U.S. patent application number 15/558957 was filed with the patent office on 2018-08-30 for modulation of tumor immunity by protein-mediated 02 delivery.
The applicant listed for this patent is OMNIOX, INC.. Invention is credited to Stephen P. L. CARY, Ana KRTOLICA, Natacha LE MOAN, Kevin G. LEONG, Jonathan A. WINGER.
Application Number | 20180243364 15/558957 |
Document ID | / |
Family ID | 56297072 |
Filed Date | 2018-08-30 |
United States Patent
Application |
20180243364 |
Kind Code |
A1 |
CARY; Stephen P. L. ; et
al. |
August 30, 2018 |
MODULATION OF TUMOR IMMUNITY BY PROTEIN-MEDIATED 02 DELIVERY
Abstract
The invention provides methods to modulate hypoxia-mediated
tumor immunity by administration of an O.sub.2 carrier polypeptide
(e.g., an H-NOX protein). The methods of the invention target both
hypoxia inducible factor 1 alpha (HIF-1.alpha.) pathways and
non-HIF-1.alpha. pathways of tumor immunity. Such methods are
useful in the treatment of a wide variety of cancers and may be
used alone or in combination with other anti-cancer therapies.
Inventors: |
CARY; Stephen P. L.; (San
Mateo, CA) ; KRTOLICA; Ana; (San Francisco, CA)
; LE MOAN; Natacha; (San Francisco, CA) ; WINGER;
Jonathan A.; (Oakland, CA) ; LEONG; Kevin G.;
(Millbrae, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OMNIOX, INC. |
San Carlos |
CA |
US |
|
|
Family ID: |
56297072 |
Appl. No.: |
15/558957 |
Filed: |
March 17, 2016 |
PCT Filed: |
March 17, 2016 |
PCT NO: |
PCT/US2016/022981 |
371 Date: |
September 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62134523 |
Mar 17, 2015 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61N 5/10 20130101; A61N
2005/1098 20130101; A61K 39/39 20130101; A61K 47/60 20170801; A61K
38/164 20130101; A61P 35/00 20180101; A61K 45/06 20130101 |
International
Class: |
A61K 38/16 20060101
A61K038/16; A61K 45/06 20060101 A61K045/06; A61K 47/60 20060101
A61K047/60; A61P 35/00 20060101 A61P035/00; A61K 39/39 20060101
A61K039/39 |
Claims
1. A method for treating cancer in an individual comprising
administering to the individual an effective amount of an O.sub.2
carrier polypeptide.
2. The method of claim 1, wherein the cancer is brain cancer,
glioblastoma, bone cancer, pancreatic cancer, skin cancer, cancer
of the head or neck, melanoma, lung cancer, uterine cancer, ovarian
cancer, colorectal cancer, anal cancer, liver cancer,
hepatocellular carcinoma, stomach cancer, testicular cancer,
endometrial cancer, cervical cancer, Hodgkin's Disease,
non-Hodgkin's lymphoma, esophageal cancer, intestinal cancer,
thyroid cancer, adrenal cancer, bladder cancer, kidney cancer,
breast cancer, multiple myeloma, sarcoma, or squamous cell
cancer.
3. A method for modulating tumor immunity in an individual with a
tumor comprising administering to the individual an effective
amount of an O.sub.2 carrier polypeptide.
4. The method of claim 3, wherein the modulating tumor immunity
comprises enhancing an immune response to the tumor.
5. A method for increasing lymphocyte infiltration to a tumor in an
individual comprising administering to the individual an effective
amount of an O.sub.2 carrier polypeptide.
6. The method of claim 5, wherein the increase in lymphocyte
infiltration to the tumor comprises an increase in infiltration of
one or more of CD4 cells, CD8 cells, or NK cells.
7. The method of claim 5 or 6, wherein the increase in lymphocyte
infiltration to the tumor is accompanied by inhibition of one or
more of Treg cells, tumor associated macrophages or myeloid derived
suppressor cells in the tumor.
8. The method of any one of claims 5-7, wherein the increase in
lymphocyte infiltration to the tumor is accompanied by an increase
in MHC1 expression on the tumor cells.
9. A method for decreasing expression of hypoxia inducible factor
1.alpha. (HIF-1.alpha.) and/or 2.alpha. (HIF-2.alpha.) in a tumor
in an individual comprising administering to the individual an
effective amount of an O.sub.2 carrier polypeptide.
10. A method for decreasing expression of programmed death ligand-1
(PD-L1) in a tumor in an individual comprising administering to the
individual an effective amount of an O.sub.2 carrier
polypeptide.
11. A method for decreasing expression of A2A adenosine receptor
(A2AR) in a tumor in an individual comprising administering to the
individual an effective amount of an O.sub.2 carrier
polypeptide.
12. The method of any one of claims 3-11, wherein the tumor is a
brain tumor, a glioblastoma, a bone tumor, a pancreatic tumor, a
skin tumor, a tumor of the head or neck, a melanoma, a lung tumor,
a uterine tumor, an ovarian tumor, a colorectal tumor, an anal
tumor, a liver tumor, a hepatocellular carcinoma, a stomach tumor,
a testicular tumor, an endometrial tumor, a cervical tumor, a
vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an
esophageal tumor, an intestinal tumor, a thyroid tumor, an adrenal
tumor, a bladder tumor, a kidney tumor, breast tumor, a multiple
myeloma tumor, a sarcoma, or a squamous cell tumor.
13. The method of any one of claims 1-12, wherein the individual is
a mammal.
14. The method of claim 13, wherein the mammal is a human.
15. The method of claim 14, wherein the mammal is a pet, a
laboratory research animal, or a farm animal.
16. The method of claim 15, wherein the pet, research animal or
farm animal is a dog, a cat, a horse, a monkey, a rabbit, a rat, a
mouse, a guinea pig, a hamster, a pig, or a cow.
17. The method of any one of claims 1-16, wherein the O.sub.2
carrier polypeptide is administered by intravenous, intra-arterial,
intratumoral, intravesicular, inhalation, intraperitoneal,
intrapulmonary, intramuscular, subcutaneous, intra-tracheal,
transmucosal, intraocular, intrathecal, or transdermal
administration.
18. The method of any one of claims 1-17, wherein administration of
the O.sub.2 carrier polypeptide is repeated.
19. The method of claim 18, wherein administration of the O.sub.2
carrier polypeptide is repeated daily, twice a day or about 1-4
times a week from about 4 weeks to about 8 weeks.
20. The method of claim 18 or 19 wherein the O.sub.2 carrier
polypeptide is administered every four, every 8, every 12 or every
24 hours for a period of about one to about 10 days.
21. The method of any one of claims 1-20, wherein the O.sub.2
carrier polypeptide is administered as a bolus.
22. The method of any one of claims 1-20, wherein the O.sub.2
carrier polypeptide is administered by infusion.
23. The method of claim 22, wherein the 02 carrier polypeptide is
infused in the individual for about 15 minutes, about 30 minutes,
about 1 hour, about 2 hours, about 3 hours, about 6 hours, about 12
hours or about 24 hours.
24. The method of any one of claims 1-23, wherein the O.sub.2
carrier polypeptide is administered in combination with radiation
therapy.
25. The method of claim 24, wherein the radiation therapy is
administered to the individual 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16,
18, 20 or 24 hours after the O.sub.2 carrier polypeptide is
administered.
26. The method of claim 24 or 25, wherein the radiation is
X-radiation.
27. The method of claim 26, wherein the X-radiation is administered
at about 0.5 gray to about 75 gray.
28. The method of any one of claims 24-27, wherein the
administration of the O.sub.2 carrier polypeptide and/or the
administration of the radiation is repeated.
29. The method of claim 28, wherein the administration is repeated
any number of times between about two times to about forty times or
more.
30. The method of claim 28 or 29, wherein the administration is
repeated after one week, two weeks, three weeks, or four weeks or
more.
31. The method of any one of claims 1-23, wherein the O.sub.2
carrier polypeptide is administered in combination with
chemotherapy or immunotherapy.
32. The method of claim 31, wherein the chemotherapy comprises a
cytotoxin.
33. The method of claim 32, wherein the administration of the
O.sub.2 carrier polypeptide and/or the administration of the
chemotherapy is repeated.
34. The method of claim 31, wherein the immunotherapy is one or
more of an anticancer vaccine, an adoptive immune cell therapy or
an agent that targets an immune checkpoint regulator.
35. The method of claim 31 or 34, wherein the immunotherapy targets
one or more of CTLA-4, PD1, PD-L1, or an immune checkpoint
regulator.
36. The method of claim 31 or 34, wherein the adoptive immude
therapy is a chimeric antigen receptor expressing T cell or an
engineered TCR-T cell.
37. The method of any one of claims 31 or 34-36, wherein the
administration of the O.sub.2 carrier polypeptide and/or the
administration of the immunotherapy is repeated.
38. The method of any one of claims 1-37, wherein the O.sub.2
carrier polypeptide is in a pharmaceutical composition.
39. The method of claim 38, wherein the pharmaceutical composition
further comprises a pharmaceutically acceptable carrier.
40. The method of any one of claims 1-39 wherein the O.sub.2
carrier polypeptide is an H-NOX protein.
41. A method for treating cancer in an individual comprising
administering to the individual an effective amount of an H-NOX
protein.
42. The method of claim 41, wherein the cancer is brain cancer,
glioblastoma, bone cancer, pancreatic cancer, skin cancer, cancer
of the head or neck, melanoma, lung cancer, uterine cancer, ovarian
cancer, colorectal cancer, anal cancer, liver cancer,
hepatocellular carcinoma, stomach cancer, testicular cancer,
endometrial cancer, cervical cancer, Hodgkin's Disease,
non-Hodgkin's lymphoma, esophageal cancer, intestinal cancer,
thyroid cancer, adrenal cancer, bladder cancer, kidney cancer,
breast cancer, multiple myeloma, sarcoma, or squamous cell
cancer.
43. A method for modulating tumor immunity in an individual with a
tumor comprising administering to the individual an effective
amount of an H-NOX protein.
44. The method of claim 43, wherein the modulating tumor immunity
comprises enhancing an immune response to the tumor.
45. A method for increasing lymphocyte infiltration to a tumor in
an individual comprising administering to the individual an
effective amount of an H-NOX protein.
46. The method of claim 45, wherein the increase in lymphocyte
infiltration to the tumor comprises an increase in infiltration of
one or more of CD4 cells, CD8 cells, or NK cells.
47. The method of claim 45 or 46, wherein the increase in
lymphocyte infiltration to the tumor is accompanied by inhibition
of one or more of Treg cells, tumor associated macrophages or
myeloid derived suppressor cells in the tumor.
48. The method of any one of claims 45-47, wherein the increase in
lymphocyte infiltration to the tumor is accompanied by an increase
in MHC1 expression on the tumor cells.
49. A method for decreasing expression of HIF-1.alpha. and/or
HIF-2.alpha. in a tumor in an individual comprising administering
to the individual an effective amount of an H-NOX protein.
50. A method for decreasing expression of PD-L1 in a tumor in an
individual comprising administering to the individual an effective
amount of an H-NOX protein.
51. A method for decreasing expression of A2AR in a tumor in an
individual comprising administering to the individual an effective
amount of an H-NOX protein.
52. The method of any one of claims 41-51, wherein the tumor is a
brain tumor, a glioblastoma, a bone tumor, a pancreatic tumor, a
skin tumor, a tumor of the head or neck, a melanoma, a lung tumor,
a uterine tumor, an ovarian tumor, a colorectal tumor, an anal
tumor, a liver tumor, a hepatocellular carcinoma, a stomach tumor,
a testicular tumor, an endometrial tumor, a cervical tumor, a
vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an
esophageal tumor, an intestinal tumor, a thyroid tumor, an adrenal
tumor, a bladder tumor, a kidney tumor, a breast tumor, a multiple
myeloma tumor, a sarcoma, or a squamous cell tumor.
53. The method of any one of claims 41-52, wherein the individual
is a mammal.
