 mg/kg daily for 14 days from
disease onset (defined as a clinical score of 2 in one or more
paws).
[0586] Termination: Animals were terminated 14 days post disease
onset by exposure to rising concentration of carbon dioxide. Mouse
paws were excised post mortem, fixed in 10% buffered formalin and
decalcified. Decalcified paws were routinely processed and then
embedded in paraffin blocks. Serial sections were cut and stained
with hematoxylin and eosin for histologic analysis, as well as CD31
staining for quantitation of microvessel density in the
synovium.
[0587] Results: Clinical Signs of Disease (Arthritis Score)
[0588] FIG. 15A shows the arthritic score mean (+/-standard error
of the mean) against days from disease onset (i.e. days of
treatment) (open squares=PBS, open triangles=isotype control,
closed squares=0.1 mg/kg 3.19.3, closed triangles=1 mg/kg 3.19.3,
closed circles=10 mg/kg 3.19.3 and open circles=prednisolone).
Dose-dependent reductions in clinical signs of disease progression
(arthritic score) and were observed. There was a significant
reduction at doses of 1 and 10 mg/kg. Area under the curve (AUC)
for clinical disease progression was calculated for each animal
from disease onset (FIG. 15B), Table 12. Unless otherwise stated,
statistical analysis was by one-way ANOVA with Dunnett's post-hoc
comparison to vehicle controls. P<0.05 was considered to be
statistically significant throughout the study.
TABLE-US-00013 TABLE 12 AUC of Clinical Score of CIA mice treated
with 3.19.3 Treatment AUC % inhibition PBS Vehicle ip 118 +/- 10
Human IgG 10 mg/kg ip 102 +/- 13 12% +/- 11% 3.19.3 0.1 mg/kg ip 99
+/- 15 16% +/- 13% 3.19.3 1 mg/kg ip 46 +/- 9 53% +/- 7%* 3.19.3 10
mg/kg ip 67 +/- 11 44% +/- 9%* Prednisolone 3 mg/kg po 41 +/- 08
78% +/- 7%* *= P < 0.05 ANOVA - one way, post hoc Dunnets v.
vehicle
[0589] Histolopathological evaluation of CIA model treatment
groups: Results showed evidence of a dose-dependent anti-arthritic
effect following administration of 3.19.3 on all parameters
evaluated including synovial hyperplasmia (FIG. 15 C), synovitis
(FIG. 15 D), pannus (FIG. 15 E), synovial fibrosis (FIG. 15F), and
periostitis (FIG. 15G). Histologically, there were no significant
differences between the isotype control-treated group and the PBS
vehicle group (FIGS. 15C-G).
[0590] Immunohistological evaluation of CD31+ (microvessel density)
in synovium: Results showed significant reduction in microvessel
density in the synovium at doses of 1 and 10 mg/kg as well as with
prednisolone. There was no effect with 0.1 mg/kg 3.19.3 treatment
(FIG. 15H).
[0591] Overall Conclusions: This study demonstrates that
neutralization of angiopoietin-2 is efficacious in ameliorating
collagen-induced arthritis in male DBA/1 mice. This study assessed
efficacy of 3.19.3 at a dose of 0.1, 1 and 10 mg/kg, and
demonstrated dose-dependent reductions in both clinical signs of
disease progression (arthritic score) and histological assessment
of synovitis and joint destruction, as well as microvessel density
in the synovium. The prednisolone-treated group showed a marked
reduction in the incidence and severity of all measures.
Example 12
Anti-Ang-2 Antibodies+Anti-TNF.alpha. Agents in Combination are
Effective in a Prophylactic Model of Arthritis
[0592] In the Example, the anti-Ang-2 antibody MEDI1/5 as a
standalone agent or in combination with the anti-TNF.alpha. agent
ENBREL.RTM. was studied in a prophylactic model of arthritis. More
specifically, the effect the MEDI1/5 antibody+/-ENBREL.RTM. on the
clinical disease progression in the glucose 6 phosphate isomerase
(G6PI) arthritis disease model.
[0593] Materials and Methods
[0594] Preparation of G6PI emulsion: On the day of immunization,
FCA (Freund's complete adjuvant) was prepared by grinding M.
tuberculosis (Difco) into IFA (Chondrex) to make a 1 mg/mL stock.
Human G6PI was set to a concentration of 3 mg/mL in phosphate
buffered saline (Gibco). G6PI was emulsified, by sonication, with
equal volume of FCA to give an emulsion of 1.5 mg/mL.