54. The method of claim 53, wherein the mammal is a human.
55. The method of claim 52, wherein the mammal is a pet, a
laboratory research animal, or a farm animal.
56. The method of claim 55, wherein the pet, research animal or
farm animal is a dog, a cat, a horse, a monkey, a rabbit, a rat, a
mouse, a guinea pig, a hamster, a pig, or a cow.
57. The method of any one of claims 41-56, wherein the H-NOX
protein is administered by intravenous, intra-arterial,
intratumoral, intravesicular, inhalation, intraperitoneal,
intrapulmonary, intramuscular, subcutaneous, intra-tracheal,
transmucosal, intraocular, intrathecal, or transdermal
administration.
58. The method of any one of claims 41-57, wherein administration
of the H-NOX protein is repeated.
59. The method of claim 58, wherein administration of the H-NOX
protein is repeated daily or twice a day from about 4 weeks to
about 8 weeks.
60. The method of claim 58 or 59 wherein the H-NOX protein is
administered every four, every 8, every 12 or every 24 hours for a
period of about one to about 10 days.
61. The method of any one of claims 41-60, wherein the H-NOX
protein is administered as a bolus.
62. The method of any one of claims 41-60, wherein the H-NOX
protein is administered by infusion.
63. The method of claim 62, wherein the H-NOX protein is infused in
the individual for about 15 minutes, about 30 minutes, about 1
hour, about 1 hour, about 2 hours, about 3 hours, about 6 hours,
about 12 hours or about 24 hours.
64. The method of any one of claims 41-63, wherein the H-NOX
protein is administered in combination with radiation therapy.
65. The method of claim 64, wherein the radiation therapy is
administered to the individual 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16,
18, 20, 22 or 24 hours after the H-NOX protein is administered.
66. The method of claim 64 or 65, wherein the radiation is
X-radiation.
67. The method of claim 66, wherein the X-radiation is administered
at about 0.5 gray to about 75 gray.
68. The method of any one of claims 64-67, wherein the
administration of the H-NOX protein and/or the administration of
the radiation is repeated.
69. The method of claim 68, wherein the administration is repeated
any number of times between about two times to about forty times or
more.
70. The method of claim 68 or 69, wherein the administration is
repeated after one week, two weeks, three weeks, or four weeks or
more.
71. The method of any one of claims 41-63, wherein the H-NOX
protein is administered in combination with chemotherapy or
immunotherapy.
72. The method of claim 71, wherein the chemotherapy comprises a
cytotoxin.
73. The method of claim 72, wherein the administration of the H-NOX
protein and/or the administration of the chemotherapy is
repeated.
74. The method of claim 71, wherein the immunotherapy is one or
more of an anticancer vaccine, an adoptive immune cell therapy or
an agent that targets an immune checkpoint regulator.
75. The method of claim 71 or 74, wherein the immunotherapy targets
one or more of CTLA-4, PD1, PD-L1, or an immune checkpoint
regulator.
76. The method of claim 71, 74 or 75, wherein the adoptive immude
therapy is a chimeric antigen receptor expressing T cell or an
engineered TCR-T cell.
77. The method of any one of claims 71, or 74-76, wherein the
administration of the H-NOX protein and/or the administration of
the immunotherapy is repeated.
78. The method of any one of claims 41-77, wherein the H-NOX
protein is a T. tengcongensis H-NOX, a L. pneumophilia 2 H-NOX, a
H. sapiens .beta.1, a R. norvegicus .beta.1, a C. lupus H-NOX, a D.
melangaster .beta.1, a D. melangaster CG14885-PA, a C. elegans
GCY-35, a N. punctiforme H-NOX, C. crescentus H-NOX, a S.
oneidensis H-NOX, or C. acetobutylicum H-NOX.
79. The method of any one of claims 41-77, wherein the H-NOX
protein comprises a H-NOX domain corresponding to the H-NOX domain
of T. tengcongensis set forth in SEQ ID NO:2.
80. The method of any one of claims 41-78, wherein the H-NOX
comprises one or more distal pocket mutations.
81. The method of claim 80, wherein the distal pocket mutation is
an amino acid substitution at a site corresponding to L144 of T.
tengcongensis H-NOX.
82. The method of claim 80 or 81, wherein the H-NOX is a T.
tengcongensis H-NOX comprising an amino acid substitution at
position 144.
83. The method of claim 82, wherein the amino acid substitution at
position 144 is an L144F substitution.
84. The method of any one of claims 41-83, wherein the H-NOX
protein is a polymeric H-NOX protein.
85. The method of claim 84, wherein the polymeric H-NOX protein
comprises monomers, wherein the monomers comprise an H-NOX domain
and a polymerization domain.
86. The method of claim 85, wherein the H-NOX domain is covalently
linked to the polymerization domain.
87. The method of any one of claims 84-86, wherein the polymeric
H-NOX protein is a trimeric H-NOX protein.
88. The method of claim 87, wherein the trimeric H-NOX protein
comprises one or more trimerization domains.
89. The method of claim 88, wherein the trimeric H-NOX protein
comprises three monomers, wherein the monomers comprise an H-NOX
domain and a trimerization domain, wherein the trimerization domain
is a bacteriophage T4 trimerization domain.
90. The method of claim 88 or 89, wherein the trimerization domain
is a foldon domain.
91. The method of claim 90, wherein the foldon domain comprises the
amino acid sequence of SEQ ID NO:4.
92. The method of any one of claims 41-91, wherein the H-NOX
protein is fused to an Fc domain of an immunoglobulin.
93. The method of any one of claims 41-92, wherein the H-NOX
protein is covalently bound to polyethylene glycol.
94. The method of any one of claims 41-93, wherein the O.sub.2
dissociation constant of the H-NOX protein is within 2 orders of
magnitude of that of hemoglobin, and wherein the NO reactivity of
the H-NOX protein is at least 10-fold lower than that of
hemoglobin.
95. The method of any one of claims 41-94, wherein the O.sub.2
dissociation constant of the polymeric H-NOX protein is between
about 1 nM and about 1000 nM at 20.degree. C.
96. The method of any one of claims 41-95, wherein the O.sub.2
dissociation constant of the H-NOX protein is between about 1 .mu.M
and about 10 .mu.M at 20.degree. C.
97. The method of any one of claims 41-96, wherein the Oz
dissociation constant of the H-NOX protein is between about 10
.mu.M and about 50 .mu.M at 20.degree. C.
98. The method of any one of claims 41-97, wherein the NO
reactivity of the H-NOX protein is less than about 700 s.sup.-1 at
20.degree. C.
99. The method of any one of claims 41-98, wherein the NO
reactivity of the H-NOX protein is at least 100-fold lower than
that of hemoglobin.
100. The method of claim 99, wherein the NO reactivity of the H-NOX
protein is at least 1,000-fold lower than that of hemoglobin.
101. The method of any one of claims 41-100, wherein the k.sub.off
for oxygen of the H-NOX protein is less than or equal to about 0.65
s.sup.-1 at 20.degree. C.
102. The method of any one of claims 41-101, wherein the k.sub.off
for oxygen of the H-NOX protein is between about 0.21 s.sup.-1 and
about 0.65 s.sup.-1 at 20.degree. C.
103. The method of any one of claims 41-102, wherein the k.sub.off
for oxygen of the H-NOX protein is between about 1.35 s.sup.-1 and
about 2.9 s.sup.-1 at 20.degree. C.
104. The method of any one of claims 41-103, wherein the rate of
heme autoxidation of the H-NOX protein is less than about 1
h.sup.-1 at 37.degree. C.
105. The method of any one of claims 41-104, wherein the H-NOX
protein is in a pharmaceutical composition.
106. The method of claim 105, wherein the pharmaceutical
composition further comprises a pharmaceutically acceptable
carrier.
107. Use of an O.sub.2 carrier protein for treating cancer in an
individual.
108. The use of claim 107, wherein the cancer is brain cancer,
glioblastoma, bone cancer, pancreatic cancer, skin cancer, cancer
of the head or neck, melanoma, lung cancer, uterine cancer, ovarian
cancer, colorectal cancer, anal cancer, liver cancer,
hepatocellular carcinoma, stomach cancer, testicular cancer,
endometrial cancer, cervical cancer, Hodgkin's Disease,
non-Hodgkin's lymphoma, esophageal cancer, intestinal cancer,
thyroid cancer, adrenal cancer, bladder cancer, kidney cancer,
breast cancer, multiple myeloma, sarcoma, or squamous cell
cancer.
109. Use of an O.sub.2 carrier protein for modulating tumor
immunity in an individual.
110. The use of claim 109, wherein the modulating tumor immunity
comprises enhancing an immune response to the tumor.
111. Use of an O.sub.2 carrier polypeptide for increasing
lymphocyte infiltration to a tumor in an individual.
112. The use of claim 111, wherein the increase in lymphocyte
infiltration to the tumor comprises an increase in infiltration of
one or more of CD4 cells, CD8 cells, or NK cells.
113. The use of claim 111 or 112, wherein the increase in
lymphocyte infiltration to the tumor is accompanied by inhibition
of one or more of Treg cells, tumor associated macrophages or
myeloid derived suppressor cells in the tumor.
114. The use of any one of claims 111-113, wherein the increase in
lymphocyte infiltration to the tumor is accompanied by an increase
in MHC1 expression on the tumor cells.
115. Use of an O.sub.2 carrier polypeptide for decreasing
expression of HIF-1.alpha. and/or HIF-2.alpha. in a tumor in an
individual.
116. Use of an O.sub.2 carrier polypeptide for decreasing
expression of PD-L1 in a tumor in an individual.
117. Use of an O.sub.2 carrier polypeptide for decreasing
expression of A2AR in a tumor in an individual.
118. The use of any one of claims 109-117, wherein the tumor is a
brain tumor, a glioblastoma, a bone tumor, a pancreatic tumor, a
skin tumor, a tumor of the head or neck, a melanoma, a lung tumor,
a uterine tumor, an ovarian tumor, a colorectal tumor, an anal
tumor, a liver tumor, a hepatocellular carcinoma, a stomach tumor,
a testicular tumor, an endometrial tumor, a cervical tumor, a
vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an
esophageal tumor, an intestinal tumor, a thyroid tumor, an adrenal
tumor, a bladder tumor, a kidney tumor, a breast tumor, a multiple
myeloma tumor, a sarcoma, or a squamous cell tumor.
119. The use of any one of claims 107-118, wherein the individual
is a mammal.
120. The use of claim 119, wherein the mammal is a human.
121. The use of any one of claims 107-120, wherein the O.sub.2
carrier polypeptide is an H-NOX protein.
122. The use of claim 121, wherein the H-NOX protein is a T.
tengcongensis H-NOX, a L. pneumophilia 2 H-NOX, a H. sapiens
.beta.1, a R. norvegicus .beta.1, a C. lupus H-NOX domain, a D.
melangaster .beta.1, a D. melangaster CG14885-PA, a C. elegans
GCY-35, a N. punctiforme H-NOX, C. crescentus H-NOX, a S.
oneidensis H-NOX, or C. acetobutylicum H-NOX.
123. The use of any one of claims 121-122, wherein the H-NOX
protein comprises a H-NOX domain corresponding to the H-NOX domain
of T. tengcongensis set forth in SEQ ID NO:2.
124. The use of any one of claims 121-123, wherein the H-NOX
comprises one or more distal pocket mutations.
125. The use of claim 124, wherein the distal pocket mutation is an
amino acid substitution at a site corresponding to L144 of T.
tengcongensis H-NOX.
126. The use of claim 124 or 125, wherein the H-NOX is a T.
tengcongensis H-NOX comprising an amino acid substitution at
position 144.
127. The use of claim 126, wherein the amino acid substitution at
position 144 is an L144F substitution.
128. The use of any one of claims 121-127, wherein the H-NOX
protein is a polymeric H-NOX protein.
129. The use of claim 128, wherein the polymeric H-NOX protein
comprises monomers, wherein the monomers comprise an H-NOX domain
and a polymerization domain.
130. The use of claim 129, wherein the H-NOX domain is covalently
linked to the polymerization domain.
131. The use of any one of claims 128-130, wherein the polymeric
H-NOX protein is a trimeric H-NOX protein.
132. The use of claim 131, wherein the trimeric H-NOX protein
comprises one or more trimerization domains.
133. The use of claim 132, wherein the trimeric H-NOX protein
comprises three monomers, wherein the monomers comprise an H-NOX
domain and a trimerization domain, wherein the trimerization domain
is a bacteriophage T4 trimerization domain.
134. The use of claim 132 or 133, wherein the trimerization domain
is a foldon domain.
135. The use of claim 134, wherein the foldon domain comprises the
amino acid sequence of SEQ ID NO:4.
136. The use of any one of claims 131-135, wherein the H-NOX
protein is fused to an Fc domain of an immnunoglobulin.
137. The use of any one of claims 121-136, wherein the H-NOX
protein is covalently bound to polyethylene glycol.
138. A kit for modulating tumor immunity in an individual
comprising an O.sub.2 carrier protein for use in the method of any
one of claims 1-106.
139. The kit of claim 138, wherein the kit further comprises one or
more of a vial, a vessel, an ampule, a bottle, a jars, or flexible
packaging.
140. The kit of claim 138 or 139, wherein the kit further comprises
one or more buffers.
141. The kit of any one of claims 138-140, wherein the kit further
comprises instructions for use.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 62/134,523, filed Mar. 17, 2015, the
disclosure of which is hereby incorporated by reference in its
entirety for all purposes.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
627042000940SeqList.txt, date recorded: Mar. 17, 2016, size: 41
KB).
TECHNICAL FIELD
[0003] This application pertains to the modulation of tumor
immunity by delivering oxygen to the tumor by way of a protein
O.sub.2 carrier polypeptide; for example an H-NOX protein.
BACKGROUND OF THE INVENTION
[0004] The hypoxic tumor microenvironment suppresses the host's
immune anti-tumor defenses by modulating multiple signaling
pathways including, but not limited to, hypoxia inducible factor
(HIF-1) signaling (Codo et al., 2014 Oncotarget, 5(17), 7651-7662;
Lee, Mace, & Repasky, 2010 Int J Hyperthermia, 26(3), 232-246;
Wei et al., 2011 PLoS One, 6(1), e16195). Major hypoxia
immunomodulating pathways are summarized in FIG. 1. Briefly, HIF-1
has been shown to: a) activate adenosinergic A2 and PD-L1 pathways
that inhibit recruitment and activation of helper and killer
T-cells and NK cells, key effectors of anti-tumor responses (Noman
et al., 2014 J Exp Med, 211(5), 781-790; Ohta et al., 2006 Proc
Natl Acad Sci USA, 103(35), 13132-13137); b) recruit and activate
inhibitory regulatory T cells (Treg), tumor associated macrophages
(TAM) and other myeloid-derived suppressor cells (MDSC) (Chaturvedi
et al., 2014 Proc Natl Acad Sci USA, 111(20), E2120-2129; Corzo et
al., 2010 J Exp Med, 207(11), 2439-2453; Wei et al., 2011); c)
directly inhibit the ability of tumor cells to be recognized by
immune system (Siemens et al., 2008 Cancer Res, 68(12), 4746-4753).
In addition, HIF-1-dependent and -independent epigenetic mechanisms
contribute to inhibition of anti-tumor immune-responses and enhance
tumor growth, angiogenesis and metastasis (Codo et al., 2014;
Mimura et al., 2011 J Pharmacol Sci, 115(4), 453-458).
[0005] In mouse metastatic tumor models, continuous supplemental
oxygenation has been shown to inhibit tumor growth and prevent
tumor's immune escape through inhibition of A2AR (A2A adenosine
receptor) adenosinergic pathway leading to T and NK cell activation
(Hatfield et al., 2015 Sci Transl Med, 7(277), 277ra230).
Specifically, continuous treatment with 60% respiratory oxygen of
mice bearing MCA205, B16 or 4T1 pulmonary metastases resulted in
>2-fold decrease in number of metastatic foci and enhanced
survival. These data correlated with decrease in tumor and
lymphocyte hypoxia, increased activated CD8 T cell (CD8+CD69+CD44+)
tumor infiltration, upregulation of immunostimulating cytokines and
chemokines and were dependent on intact A2AR signaling. At the same
time respiratory hyperoxia was shown to reduce the number and
suppressive the activity of Treg in pulmonary tumor
microenvironment (TME) due to reduced Foxp3, CD39/CD73 (adenosine
generating enzymes upstream of A2AR) and CTLA-4 expression.
Finally, tumor regression induced by dual CTLA-4/PD-1 blockade of
pulmonary tumors was enhanced by continuous respiratory
hyperoxia.
[0006] Despite convincing pre-clinical evidence demonstrating the
capacity of tumor oxygenation to reverse immunosuppressive TME and
inhibit tumor growth, in human clinical trials supplemental
oxygenation using hyperbaric or normobaric oxygen yielded limited
effects (Overgaard, 2007 J Clin Oncol, 25(26), 4066-4074). This is
likely due to the inability of soluble oxygen to effectively
diffuse beyond .about.80 .mu.m from blood vessels, limiting its
penetration deep into hypoxic tumor tissue. Therefore, the need
exists for oxygen delivery agents that penetrate into patients'
tumors to transport oxygen beyond the normal diffusion limits, and
thereby oxygenate hypoxic microenvironments to impede
immunosuppressive pathways. This will result in maximal stimulation
of anti-tumor immune responses, both alone and in combination with
other immune checkpoint inhibitors and other cancer immunotherapy
approaches.
[0007] H-NOX proteins (named for Heme-Nitric oxide and OXygen
binding domain) are members of a highly-conserved,
well-characterized family of hemoproteins (Iyer, L M et al. (2003)
BMC Genomics 4(1):5; Karow, D S et al. (2004) Biochemistry
43(31):10203-10211; Boon, E M et al. (2005) Nature Chem. Biol.
1:53-59; Boon, E M et al. (2005) Curr. Opin. Chem. Biol.
9(5):441-446; Boon, E M et al. (2005) J. Inorg. Biochem.
99(4):892-902; Cary, S P et al. (2005) Proc Natl Acad Sci USA
102(37):13064-9; Karow D S et al. (2005) Biochemistry
44(49):16266-74; Cary, S P et al. (2006) Trends Biochem Sci
31(4):231-9; Boon, E M et al. (2006) J Biol Chem 281(31):21892-902;
Winger, J A et al. (2007) J Biol Chem. 282(2):897-907). H-NOX
proteins are nitric-oxide-neutral, unlike previous hemoglobin-based
oxygen carriers, H-NOX do not scavenge circulating nitric oxide
(NO), and thus are not associated with hypertensive or renal side
effects. The intrinsic low NO reactivity (and high NO stability)
makes wild-type and mutant H-NOX proteins desirable blood
substitutes because of the lower probability of inactivation of
H-NOX proteins by endogenous NO and the lower probability of
scavenging of endogenous NO by H-NOX proteins. Importantly, the
presence of a distal pocket tyrosine in some H-NOX proteins
(Pellicena, P. et al. (2004) Proc Natl. Acad Sci USA
101(35):12854-12859) is suggestive of undesirable, high NO
reactivity, contraindicating use as a blood substitute. For
example, by analogy, a Mycobacterium tuberculosis hemoglobin
protein, with a structurally analogous distal pocket tyrosine,
reacts extremely rapidly with NO, and is used by the Mycobacterium
to effectively scavenge and avoid defensive NO produced by an
infected host (Ouellet, H. et al. (2002) Proc. Natl. Acad. Sci. USA
99(9):5902-5907). However, it was surprisingly discovered that
H-NOX proteins actually have a much lower NO reactivity than that
of hemoglobin making their use as blood substitutes possible.
[0008] H-NOX proteins for the delivery of O.sub.2 and/or NO for
therapeutic and other uses are described in U.S. Pat. Nos.
8,404,631 and 8,404,632; WO 2007/139791, WO 2007/139767 and WO
2014/107171; and U.S. patent application Ser. No. 14/530,569, the
contents of each is incorporated by reference in its entirety.
[0009] All references cited herein, including patent applications
and publications, are incorporated herein by reference in their
entirety.
BRIEF SUMMARY OF THE INVENTION
[0010] The invention provides methods for modulating tumor immunity
in an individual with a tumor comprising administering to the
individual an effective amount of an O.sub.2 carrier polypeptide.
In some embodiments, the invention provides methods for enhancing
an immune response to the tumor. In some embodiments, the invention
provides methods for increasing lymphocyte infiltration to a tumor
in an individual comprising administering to the individual an
effective amount of an O.sub.2 carrier polypeptide. In some
embodiments, the increase in lymphocyte infiltration to the tumor
comprises an increase in infiltration of one or more of CD4 cells,
CD8 cells, or NK cells. In some embodiments, the increase in
lymphocyte infiltration to the tumor is accompanied by inhibition
of one or more of Treg cells, tumor associated macrophages or
myeloid derived suppressor cells in the tumor. In some embodiments,
the increase in lymphocyte infiltration to the tumor is accompanied
by an increase in MHC1 expression on the tumor cells. In some
embodiments, the modulating of tumor immunity comprises increasing
antigen processing. In some embodiments, the modulating of tumor
immunity comprises increasing the presentation capabilities of
dendritic cells (DC).
[0011] In some embodiments, the invention provides methods for
decreasing expression of hypoxia inducible factor 1.alpha.
(HIF-1.alpha.) and/or hypoxia inducible factor 2.alpha.
(HIF-2.alpha.) in a tumor in an individual comprising administering
to the individual an effective amount of an O.sub.2 carrier
polypeptide. In some embodiments, the invention provides methods
for decreasing expression of programmed death ligand-1 (PD-L1) in a
tumor in an individual comprising administering to the individual
an effective amount of an O.sub.2 carrier polypeptide. In some
embodiments, the invention provides methods for decreasing
expression of A2A adenosine receptor (A2AR) in a tumor in an
individual comprising administering to the individual an effective
amount of an O.sub.2 carrier polypeptide.
[0012] In some embodiments of the above embodiments, the tumor is a
brain tumor, a glioblastoma, a bone tumor, a pancreatic tumor, a
skin tumor, a tumor of the head or neck, a melanoma, a lung tumor,
a uterine tumor, an ovarian tumor, a colorectal tumor, a liver
tumor, a hepatocellular carcinoma, a stomach tumor, a testicular
tumor, an endometrial tumor, a cervical tumor, a vaginal tumor, a
Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an esophageal tumor,
an intestinal tumor, a thyroid tumor, an adrenal tumor, a bladder
tumor, a kidney tumor, breast tumor, a multiple myeloma tumor, a
sarcoma, or a squamous cell tumor.
[0013] In some aspects, the invention provides methods for treating
cancer in an individual comprising administering to the individual
an effective amount of an O.sub.2 carrier polypeptide. In some
embodiments, the cancer is brain cancer, glioblastoma, bone cancer,
pancreatic cancer, skin cancer, cancer of the head or neck,
melanoma, lung cancer, uterine cancer, ovarian cancer, colorectal
cancer, anal cancer, liver cancer, hepatocellular carcinoma,
stomach cancer, testicular cancer, endometrial cancer, cervical
cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, esophageal
cancer, intestinal cancer, thyroid cancer, adrenal cancer, bladder
cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma,
anal cancer or squamous cell cancer.
[0014] In some embodiments of the above aspects and embodiments,
the individual is a mammal. In further embodiments, the mammal is a
human (e.g., a human patient). In other embodiments, the mammal is
a pet, a laboratory research animal, or a farm animal. In some
embodiments, the pet, research animal or farm animal is a dog, a
cat, a horse, a monkey, a rabbit, a rat, a mouse, a guinea pig, a
hamster, a pig, or a cow.