[0595] Induction of arthritis: On day 0, male DBA/1J mice (9-10
week old, Jackson Laboratories) were administered 0.2 mL (300 .mu.g
G6PI) over two sites at the base of the tail via subcutaneous
injections.
[0596] Assessment of arthritis: Observations for clinical signs of
disease were carried out daily from day 0 post immunization,
whereby the animals were removed from their micro-environment, and
scored using the scoring system outlined in Table 13 below.
TABLE-US-00014 TABLE 13 Scoring system for hind and forepaws Score
Description 0 Normal 0.5 Eruthema and slight swelling 1 Swelling of
digits only 1.5 Local mild/moderate swelling 2 Severe local
swelling of major joint 2.5 Severe local swelling of major joint
and other 3 Severe swelling of entire paw 3.5 Severe swelling and
partial or total ankylosis
[0597] Dosing: Animals were randomly assigned to treatment groups,
as outlined in Table 14 below.
TABLE-US-00015 TABLE 14 Treatment groups Group Treatment No. of
animals 1 10 mg/kg Human IgG1 control antibody i.p. 8 2 10 mg/kg
MEDI1/5 i.p. 8 3 1 mg/kg ENBREL .RTM. i.p. 8 4 4 mg/kg ENBREL .RTM.
i.p. 8 5 10 mg/kg MEDI1/5 i.p + 1 mg/kg ENBREL .RTM. 8 i.p. 6 10
mg/kg MEDI1/5 i.p + 1 mg/kg ENBREL .RTM. 8 i.p.
[0598] Animals in the enbrel treatment group were dosed daily with
10 mL/kg from days 0-9, then every 3 days until termination on day
16. All other treatments were administered every 3 days.
[0599] Termination: Animals were terminated 16 days post
immunization by exposure to rising concentration of carbon dioxide.
Mouse paws were excised post mortem, fixed in 10% buffered
formalin, assessed for bone mineral density and then decalcified.
Decalcified paws were routinely processed and then embedded in
paraffin blocks. Serial sections were cut and stained with
hematoxylin and eosin for histologic analysis. The histological
analysis was scored by the following schedule: [0600] Total joint
scores [0601] Both hind knee and ankle joints scored for: [0602]
> Inflammation [0603] > Bone damage [0604] > Pannus
formation [0605] > Cartilage damage [0606] Score of 0-5 [0607]
> 0=normal [0608] > 1=minimal [0609] > 2=mild [0610] >
3=moderate [0611] > 4=marked [0612] > 5=severe
[0613] Bone Mineral Density
[0614] Bone mineral density was assessed in the stifle joint of the
hind limbs using DEXA imaging (GE Piximus). Following collection,
hind limbs were placed in 10% neutral buffered saline for 4 days.
Just prior to imaging, limbs were placed in 70% ethanol, then
allowed to dry.
[0615] Results:
[0616] Reductions in clinical signs of disease progression
(arthritic score) (FIGS. 16A (closed circles=isotype control,
closed diamond=10 mg/kg MEDI1/5, open diamond 1 mg/kg etanercept,
grey diamond=combination of 10 mg/kg MEDI1/5 with 1 mg/kg
etanercept, open square=4 mg/kg enbrel, grey square=combination of
10 mg/kg MEDI1/5 with 4 mg/kg etanercept) and 16B) were observed
with either etanercept or MEDI1/5 treatment. There was a further
reduction in clincal score when MEDI1/5 was administered in
combination with the lower dose of etanercept (One-Way ANOVA with
Dunnett's post-hoc comparison to isotype control). All joints in
the isotype control antibody treated animals showed evidence of
disease, whereas MEDI1/5 or 1 mg/kg etanercept treatments resulted
in 31% or 22% of the joints showing no signs of disease,
respectively. When the 10 mg/kg MEDI1/5 and 1 mg/kg etanercept were
administered in combination, 50% of the joints of the animals in
this group were disease free, comparable to the level of protection
provided by the high dose (4 mg/kg) of enbrel (53%) (Table 15).
Histological assessment of synovitis and joint destruction (FIG.
16C) supported the clinical score results as did the protection
from loss of bone mineral density (FIG. 16D).