[0015] In some embodiments of the above aspects and embodiments,
the O.sub.2 carrier polypeptide is administered by intravenous,
intra-arterial, intratumoral, intravesicular, inhalation,
intraperitoneal, intrapulmonary, intramuscular, subcutaneous,
intra-tracheal, transmucosal, intraocular, intrathecal, or
transdermal administration. In some embodiments, administration of
the O.sub.2 carrier polypeptide is repeated. In some embodiments,
administration of the O.sub.2 carrier polypeptide is repeated daily
or twice a day from about 4 weeks to about 8 weeks. In some
embodiments, the O.sub.2 carrier polypeptide is administered every
four, every 8, every 12 or every 24 hours for a period of about one
to about 10 days. In some embodiments, the O.sub.2 carrier
polypeptide is administered as a bolus. In other embodiments, the
O.sub.2 carrier polypeptide is administered by infusion. In some
embodiments, the O.sub.2 carrier polypeptide is infused in the
individual for about 15 minutes, about 30 minutes, about 1 hour,
about 2 hours, about 3 hours, about 6 hours, about 12 hours, about
24 hours or more than 24 hours.
[0016] In some embodiments, the invention provides methods to
modulate tumor immunity or to treat cancer in an individual wherein
an O.sub.2 carrier polypeptide is administered in combination with
radiation therapy. In some embodiments, the radiation therapy is
administered to the individual 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16,
18, 20 or 24 hours after the O.sub.2 carrier polypeptide is
administered. In some embodiments, the radiation is X-radiation. In
some embodiments, the X-radiation is administered at about 0.5 gray
to about 75 gray. In some embodiments, the administration of the
O.sub.2 carrier polypeptide and/or the administration of the
radiation is repeated. In some embodiments, the administration is
repeated more than about any of two, three, four times, five times,
ten times, 15 times, 20 times, 25 times or 30 times. In some
embodiments, the administration is repeated after one week, two
weeks, three weeks, or four weeks.
[0017] In some embodiments, the invention provides methods to
modulate tumor immunity or to treat cancer in an individual wherein
an O.sub.2 carrier polypeptide is administered in combination with
chemotherapy or immunotherapy. In some embodiments, the
chemotherapy comprises a cytotoxin. In some embodiments, the
administration of the O.sub.2 carrier polypeptide and/or the
administration of the chemotherapy is repeated. In some
embodiments, the immunotherapy is one or more of an anticancer
vaccine, an adoptive immune cell therapy or an agent that targets
an immune checkpoint regulator. In some embodiments, the
immunotherapy targets one or more of CTLA-4, PD1, PD-L1, or an
immune checkpoint regulator. In some embodiments, the adoptive
immune therapy is a chimeric antigen receptor expressing T cell or
an engineered TCR-T cell. In some embodiments, the immune therapy
is an oncolytic virus or a Bispecific T cell Engager (BiTE). In
some embodiments, the administration of the O.sub.2 carrier
polypeptide and/or the administration of the immunotherapy is
repeated.
[0018] In some embodiments of the above embodiments, the O.sub.2
carrier polypeptide is in a pharmaceutical composition. In some
embodiments, the pharmaceutical composition further comprises a
pharmaceutically acceptable carrier. In some embodiments of any of
the above embodiments, the O.sub.2 carrier polypeptide is an H-NOX
protein.
[0019] In some aspects, the invention provides methods for
modulating tumor immunity in an individual with a tumor comprising
administering to the individual an effective amount of an H-NOX
protein. In some embodiments, the invention provides methods for
enhancing an immune response to the tumor. In some embodiments, the
invention provides methods for increasing leucocyte infiltration to
a tumor in an individual comprising administering to the individual
an effective amount of an H-NOX protein. In some embodiments, the
invention provides methods for increasing lymphocyte infiltration
to a tumor in an individual comprising administering to the
individual an effective amount of an H-NOX protein. In some
embodiments, the increase in lymphocyte infiltration to the tumor
comprises an increase in infiltration of one or more of CD4 cells,
CD8 cells, or NK cells. In some embodiments, the increase in
lymphocyte infiltration to the tumor is accompanied by inhibition
of one or more of Treg cells, tumor associated macrophages or
myeloid derived suppressor cells in the tumor. In some embodiments,
the increase in lymphocyte infiltration to the tumor is accompanied
by an increase in MHC1 expression on the tumor cells. In some
embodiments, the modulating of tumor immunity comprises increasing
antigen processing. In some embodiments, the modulating of tumor
immunity comprises increasing lymphocyte activation. In some
embodiments, the modulating of tumor immunity comprises increasing
the presentation capabilities of dendritic cells (DC).
[0020] In some embodiments, the invention provides methods for
decreasing expression of hypoxia inducible factor 1.alpha.
(HIF-1.alpha.) and/or hypoxia inducible factor 2.alpha.
(HIF-2.alpha.) in a tumor in an individual comprising administering
to the individual an effective amount of an H-NOX protein. In some
embodiments, the invention provides methods for decreasing
expression of programmed death ligand-1 (PD-L1) in a tumor in an
individual comprising administering to the individual an effective
amount of an H-NOX protein. In some embodiments, the invention
provides methods for decreasing expression of A2A adenosine
receptor (A2AR) in a tumor in an individual comprising
administering to the individual an effective amount of an H-NOX
protein.
[0021] In some embodiments of the above embodiments, the tumor is a
brain tumor, a glioblastoma, a bone tumor, a pancreatic tumor, a
skin tumor, a tumor of the head or neck, a melanoma, a lung tumor,
a uterine tumor, an ovarian tumor, a colorectal tumor, an anal
tumor, a liver tumor, a hepatocellular carcinoma, a stomach tumor,
a testicular tumor, an endometrial tumor, a cervical tumor, a
vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an
esophageal tumor, an intestinal tumor, a thyroid tumor, an adrenal
tumor, a bladder tumor, a kidney tumor, breast tumor, a multiple
myeloma tumor, a sarcoma, or a squamous cell tumor.
[0022] In some aspects, the invention provides methods for treating
cancer in an individual comprising administering to the individual
an effective amount of an H-NOX protein. In some embodiments, the
cancer is brain cancer, glioblastoma, bone cancer, pancreatic
cancer, skin cancer, cancer of the head or neck, melanoma, lung
cancer, uterine cancer, ovarian cancer, colorectal cancer, anal
cancer, liver cancer, hepatocellular carcinoma, stomach cancer,
testicular cancer, endometrial cancer, cervical cancer, Hodgkin's
Disease, non-Hodgkin's lymphoma, esophageal cancer, intestinal
cancer, thyroid cancer, adrenal cancer, bladder cancer, kidney
cancer, breast cancer, multiple myeloma, sarcoma or squamous cell
cancer.
[0023] In some embodiments of the above aspects and embodiments,
the individual is a mammal. In further embodiments, the mammal is a
human (e.g., a human patient). In other embodiments, the mammal is
a pet, a laboratory research animal, or a farm animal. In some
embodiments, the pet, research animal or farm animal is a dog, a
cat, a horse, a monkey, a rabbit, a rat, a mouse, a guinea pig, a
hamster, a pig, or a cow.
[0024] In some embodiments of the above aspects and embodiments,
the H-NOX protein is administered by intravenous, intra-arterial,
intratumoral, intravesicular, inhalation, intraperitoneal,
intrapulmonary, intramuscular, subcutaneous, intra-tracheal,
transmucosal, intraocular, intrathecal, or transdermal
administration. In some embodiments, administration of the H-NOX
protein is repeated. In some embodiments, administration of the
H-NOX protein is repeated daily or twice a day from about 4 weeks
to about 8 weeks. In some embodiments, the H-NOX protein is
administered every four, every 8, every 12, every 24 hours, or
every 48 hours for a period of about one to about 10 days. In some
embodiments, the H-NOX protein is administered as a bolus. In other
embodiments, the H-NOX protein is administered by infusion. In some
embodiments, the H-NOX protein is infused in the individual for
about 15 minutes, about 30 minutes, about 1 hour, about 2 hours,
about 3 hours, about 6 hours, about 12 hours, about 24 hours or
more than 24 hours.
[0025] In some embodiments, the invention provides methods to
modulate tumor immunity or to treat cancer in an individual wherein
an H-NOX protein is administered in combination with radiation
therapy. In some embodiments, the radiation therapy is administered
to the individual 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or 24
hours after the H-NOX protein is administered. In some embodiments,
the radiation is X-radiation. In some embodiments, the X-radiation
is administered at about 0.5 gray to about 75 gray. In some
embodiments, the administration of the H-NOX protein and/or the
administration of the radiation is repeated. In some embodiments,
the administration is repeated more than about any of two, three,
four times, five times, ten times, 15 times, 20 times, 25 times, 30
times or 40 times. In some embodiments, the administration is
repeated after one week, two weeks, three weeks, or four weeks or
more.
[0026] In some embodiments, the invention provides methods to
modulate tumor immunity or to treat cancer in an individual wherein
an H-NOX protein is administered in combination with chemotherapy
or immunotherapy. In some embodiments, the chemotherapy comprises a
cytotoxin. In some embodiments, the administration of the H-NOX
protein and/or the administration of the chemotherapy is repeated.
In some embodiments, the immunotherapy is one or more of an
anticancer vaccine, an adoptive immune cell therapy or an agent
that targets an immune checkpoint regulator. In some embodiments,
the immunotherapy targets one or more of CTLA-4, PD1, PD-L1, or an
immune checkpoint regulator. In some embodiments, the adoptive
immude therapy is a chimeric antigen receptor expressing T cell or
an engineered TCR-T cell. In some embodiments, the immune therapy
is an oncolytic virus or a Bispecific T cell Engager (BiTE). In
some embodiments, the administration of the H-NOX protein and/or
the administration of the immunotherapy is repeated.
[0027] In some embodiments of the above aspects and embodiments,
the H-NOX protein is a T. tengcongensis H-NOX, a L. pneumophilia 2
H-NOX, a H. sapiens .beta.1, a R. norvegicus .beta.1, a C. lupus
H-NOX, a D. melangaster .beta.1, a D. melangaster CG14885-PA, a C.
elegans GCY-35, a N. punctiforme H-NOX, C. crescentus H-NOX, a S.
oneidensis H-NOX, or C. acetobutylicum H-NOX. In some embodiments,
the H-NOX protein comprises a H-NOX domain corresponding to the
H-NOX domain of T. tengcongensis set forth in SEQ ID NO:2.
[0028] In some embodiments, the H-NOX comprises one or more distal
pocket mutations. In some embodiments, the distal pocket mutation
is an amino acid substitution at a site corresponding to L144 of T.
tengcongensis H-NOX. In some embodiments, the H-NOX is a T.
tengcongensis H-NOX comprising an amino acid substitution at
position 144. In some embodiments, the amino acid substitution at
position 144 is an L144F substitution.
[0029] In some embodiments, the H-NOX protein is a polymeric H-NOX
protein. In some embodiments, the polymeric H-NOX protein comprises
monomers, wherein the monomers comprise an H-NOX domain and a
polymerization domain. In some embodiments, the H-NOX domain is
covalently linked to the polymerization domain. In some
embodiments, the polymeric H-NOX protein is a trimeric H-NOX
protein. In some embodiments, the trimeric H-NOX protein comprises
one or more trimerization domains. In some embodiments, the
trimeric H-NOX protein comprises three monomers, wherein the
monomers comprise an H-NOX domain and a trimerization domain,
wherein the trimerization domain is a bacteriophage T4
trimerization domain. In some embodiments, the trimerization domain
is a foldon domain. In some embodiments, the foldon domain
comprises the amino acid sequence of SEQ ID NO:4.
[0030] In some embodiments, the H-NOX protein is fused to an Fc
domain of an immunoglobulin. In some embodiments, the H-NOX protein
is covalently bound to polyethylene glycol.
[0031] In some embodiments, the O.sub.2 dissociation constant of
the H-NOX protein is within 2 orders of magnitude of that of
hemoglobin, and wherein the NO reactivity of the H-NOX protein is
at least 10-fold lower than that of hemoglobin. In some
embodiments, the O.sub.2 dissociation constant of the polymeric
H-NOX protein is between about 1 nM and about 1000 nM at 20.degree.