TABLE-US-00016 TABLE 15 Disease free joints observed in MEDI1/5
treated arthritic mice Treatment % Disease free joints 10 mg/ml
Isotype control 0% 10 mg/kg MEDI1/5 31% 1 mg/kg etanercept 22% 10
mg/kg MEDI1/5 + 1 mg/kg etanercept 50% 4 mg/kg etanercept 53% 10
mg/kg MEDI1/5 + 1 mg/kg etanercept 56%
[0617] Conclusions:
[0618] This study demonstrates that neutralization of
angiopoietin-2 is efficacious in inhibiting the development of
G6PI-induced arthritis in male DBA/1J mice comparable to the
anti-TNF comparator, etanercept. This study also assessed efficacy
of MEDI1/5 combined with etanercept and demonstrated that
combination treatment with the lower dose of enbrel provides
further efficacy in both clinical signs of disease progression
(arthritic score) and histological assessment of synovitis and
joint destruction, as well as loss of bone mineral density, when
administered prior to onset of disease.
Example 13
Anti-Ang-2 Antibodies+Anti-TNF.alpha. Agents in Combination are
Effective in a Therapeutic Model of Arthritis
[0619] The effect of treatment with MEDI 1/5 with or without
etanercept was evaluated following onset of clinical disease in the
glucose 6 phosphate isomerase (G6PI) arthritis disease model.
G6PI-induced arthritis was induced in male DBA/1J mice and animals
dosed therapeutically with test treatments.
[0620] Materials and Methods: The mice in this study were prepared
similarly as presented in Example 12.
[0621] Dosing: This was a rolling admissions study: once an animal
reached a clinical score of 3.5 to 5.0, it was randomly assigned to
treatment groups (as outlined below) and dosing began. The day of
initial dosing became study day 0 for that animal. Treatment groups
were similar to those presented in Example 12.
[0622] Termination: Study was terminated 12 days post treatment
initiation. No further endpoints were assessed on these
animals.
[0623] Results: When administered in a therpeutic approach
following the onset of clinical disease, modest reductions in
clinical signs of disease progression (arthritic score) were
observed with MEDI1/5 treatment, while both doses of etanercept
tested had no effect on disease progression. There was a more
dramatic inhibition of progression of disease when MEDI1/5 (10
mg/kg) was administered in combination with the higher dose of
etanercept (4 mg/kg) (FIG. 17A (closed circles=isotype control,
closed diamond=MEDI1/5, open diamond=1 mg/kg etanercept, grey
diamond=combination of MEDI1/5 with 1 mg/kg etanercept, open
square=4 mg/kg etanercept, grey square=combination of MEDI1/5 with
4 mg/kg etanercept).
[0624] Conclusions: This study demonstrates that neutralization of
angiopoietin-2 is efficacious in ameliorating G6PI-induced
arthritis in male DBA/1 mice comparable to the anti-TNF comparator,
etanercept, when administered following the onset of clinical signs
of disease. This study also assessed efficacy of MEDI1/5 combined
with etanercept and demonstrated that combination treatment
provides enhanced efficacy over either agent alone.
Sequence CWU 1
1
81215PRTArtificialsynthetic construct 1Glu Ile Val Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser 20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile
Cys Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser
Ser Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185
190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
2123PRTArtificialSynthetic construct 2Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30 Gly Met His
Trp Gly Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn
Trp Phe Asp Pro 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 3215PRTArtificialSynthetic construct 3Glu Ile Val Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser 20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Thr Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe
Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Ser Ser Ser Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu
Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
4215PRTArtificialSynthetic construct 4Glu Ile Val Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser 20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile
Asn Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser
Ser Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185
190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
5215PRTArtificialSynthetic construct 5Glu Ile Val Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser 20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile
Asp Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser
Ser Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185
190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
6215PRTArtificialSynthetic construct 6Glu Ile Val Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser 20 25 30 Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile
Ala Gly Ala Ser Ser Trp Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu
65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser
Ser Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys
Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185
190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
7123PRTArtificialSynthetic construct 7Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Thr Asn Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Val Ile Ser His Asp Gly Asn Asn Lys Tyr Tyr Val Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Ile Asp Phe Trp Ser Gly Leu Asn
Trp Phe Asp Pro 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 8215PRTArtificialSynthetic construct 8Glu Ile Val Leu
Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Thr Gly Ser 20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35
40 45 Ile Thr Gly Ala Ser Ser Trp Ala Thr Gly Ile Ala Asp Arg Phe
Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Ser Ser Ser Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu
Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165
170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215
* * * * *