C. In some embodiments, the O.sub.2 dissociation constant of the
H-NOX protein is between about 1 .mu.M and about 10 .mu.M at
20.degree. C. In some embodiments, the O.sub.2 dissociation
constant of the H-NOX protein is between about 10 .mu.M and about
50 .mu.M at 20.degree. C. In some embodiments, the NO reactivity of
the H-NOX protein is less than about 700 s.sup.-1 at 20.degree. C.
In some embodiments, the NO reactivity of the H-NOX protein is at
least 100-fold lower than that of hemoglobin. In some embodiments,
the NO reactivity of the H-NOX protein is at least 1,000-fold lower
than that of hemoglobin. In some embodiments, the k.sub.off for
oxygen of the H-NOX protein is less than or equal to about 0.65
s.sup.-1 at 20.degree. C. In some embodiments, the k.sub.off for
oxygen of the H-NOX protein is between about 0.21 s.sup.-1 and
about 0.65 s.sup.-1 at 20.degree. C. In some embodiments, the
k.sub.off for oxygen of the H-NOX protein is between about 1.35
s.sup.-1 and about 2.9 s.sup.-1 at 20.degree. C. In some
embodiments, the rate of heme autoxidation of the H-NOX protein is
less than about 1 h.sup.-1 at 37.degree. C.
[0032] In some embodiments of the above embodiments, the H-NOX
protein is in a pharmaceutical composition. In some embodiments,
the pharmaceutical composition further comprises a pharmaceutically
acceptable carrier.
[0033] In some aspects the invention provides the use of an O.sub.2
carrier protein for modulating tumor immunity in an individual. In
some embodiments, the modulating tumor immunity comprises enhancing
an immune response to the tumor. In some embodiments, the invention
provides the use of an O.sub.2 carrier polypeptide for increasing
leucocyte infiltration to a tumor in an individual. In some
embodiments, the invention provides the use of an O.sub.2 carrier
polypeptide for increasing lymphocyte infiltration to a tumor in an
individual. In some embodiments, the increase in lymphocyte
infiltration to the tumor comprises an increase in infiltration of
one or more of CD4 cells, CD8 cells, or NK cells. In some
embodiments, the increase in lymphocyte infiltration to the tumor
is accompanied by inhibition of one or more of Treg cells, tumor
associated macrophages or myeloid derived suppressor cells in the
tumor. In some embodiments, the increase in leucocyte infiltration
to the tumor is accompanied by an increase in MHC1 expression on
the tumor cells. In some embodiments, the increase in lymphocyte
infiltration to the tumor is accompanied by an increase in MHC1
expression on the tumor cells.
[0034] In some embodiments, the invention provides the use of an
O.sub.2 carrier polypeptide for decreasing expression of
HIF-1.alpha. and/or HIF-2.alpha. in a tumor in an individual. In
some embodiments, the invention provides the use of an O.sub.2
carrier polypeptide for decreasing expression of PD-L1 in a tumor
in an individual. In some embodiments, the invention provides the
use of an O.sub.2 carrier polypeptide for decreasing expression of
A2AR in a tumor in an individual.
[0035] In some embodiments of the above uses, the tumor is a brain
tumor, a glioblastoma, a bone tumor, a pancreatic tumor, a skin
tumor, a tumor of the head or neck, a melanoma, a lung tumor, a
uterine tumor, an ovarian tumor, a colorectal tumor, an anal tumor,
a liver tumor, a hepatocellular carcinoma, a stomach tumor, a
testicular tumor, an endometrial tumor, a cervical tumor, a vaginal
tumor, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an
esophageal tumor, an intestinal tumor, a thyroid tumor, an adrenal
tumor, a bladder tumor, a kidney tumor, a breast tumor, a multiple
myeloma tumor, a sarcoma, or a squamous cell tumor.
[0036] In some embodiments, the invention provides the use of an
O.sub.2 carrier protein for treating cancer in an individual. In
some embodiments, the cancer is brain cancer, glioblastoma, bone
cancer, pancreatic cancer, skin cancer, cancer of the head or neck,
melanoma, lung cancer, uterine cancer, ovarian cancer, colorectal
cancer, anal cancer, liver cancer, hepatocellular carcinoma,
stomach cancer, testicular cancer, endometrial cancer, cervical
cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, esophageal
cancer, intestinal cancer, thyroid cancer, adrenal cancer, bladder
cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma, or
squamous cell cancer.
[0037] In some embodiments of the above uses, the individual is a
mammal. In some embodiments, the mammal is a human.
[0038] In some embodiments of the above uses, the O.sub.2 carrier
polypeptide is an H-NOX protein. In some embodiments, the H-NOX
protein is a T. tengcongensis H-NOX, a L. pneumophilia 2 H-NOX, a
H. sapiens .beta.1, a R. norvegicus .beta.1, a C. lupus H-NOX, a D.
melangaster (1, a D. melangaster CG14885-PA, a C. elegans GCY-35, a
N. punctiforme H-NOX, C. crescentus H-NOX, a S. oneidensis H-NOX,
or C. acetobutylicum H-NOX. In some embodiments, the H-NOX protein
comprises a H-NOX domain corresponding to the H-NOX domain of T.
tengcongensis set forth in SEQ ID NO:2. In some embodiments, the
H-NOX comprises one or more distal pocket mutations. In some
embodiments, the distal pocket mutation is an amino acid
substitution at a site corresponding to L144 of T. tengcongensis
H-NOX. In some embodiments, the H-NOX is a T. tengcongensis H-NOX
comprising an amino acid substitution at position 144. In some
embodiments, the amino acid substitution at position 144 is an
L144F substitution.
[0039] In some embodiments, the H-NOX protein is a polymeric H-NOX
protein. In some embodiments, the polymeric H-NOX protein comprises
monomers, wherein the monomers comprise an H-NOX domain and a
polymerization domain. In some embodiments, the H-NOX domain is
covalently linked to the polymerization domain. In some
embodiments, the polymeric H-NOX protein is a trimeric H-NOX
protein. In some embodiments, the trimeric H-NOX protein comprises
one or more trimerization domains. In some embodiments, the
trimeric H-NOX protein comprises three monomers, wherein the
monomers comprise an H-NOX domain and a trimerization domain,
wherein the trimerization domain is a bacteriophage T4
trimerization domain. In some embodiments, the trimerization domain
is a foldon domain. In some embodiments, the foldon domain
comprises the amino acid sequence of SEQ ID NO:4.
[0040] In some embodiments, the H-NOX protein is fused to an Fc
domain of an immunoglobulin. In some embodiments, the H-NOX protein
is covalently bound to polyethylene glycol.
[0041] In some aspects, the invention provides kits for modulating
tumor immunity in an individual comprising an O.sub.2 carrier
protein for use in the methods described herein. In some
embodiments, the kit further comprises one or more of a vial, a
vessel, an ampule, a bottle, a jars, or flexible packaging. In some
embodiments, the kit further comprises one or more buffer. In some
embodiments, the kit further comprises instructions for use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIGS. 1A and 1B shows a model of the major immunosuppressive
pathways promoted by hypoxia (FIG. 1A) and the points of
therapeutic intervention that may be exerted by O.sub.2 carrier
polypeptide treatment (FIG. 1B).
[0043] FIGS. 2A-2C show tumor oxygenation after single bolus dose
of PEGylated trimer Tt H-NOX L144F assessed by pimonidazole and
HIF-1 ELISA. FIG. 2A shows pimonidazole levels measured by
competitive ELISA. FIG. 2B shows HIF-1.alpha. levels measured by
sandwich ELISA. Graphs show quantification of pimonidazole and
HIF-1.alpha. signals after PEGylated trimer Tt H-NOX L144F
administration. Mean values+/-SEM. ***p<0.001, **p<0.01 by
One way ANOVA and Bonferroni's post-hoc tests. FIG. 2C shows
assessment of tumors for the accumulation of PEGylated trimer Tt
H-NOX L144F by sandwich H-NOX ELISA and results expressed per gram
of tumor tissue.
[0044] FIGS. 3A-3D show direct measurements of tumor tissue
oxygenation following PEGylated trimer Tt H-NOX L144F
administration. Tumors were treated either with PEGylated trimer TL
H-NOX L144F (FIG. 3A), non-functional control Tt H-NOX protein
(FIG. 3C), or with 100% oxygen starting at pO.sub.2=0.44 mmHg (FIG.
3B) with 100% oxygen starting at pO.sub.2=5 mmHg (FIG. 3D).
[0045] FIG. 4 shows enhancement of radiation efficacy following
PEGylated trimer Tt H-NOX L144F treatment of mice bearing H460
tumors. Mice bearing H460 subcutaneous xenograft tumors (150-300
mm.sup.3) were either pre-treated with PEGylated trimer Tt H-NOX
L144F or treated with 10 Gy alone, irradiated, tumors extracted and
processed for clonogenic assay. Cell numbers were counted 7 days
later in triplicate samples from each tumor. Each dot on the graph
represents average surviving fraction for one tumor.
[0046] FIGS. 5A-5C show PEGylated trimer Tt H-NOX L144F
downregulates HIF-1.alpha. targets involved in immunosuppression.
Mice bearing H460 subcutaneous xenograft tumors (150-300 mm.sup.3)
were either pre-treated with PEGylated trimer Tt H-NOX L144For
treated with vehicle alone, and harvested for qRT-PCR analysis.
FIG. 5A shows expression of VEGF. FIG. 5B shows expression of
GLUT1. FIG. 5C shows expression of PD-L1.
[0047] FIG. 6A shows the nucleic acid (SEQ ID NO:5) and amino acid
sequence (SEQ ID NO:6) of the foldon domain of bacteriophage T4
fibritin fused to the C-terminus of a Thermoanaerobacter
tengcongensis L144F H-NOX sequence and including the His6 tag. FIG.
6B shows the nucleic acid (SEQ ID NO:7) and amino acid sequence
(SEQ ID NO:8) of the L144F H-NOX-foldon monomer without a His6
tag.
[0048] FIGS. 7A-7C show representative images of tumor hypoxia and
T cell infiltration is B16F10 subcutaneous tumors (FIG. 7A), CT26
subcutaneous tumors (FIG. 7B) and GL261 intracranial tumors (FIG.
7C). Hypoxia (top panels) and T cell infiltration (middle panels)
is shown by immunohistochemistry. Bottom panels show results of
quantitative analysis of multiple tumor sections. Significantly
fewer CD4 and CD8 T cells infiltrate the hypoxic regions of
tumors.
[0049] FIG. 8 shows quantification of CD8 T cells in hypoxic areas
of tumors after H-NOX treatment (OMX) or vehicle control treatment
(Veh). Representative images are shown. Hypoxic areas were labeled
with pimondazole by immunohistochemical analysis. Following OMX
treatment there is an increase in CD4 (data not shown) and CD8 T
cell infiltration into regions of tumors that were hypoxic prior to
OMX administration.
[0050] FIGS. 9A and 9B show quantification of T cells in normoxic
and hypoxic areas of tumors after H-NOX treatment (OMX) or vehicle
control treatment (Veh). Both CD4 and CD8 T cells were evaluated.
Tumor areas evaluated included areas on the periphery of the tumor
and in the tumor center. Results of quantitative image analysis of
multiple sections are shown in FIG. 9A and representative images in
FIG. 9B. Hypoxic areas were labelled using immunohistochemical
analysis of carbonic anhydrase IX (CAIX) expression. Following OMX
treatment, there is an increase in CD4 and CD8 T cell infiltration
into regions of tumors that were hypoxic prior to OMX
administration.
[0051] FIG. 10 shows the results of immunohistochemistry for
hypoxia (pimondazole) and CD3 vessels is GL261 tumor model.
[0052] FIG. 11 shows immunohistochemical analysis of H-NOX tumor
penetration, tumor hypoxia and CD8 T cell localization in canine
oral melanoma tumors. Tissues were stained with hematoxylin and
eosin (H&E), DNA interchelating dye (DAPI) and with anti-H-NOX
(OMX), -carbonic anhydrase IX (CAIX) and -CD8 antibodies to assess
CD8 lymphocyte localization in tumor regions that were hypoxic
prior to H-NOX (OMX) treatment. Images reveal CD8 positive T cells
localized throughout regions of the tumor that were hypoxic prior
to H-NOX (OMX) treatment (CAIX positive).
[0053] FIGS. 12A-12K show that larger tumor size correlates with
enhanced hypoxia and reduced lymphocyte infiltration in
subcutaneous 4T1-Luc syngeneic mouse tumors. FIG. 12A shows tumor
volumes on day 10 and day 14 post-implant. FIG. 12B shows fraction
of lymphocytes within the viable cell population. FIG. 12C shows
the absolute lymphocyte cell numbers within the viable population.
FIG. 12D shows a negative correlation between tumor volume and
percentage lymphocytes. FIG. 12E shows a positive correlation
between tumor volume and percentage hypoxia. FIG. 12F shows a
negative correlation between percentage hypoxia and percentage
lymphocytes. FIG. 12G shows a negative correlation between tumor
volume and percentage CD3-positive T cells. FIG. 12H shows a
negative correlation between tumor volume and percentage
CD4-positive T cells. FIG. 12I shows a negative correlation between
tumor volume and percentage CD8-positive T cells. FIG. 12J shows a
negative correlation between tumor volume and percentage
CD3-CD4-double-positive T cells. FIG. 12K shows a negative
correlation between tumor volume and percentage
CD3-CD8-double-positive T cells.
[0054] FIGS. 13A-13F shows that hypoxic tumor regions are
immunosuppressive and exhibit reduced T cell infiltration in
subcutaneous 4T1-Luc syngeneic mouse tumors. Immunofluorescence
staining of tumor region #1 for (FIG. 13A) pimonidazole-positive
hypoxic areas and (FIG. 13B) CD8-positive T cells, counterstained
with (FIG. 13C) DAPI to highlight nuclei. Immunofluorescence
staining of tumor region #2 for (FIG. 13D) pimonidazole-positive
hypoxic areas and (FIG. 13E) CD4-positive T cells, counterstained
with (FIG. 13F) DAPI to highlight nuclei.
DETAILED DESCRIPTION OF THE INVENTION
[0055] The present invention provides methods for treating cancer
in an individual comprising administering to the individual an
effective amount of an O.sub.2 carrier polypeptide such as an H-NOX
protein. In certain aspects, the invention provides methods for
modulating hypoxia-mediated tumor immunity in an individual
comprising administering to the individual an effective amount of
an O.sub.2 carrier polypeptide, such as an H-NOX protein. The
O.sub.2 carrier polypeptide is delivered to the tumor where it
enhances an immune response to the tumor. Enhancement of an immune
response to the tumor may be mediated by targeting hypoxia
inducible factor 1.alpha. (HIF-1.alpha.)-mediated pathways of tumor
immunity and/or non-HIF-1.alpha.-mediated pathways of tumor
immunity. In some aspects, the invention provides methods for
increasing lymphocyte infiltration to a tumor in an individual
comprising administering to the individual an effective amount of
an O.sub.2 carrier polypeptide. In some embodiments, the increase
in lymphocyte infiltration to the tumor comprises an increase in
infiltration of one or more of CD4 cells, CD8 cells, or NK cells.
In some embodiments, the increase in lymphocyte infiltration to the
tumor is accompanied by inhibition of one or more of Treg cells,
tumor associated macrophages or myeloid derived suppressor cells in
the tumor. In some embodiments, the increase in lymphocyte
infiltration to the tumor is accompanied by an increase in MHC1
expression on the tumor cells. In some embodiments, the invention
provides methods for decreasing expression of hypoxia inducible
factor 1.alpha. (HIF-1.alpha.) in a tumor in an individual
comprising administering to the individual an effective amount of
an O.sub.2 carrier polypeptide (e.g., an H-NOX protein). In some
embodiments, the invention provides methods for decreasing
expression of programmed death ligand-1 (PD-L1) in a tumor in an
individual comprising administering to the individual an effective
amount of an O.sub.2 carrier polypeptide (e.g., an H-NOX protein).
In some embodiments, the invention provides methods for decreasing
expression of A2A adenosine receptor (A2AR) in a tumor in an
individual comprising administering to the individual an effective
amount of an O.sub.2 carrier polypeptide (e.g., an H-NOX
protein).
Definitions
[0056] Unless defined otherwise, the meanings of all technical and
scientific terms used herein are those commonly understood by one
of skill in the art to which this invention belongs. One of skill
in the art will also appreciate that any methods and materials
similar or equivalent to those described herein can also be used to
practice or test the invention.
[0057] For use herein, unless clearly indicated otherwise, use of
the terms "a", "an," and the like refers to one or more.
[0058] In this application, the use of "or" means "and/or" unless
expressly stated or understood by one skilled in the art. In the
context of a multiple dependent claim, the use of "or" refers back
to more than one preceding independent or dependent claim.
[0059] Reference to "about" a value or parameter herein includes
(and describes) embodiments that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X."
[0060] It is understood that aspect and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
[0061] The terms "polypeptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues, and
are not limited to a minimum length. Such polymers of amino acid
residues may contain natural or non-natural amino acid residues,
and include, but are not limited to, peptides, oligopeptides,
dimers, trimers, and polymers of amino acid residues. Both
full-length proteins and fragments thereof are encompassed by the
definition. The terms also include post-expression modifications of
the polypeptide, for example, glycosylation, sialylation,
acetylation, phosphorylation, and the like. Furthermore, for
purposes of the present invention, a "polypeptide" refers to a
protein which includes modifications, such as deletions, additions,
and substitutions (generally conservative in nature), to the native
sequence, as long as the protein maintains the desired activity.
These modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through mutations of
hosts which produce the proteins or errors due to PCR
amplification. As used herein, a protein may include two or more
subunits, covalently or non-covalently associated; for example, a
protein may include two or more associated monomers.
[0062] The terms "nucleic acid molecule", "nucleic acid" and
"polynucleotide" may be used interchangeably, and refer to a
polymer of nucleotides. Such polymers of nucleotides may contain
natural and/or non-natural nucleotides, and include, but are not
limited to, DNA, RNA, and PNA. "Nucleic acid sequence" refers to
the linear sequence of nucleotides that comprise the nucleic acid
molecule or polynucleotide.
[0063] As used herein, the term "hypoxia inducible factor" or "HIF"
refers to a family of transcription factor that respond to
decreases in oxygen, or hypoxia, in the cellular environment.
Members of the human HIF family include HIF-1.alpha., HIF-1.beta.,
HIF-2.alpha., HIF-2.beta., HIF3.alpha., HIF3.beta.. HIF-1 functions
as a master regulator of homeostatic responses to hypoxia by
activating transcription of many genes, including those involved in
energy metabolism, angiogenesis, apoptosis, and other genes whose
protein products increase oxygen delivery or facilitate metabolic
adaptation to hypoxia. HIF-1 plays a role in embryonic
vascularization, tumor angiogenesis and pathophysiology of ischemic
disease. Human hypoxia-inducible factor 1, alpha subunit or human
HIF-1.alpha. interacts with a number of polypeptides including but
not limited to ARNTL, ARNT, CREBB, EP300, HIF-1AN, Mdm2, NR4A, p53,
PSMA7, STAT3, UBC, VH and pVHL. Human HIF-1.alpha. is encoded by
the HIF-1A gene. The amino acid sequence of human HIF-1.alpha. is
provided by GenBank Accession no. NP_001230013 and the nucleotide
sequence of human HIF-1.alpha. mRNA is provided by GenBank
Accession No. NM_001243084. The amino acid sequence of mouse
HIF-1.alpha. is provided by GenBank Accession no. NP_010431 and the
nucleotide sequence of mouse HIF-1.alpha. mRNA is provided by
GenBank Accession No. NM_034561.
[0064] As used herein, "programmed death-ligand 1" or "PD-L1"
refers to a transmembrane protein that is part of an immune
checkpoint pathway that plays a role in suppressing the immune
system. Interaction of PDL1 with the PD1 receptor or the B7.1
receptor inhibits T cell receptor-mediated activation of IL-2 and T
cell proliferation. Human PD-L1 is encoded by the CD274 gene. The
amino acid sequence of human PD-L1 is provided by GenBank Accession
no. NP_001254635 and the nucleotide sequence of human PD-L1 mRNA is
provided by GenBank Accession No. NM_001267706. The amino acid
sequence of mouse PD-L1 is provided by GenBank Accession no.
NP_021893 and the nucleotide sequence of mouse PD-L1 mRNA is
provided by GenBank Accession No. NM_068693.
[0065] As used herein, an "adenosine A2A receptor" or "A2AR" refers
to a receptor of the G protein-coupled receptor superfamily. A2AR
is a receptor for adenosine that plays a role in oxygen consumption
and is thought to play a role in suppressing overreactive immune
cells by way or increased levels of cAMP. Human A2AR is encoded by
ADORA2A the gene. The amino acid sequence of human A2AR is provided
by GenBank Accession no. NP_000666 and the nucleotide sequence of
human A2AR mRNA is provided by GenBank Accession No. NM_000675. The
amino acid sequence of mouse A2AR is provided by GenBank Accession
no. NP_033760 and the nucleotide sequence of mouse A2AR mRNA is
provided by GenBank Accession No. NM 09630.
[0066] As used herein, an "H-NOX protein" means a protein that has
an H-NOX domain (named for Heme-Nitric oxide and OXygen binding
domain). An H-NOX protein may or may not contain one or more other
domains in addition to the H-NOX domain. In some examples, an H-NOX
protein does not comprise a guanylyl cyclase domain. An H-NOX
protein may or may not comprise a polymerization domain.
[0067] As used herein, a "polymeric H-NOX protein" is an H-NOX
protein comprising two or more H-NOX domains. The H-NOX domains may
be covalently or non-covalently associated.
[0068] As used herein, an "H-NOX domain" is all or a portion of a
protein that binds nitric oxide and/or oxygen by way of heme. The
H-NOX domain may comprise heme or may be found as an apoproprotein
that is capable of binding heme. In some examples, an H-NOX domain
includes six alpha-helices, followed by two beta-strands, followed
by one alpha-helix, followed by two beta strands. In some examples,
an H-NOX domain corresponds to the H-NOX domain of
Thermoanaerobacter tengcongensis H-NOX set forth in SEQ ID NO:2.
For example, the H-NOX domain may be at least about 10%, 15%, 20%,
25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to
the H-NOX domain of Thermoanaerobacter tengcongensis H-NOX set
forth in SEQ ID NO:2. In some embodiments, the H-NOX domain may be
10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%,
80%-90%, 90%-95%, 95%-99% or 100% identical to the H-NOX domain of
Thermoanaerobacter tengcongensis H-NOX set forth in SEQ ID
NO:2.
[0069] As used herein, a "polymerization domain" is a domain (e.g.
a polypeptide domain) that promotes the association of monomeric
moieties to form a polymeric structure. For example, a
polymerization domain may promote the association of monomeric
H-NOX domains to generate a polymeric H-NOX protein. An exemplary
polymerization domain is the foldon domain of T4 bacteriophage,
which promotes the formation of trimeric polypeptides. Other
examples of polymerization domains include, but are not limited to,
Arc, POZ, coiled coil domains (including GCN4, leucine zippers,
Velcro), uteroglobin, collagen, 3-stranded coiled colis
(matrilin-1), thrombosporins, TRPV1-C, P53, Mnt, avadin,
streptavidin, Bcr-Abl, COMP, verotoxin subunit B, CamKII, RCK, and
domains from N ethylmaleimide-sensitive fusion protein, STM3548,
KaiC, TyrR, Hcp1, CcmK4, GP41, anthrax protective antigen,
aerolysin, a-hemolysin, C4b-binding protein, Mi-CK, arylsurfatase
A, and viral capsid proteins.
[0070] As used herein, an "amino acid linker sequence" or an "amino
acid spacer sequence" is a short polypeptide sequence that may be
used to link two domains of a protein. In some embodiments, the
amino acid linker sequence is one, two, three, four, five, six,
seven, eight, nine, ten or more than ten amino acids in length.
Exemplary amino acid linker sequences include but are not limited
to a Gly-Ser-Gly sequence and an Arg-Gly-Ser sequence.
[0071] As used herein, a "His.sub.6 tag" refers to a peptide
comprising six His residues attached to a polypeptide. A His.sub.6
tag may be used to facilitate protein purification; for example,
using chromatography specific for the His.sub.6 tag. Following
purification, the His.sub.6 tag may be cleaved using an
exopeptidase.
[0072] The term "substantially similar" or "substantially the
same," as used herein, denotes a sufficiently high degree of
similarity between two or more numeric values such that one of
skill in the art would consider the difference between the two or
more values to be of little or no biological and/or statistical
significance within the context of the biological characteristic
measured by said value. In some embodiments the two or more
substantially similar values differ by no more than about any one
of 5%, 10%, 15%, 20%, 25%, or 50%.
[0073] The phrase "substantially reduced," or "substantially
different," as used herein, denotes a sufficiently high degree of
difference between two numeric values such that one of skill in the
art would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values. In some embodiments, the
two substantially different numeric values differ by greater than
about any one of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,
80%, or 90%. In some embodiment, the two substantially different
numeric values differ by about any one of 10%-20%, 20%-30%,
30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%,
95%-99% or 100%.
[0074] A "native sequence" polypeptide comprises a polypeptide
having the same amino acid sequence as a polypeptide found in
nature. Thus, a native sequence polypeptide can have the amino acid
sequence of naturally occurring polypeptide from any organism. Such
native sequence polypeptide can be isolated from nature or can be
produced by recombinant or synthetic means. The term "native
sequence" polypeptide specifically encompasses naturally occurring
truncated or secreted forms of the polypeptide (e.g., an
extracellular domain sequence), naturally occurring variant forms
(e.g., alternatively spliced forms) and naturally occurring allelic
variants of the polypeptide.
[0075] A polypeptide "variant" means a biologically active
polypeptide having at least about 80% amino acid sequence identity
with the native sequence polypeptide after aligning the sequences
and introducing gaps, if necessary, to achieve the maximum percent
sequence identity, and not considering any conservative
substitutions as part of the sequence identity. Such variants
include, for instance, polypeptides wherein one or more amino acid
residues are added, or deleted, at the N- or C-terminus of the
polypeptide. In some embodiments, a variant will have at least
about any one of 80%, 90% or 95% amino acid sequence identity with
the native sequence polypeptide. In some embodiments, a variant
will have about any one of 80%-90%, 90%-95% or 95%-99% amino acid
sequence identity with the native sequence polypeptide.
[0076] As used herein, a "mutant protein" means a protein with one
or more mutations compared to a protein occurring in nature. In one
embodiment, the mutant protein has a sequence that differs from
that of all proteins occurring in nature. In various embodiments,
the amino acid sequence of the mutant protein is at least about any
of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97, 98, 99, or
99.5% identical to that of the corresponding region of a protein
occurring in nature. In some embodiments, the amino acid sequence
of the mutant protein is at least about any of 10%-20%, 20%-30%,
30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%,
95%-99% or 100% identical to that of the corresponding region of a
protein occurring in nature. In some embodiments, the mutant
protein is a protein fragment that contains at least about any of
25, 50, 75, 100, 150, 200, 300, or 400 contiguous amino acids from
a full-length protein. In some embodiments, the mutant protein is a
protein fragment that contains about any of 25-50, 50-75, 75-100,
100-150, 150-200, 200-300, or 300-400 contiguous amino acids from a
full-length protein. Sequence identity can be measured, for
example, using sequence analysis software with the default
parameters specified therein (e.g., Sequence Analysis Software
Package of the Genetics Computer Group, University of Wisconsin
Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705).
This software program matches similar sequences by assigning
degrees of homology to various amino acids replacements, deletions,
and other modifications.
[0077] As used herein, a "mutation" means an alteration in a
reference nucleic acid or amino acid sequence occurring in nature.
Exemplary nucleic acid mutations include an insertion, deletion,
frameshift mutation, silent mutation, nonsense mutation, or
missense mutation. In some embodiments, the nucleic acid mutation
is not a silent mutation. Exemplary protein mutations include the
insertion of one or more amino acids (e.g., the insertion of 2, 3,
4, 5, 6, 7, 8, 9, or 10 amino acids), the deletion of one or more
amino acids (e.g., a deletion of N-terminal, C-terminal, and/or
internal residues, such as the deletion of at least about any of 5,
10, 15, 25, 50, 75, 100, 150, 200, 300, or more amino acids or a
deletion of about any of 5-10, 10-15, 15-25, 25-50, 50-75, 75-100,
100-150, 150-200, 200-300, or 300-400 amino acids), the replacement
of one or more amino acids (e.g., the replacement of 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 amino acids), or combinations of two or more of
the foregoing. The nomenclature used in referring to a particular
amino acid mutation first identifies the wild-type amino acid,
followed by the residue number and finally the substitute amino
acid. For example, Y140L means that tyrosine has been replaced by a
leucine at residue number 140. Likewise, a variant H-NOX protein
may be referred to by the amino acid variations of the H-NOX
protein. For example, a T. tengcongensis Y140L H-NOX protein refers
to a T. tengcongensis H-NOX protein in which the tyrosine residue
at position number 140 has been replaced by a leucine residue and a
T. tengcongensis W9F/Y140L H-NOX protein refers to a T.
tengcongensis H-NOX protein in which the tryptophan residue at
position 9 has been replaced by a phenylalanine residue and the
tyrosine residue at position number 140 has been replaced by a
leucine residue.
[0078] An "evolutionary conserved mutation" is the replacement of
an amino acid in one protein by an amino acid in the corresponding
position of another protein in the same protein family.
[0079] As used herein, "derived from" refers to the source of the
protein into which one or more mutations is introduced. For
example, a protein that is "derived from a mammalian protein"
refers to protein of interest that results from introducing one or
more mutations into the sequence of a wild-type (i.e., a sequence
occurring in nature) mammalian protein.
[0080] As used herein, "Percent (%) amino acid sequence identity"
and "homology" with respect to a peptide, polypeptide or antibody
sequence are defined as the percentage of amino acid residues in a
candidate sequence that are identical with the amino acid residues
in the specific peptide or polypeptide sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity, and not considering any
conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or MEGALIGN.TM. (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared.
[0081] As used herein, a "k.sub.off" refers to a dissociation rate,
such as the rate of release of O.sub.2 or NO from a protein. A
lower numerical lower k.sub.off indicates a slower rate of
dissociation.
[0082] As used herein, "k.sub.on" refers to an association rate,
such as the rate of binding of O.sub.2 or NO to a protein. A lower
numerical lower k.sub.on indicates a slower rate of
association.
[0083] As used herein, "dissociation constant" refers to a "kinetic
dissociation constant" or a "calculated dissociation constant." A
"kinetic dissociation constant" or "K.sub.D" is a ratio of kinetic
off-rate (k.sub.off) to kinetic on-rate (k.sub.on), such as a
K.sub.D value determined as an absolute value using standard
methods (e.g., standard spectroscopic, stopped-flow, or
flash-photolysis methods) including methods known to the skilled
artisan and/or described herein. "Calculated dissociation constant"
or "calculated K.sub.D" refers to an approximation of the kinetic
dissociation constant based on a measured k.sub.off. A value for
the k.sub.on is derived via the correlation between kinetic K.sub.D
and k.sub.off as described herein.
[0084] As used herein, "oxygen affinity" is a qualitative term that
refers to the strength of oxygen binding to the heme moiety of a
protein. This affinity is affected by both the k.sub.off and
k.sub.on for oxygen. A numerically lower oxygen K.sub.D value means
a higher affinity.
[0085] As used herein, "NO affinity" is a qualitative term that
refers to the strength of NO binding to a protein (such as binding
to a heme group or to an oxygen bound to a heme group associated
with a protein). This affinity is affected by both the k.sub.off
and k.sub.on for NO. A numerically lower NO K.sub.D value means a
higher affinity.
[0086] As used herein, "NO stability" refers to the stability or
resistance of a protein to oxidation by NO in the presence of
oxygen. For example, the ability of the protein to not be oxidized
when bound to NO in the presence of oxygen is indicative of the
protein's NO stability. In some embodiments, less than about any of
50, 40, 30, 10, or 5% of an H-NOX protein is oxidized after
incubation for about any of 1, 2, 4, 6, 8, 10, 15, or 20 hours at
20.degree. C.
[0087] As used herein, "NO reactivity" refers to the rate at which
iron in the heme of a heme-binding protein is oxidized by NO in the
presence of oxygen. A lower numerical value for NO reactivity in
units of s.sup.-1 indicates a lower NO reactivity
[0088] As used herein, an "autoxidation rate" refers to the rate at
which iron in the heme of a heme-binding protein is autoxidized. A
lower numerical autoxidation rate in units of s.sup.-1 indicates a
lower autoxidation rate.
[0089] The term "vector" is used to describe a polynucleotide that
may be engineered to contain a cloned polynucleotide or
polynucleotides that may be propagated in a host cell. A vector may
include one or more of the following elements: an origin of
replication, one or more regulatory sequences (such as, for
example, promoters and/or enhancers) that regulate the expression
of the polypeptide of interest, and/or one or more selectable
marker genes (such as, for example, antibiotic resistance genes and
genes that may be used in colorimetric assays, e.g.,
.beta.-galactosidase). The term "expression vector" refers to a
vector that is used to express a polypeptide of interest in a host
cell.
[0090] A "host cell" refers to a cell that may be or has been a
recipient of a vector or isolated polynucleotide. Host cells may be
prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells
include mammalian cells, such as primate or non-primate animal
cells; fungal cells, such as yeast; plant cells; and insect cells.
Exemplary prokaryotic cells include bacterial cells; for example,
E. coli cells.
[0091] The term "isolated" as used herein refers to a molecule that
has been separated from at least some of the components with which
it is typically found in nature or produced. For example, a
polypeptide is referred to as "isolated" when it is separated from
at least some of the components of the cell in which it was
produced. Where a polypeptide is secreted by a cell after
expression, physically separating the supernatant containing the
polypeptide from the cell that produced it is considered to be
"isolating" the polypeptide. Similarly, a polynucleotide is
referred to as "isolated" when it is not part of the larger
polynucleotide (such as, for example, genomic DNA or mitochondrial
DNA, in the case of a DNA polynucleotide) in which it is typically
found in nature, or is separated from at least some of the
components of the cell in which it was produced, e.g., in the case
of an RNA polynucleotide. Thus, a DNA polynucleotide that is
contained in a vector inside a host cell may be referred to as
"isolated".
[0092] The terms "individual" or "subject" are used interchangeably
herein to refer to an animal; for example a mammal. In some
embodiments, methods of treating mammals, including, but not
limited to, humans, rodents, simians, felines, canines, equines,
bovines, porcines, ovines, caprines, mammalian laboratory animals,
mammalian farm animals, mammalian sport animals, and mammalian
pets, are provided. In some examples, an "individual" or "subject"
refers to an individual or subject in need of treatment for a
disease or disorder.
[0093] A "disease" or "disorder" as used herein refers to a
condition where treatment is needed.
[0094] The term "cancer" refers to a malignant proliferative
disorder associated with uncontrolled cell proliferation,
unrestrained cell growth, and decreased cell death via
apoptosis.
[0095] The term "tumor" is used herein to refer to a group of cells
that exhibit abnormally high levels of proliferation and growth. A
tumor may be benign, pre-malignant, or malignant; malignant tumor
cells are cancerous. Tumor cells may be solid tumor cells or
leukemic tumor cells. The term "tumor growth" is used herein to
refer to proliferation or growth by a cell or cells that comprise a
tumor that leads to a corresponding increase in the size of the
tumor.
[0096] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. "Treatment" as used herein,
covers any administration or application of a therapeutic for
disease in a mammal, including a human. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (e.g., not worsening) state of disease,
preventing spread (e.g., metastasis) of disease, delay or slowing
of disease progression, amelioration or palliation of the disease
state, and remission (whether partial or total), whether detectable
or undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Also
encompassed by "treatment" is a reduction of pathological
consequence of a proliferative disease. The methods of the
invention contemplate any one or more of these aspects of
treatment.
[0097] In the context of cancer, the term "treating" includes any
or all of: inhibiting growth of tumor cells or cancer cells,
inhibiting replication of tumor cells or cancer cells, lessening of
overall tumor burden and ameliorating one or more symptoms
associated with the disease.
[0098] The terms "inhibition" or "inhibit" refer to a decrease or
cessation of any phenotypic characteristic or to the decrease or
cessation in the incidence, degree, or likelihood of that
characteristic. To "reduce" or "inhibit" is to decrease, reduce or
arrest an activity, function, and/or amount as compared to a
reference. In certain embodiments, by "reduce" or "inhibit" is
meant the ability to cause an overall decrease of 20% or greater.
In another embodiment, by "reduce" or "inhibit" is meant the
ability to cause an overall decrease of 50% or greater. In yet
another embodiment, by "reduce" or "inhibit" is meant the ability
to cause an overall decrease of 75%, 85%, 90%, 95%, or 99%.
[0099] As used herein, "delaying development of a disease" means to
defer, hinder, slow, retard, stabilize, suppress and/or postpone
development of the disease (such as cancer). This delay can be of
varying lengths of time, depending on the history of the disease
and/or individual being treated. As is evident to one skilled in
the art, a sufficient or significant delay can, in effect,
encompass prevention, in that the individual does not develop the
disease. For example, a late stage cancer, such as development of
metastasis, may be delayed.
[0100] A "reference" as used herein, refers to any sample,
standard, or level that is used for comparison purposes. A
reference may be obtained from a healthy and/or non-diseased
sample. In some examples, a reference may be obtained from an
untreated sample. In some examples, a reference is obtained from a
non-diseased on non-treated sample of a subject individual. In some
examples, a reference is obtained from one or more healthy
individuals who are not the subject or patient.
[0101] "Preventing," as used herein, includes providing prophylaxis
with respect to the occurrence or recurrence of a disease in a
subject that may be predisposed to the disease but has not yet been
diagnosed with the disease.
[0102] An "effective amount" of an agent refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic or prophylactic result.
[0103] A "therapeutically effective amount" of a substance/molecule
of the invention, agonist or antagonist may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the substance/molecule, agonist or
antagonist to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the substance/molecule, agonist or
antagonist are outweighed by the therapeutically beneficial
effects. A therapeutically effective amount may be delivered in one
or more administrations.
[0104] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
will be less than the therapeutically effective amount.
[0105] The terms "pharmaceutical formulation" and "pharmaceutical
composition" refer to a preparation which is in such form as to
permit the biological activity of the active ingredient(s) to be
effective, and which contains no additional components which are
unacceptably toxic to a subject to which the formulation would be
administered. Such formulations may be sterile and essentially free
of endotoxins.
[0106] A "pharmaceutically acceptable carrier" refers to a
non-toxic solid, semisolid, or liquid filler, diluent,
encapsulating material, formulation auxiliary, or carrier
conventional in the art for use with a therapeutic agent that
together comprise a "pharmaceutical composition" for administration
to a subject. A pharmaceutically acceptable carrier is non-toxic to
recipients at the dosages and concentrations employed and is
compatible with other ingredients of the formulation. The
pharmaceutically acceptable carrier is appropriate for the
formulation employed.
[0107] A "sterile" formulation is aseptic or essentially free from
living microorganisms and their spores.
[0108] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive or sequential administration in any order.
[0109] The term "concurrently" is used herein to refer to
administration of two or more therapeutic agents, where at least
part of the administration overlaps in time or where the
administration of one therapeutic agent falls within a short period
of time relative to administration of the other therapeutic agent.
For example, the two or more therapeutic agents are administered
with a time separation of no more than about 60 minutes, such as no
more than about any of 30, 15, 10, 5, or 1 minutes.
[0110] The term "sequentially" is used herein to refer to
administration of two or more therapeutic agents where the
administration of one or more agent(s) continues after
discontinuing the administration of one or more other agent(s). For
example, administration of the two or more therapeutic agents are
administered with a time separation of more than about 15 minutes,
such as about any of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days,
3 days, 1 week, 2 weeks, or 1 month.
[0111] As used herein, "in conjunction with" refers to
administration of one treatment modality in addition to another
treatment modality. As such, "in conjunction with" refers to
administration of one treatment modality before, during or after
administration of the other treatment modality to the
individual.
[0112] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0113] An "article of manufacture" is any manufacture (e.g., a
package or container) or kit comprising at least one reagent, e.g.,
a medicament for treatment of a disease or disorder (e.g., cancer),
or a probe for specifically detecting a biomarker described herein.
In certain embodiments, the manufacture or kit is promoted,
distributed, or sold as a unit for performing the methods described
herein.
H-NOX Proteins
Overview of H-NOX Protein Family
[0114] Unless otherwise indicated, any wild-type or mutant H-NOX
protein can be used in the compositions, kits, and methods as
described herein. As used herein, an "H-NOX protein" means a
protein that has an H-NOX domain (named for Heme-Nitric oxide and
OXygen binding domain). An H-NOX protein may or may not contain one
or more other domains in addition to the H-NOX domain. H-NOX
proteins are members of a highly-conserved, well-characterized
family of hemoproteins (Iyer, L. M. et al. (Feb. 3, 2003). BMC
Genomics 4(1):5; Karow, D. S. et al. (Aug. 10, 2004). Biochemistry
43(31):10203-10211; Boon, E. M. et al. (2005). Nature Chem. Biol.
1:53-59; Boon, E. M. et al. (October 2005). Curr. Opin. Chem. Biol.
9(5):441-446; Boon, E. M. et al. (2005). J. Inorg. Biochem.
99(4):892-902). H-NOX proteins are also referred to as Pfam 07700
proteins or HNOB proteins (Pfam--A database of protein domain
family alignments and Hidden Markov Models, Copyright (C) 1996-2006
The Pfam Consortium; GNU LGPL Free Software Foundation, Inc., 59
Temple Place--Suite 330, Boston, Mass. 02111-1307, USA). In some
embodiments, an H-NOX protein has, or is predicted to have, a
secondary structure that includes six alpha-helices, followed by
two beta-strands, followed by one alpha-helix, followed by two
beta-strands. An H-NOX protein can be an apoprotein that is capable
of binding heme or a holoprotein with heme bound. An H-NOX protein
can covalently or non-covalently bind a heme group. Some H-NOX
proteins bind NO but not Oz, and others bind both NO and O.sub.2.
H-NOX domains from facultative aerobes that have been isolated bind
NO but not O.sub.2. H-NOX proteins from obligate aerobic
prokaryotes, C. elegans, and D. melanogaster bind NO and O.sub.2.
Mammals have two H-NOX proteins: .beta.1 and .beta.2. An alignment
of mouse, rat, cow, and human H-NOX sequences shows that these
species share >99% identity. In some embodiments, the H-NOX
domain of an H-NOX protein or the entire H-NOX protein is at least
about any of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97,
98, 99, or 99.5% identical to that of the corresponding region of a
naturally-occurring Thermoanaerobacter tengcongensis H-NOX protein
(e.g. SEQ ID NO:2) or a naturally-occurring sGC protein (e.g., a
naturally-occurring sGC .beta.1 protein). In some embodiments, the
H-NOX domain of an H-NOX protein or the entire H-NOX protein is at
least about any of 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%,
70-80%, 80-90%, 90-95%, 95-99, or 99-99.9% identical to that of the
corresponding region of a naturally-occurring Thermoanaerobacter
tengcongensis H-NOX protein (e.g. SEQ ID NO:2) or a
naturally-occurring sGC protein (e.g., a naturally-occurring sGC 1
protein). As discussed further herein, an H-NOX protein may
optionally contain one or more mutations relative to the
corresponding naturally-occurring H-NOX protein. In some
embodiments, the H-NOX protein includes one or more domains in
addition to the H-NOX domain. In particular embodiments, the H-NOX
protein includes one or more domains or the entire sequence from
another protein. For example, the H-NOX protein may be a fusion
protein that includes an H-NOX domain and part or all of another
protein, such as albumin (e.g., human serum albumin). In some
embodiments, only the H-NOX domain is present. In some embodiments,
the H-NOX protein does not comprise a guanylyl cyclase domain. In
some embodiments, the H-NOX protein comprises a tag; for example, a
His.sub.6 tag.
Polymeric H-NOX Proteins
[0115] In some aspects, the invention provides polymeric H-NOX
proteins comprising two or more H-NOX domains. The two or more
H-NOX domains may be covalently linked or noncovalently linked. In
some embodiments, the polymeric H-NOX protein is in the form of a
dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an
octomer, a nanomer, or a decamer. In some embodiments, the
polymeric H-NOX protein comprises homologous H-NOX domains. In some
embodiments, the polymeric H-NOX protein comprises heterologous
H-NOX domains; for example, the H-NOX domains may comprises amino
acid variants of a particular species of H-NOX domain or may
comprise H-NOX domains from different species. In some embodiments,
at least one of the H-NOX domains of a polymeric H-NOX protein
comprises a mutation corresponding to an L144F mutation of T.
tengcongensis H-NOX. In some embodiments, at least one of the H-NOX
domains of a polymeric H-NOX protein comprises a mutation
corresponding to a W9F/L144F mutation of T. tengcongensis H-NOX. In
some embodiments, the polymeric H-NOX proteins comprise one or more
polymerization domains. In some embodiments, the polymeric H-NOX
protein is a trimeric H-NOX protein. In some embodiments, the
polymeric H-NOX protein comprises at least one trimerization
domain. In some embodiments, the trimeric H-NOX protein comprises
three T. tengcongensis H-NOX domains. In some embodiments the
trimeric H-NOX domain comprises three T. tengcongensis L144F H-NOX
domains (trimeric Tt H-NOX L144F). In some embodiments the trimeric
H-NOX domain comprises three T. tengcongensis W9F/L144F H-NOX
domains
[0116] In some aspects of the invention, the polymeric H-NOX
protein comprises two or more associated monomers. The monomers may
be covalently linked or noncovalently linked. In some embodiments,
monomeric subunits of a polymeric H-NOX protein are produced where
the monomeric subunits associate in vitro or in vivo to form the
polymeric H-NOX protein. In some embodiments, the monomers comprise
an H-NOX domain and a polymerization domain. In some embodiments,
the polymerization domain is covalently linked to the H-NOX domain;
for example, the C-terminus of the H-NOX domain is covalently
linked to the N-terminus or the C-terminus of the polymerization
domain. In other embodiments, the N-terminus of the H-NOX domain is
covalently linked to the N-terminus or the C-terminus of the
polymerization domain. In some embodiments, an amino acid spacer is
covalently linked between the H-NOX domain and the polymerization
domain. An "amino acid spacer" and an "amino acid linker" are used
interchangeably herein. In some embodiments, at least one of the
monomeric subunits of a polymeric H-NOX protein comprises a
mutation corresponding to an L144F mutation of T. tengcongensis
H-NOX. In some embodiments, at least one of the monomeric subunits
of a polymeric H-NOX protein comprises a mutation corresponding to
a W9F/L144F mutation of T. tengcongensis H-NOX. In some embodiments
the polymeric H-NOX protein is a trimeric H-NOX protein. In some
embodiments, the monomer of a trimeric H-NOX protein comprises an
H-NOX domain and a foldon domain of T4 bacteriophage. In some
embodiments, the monomer of a trimeric H-NOX protein comprises a T.
tengcongensis H-NOX domain and a foldon domain. In some
embodiments, the monomer of a trimeric H-NOX protein comprises a T.
tengcongensis L144F H-NOX domain and a foldon domain. In some
embodiments, the monomer of a trimeric H-NOX protein comprises a T.
tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some
embodiments, the trimer H-NOX protein comprises three monomers,
each monomer comprising a T. tengcongensis L144F H-NOX domain and a
foldon domain. In some embodiments, the H-NOX domain is linked to
the foldon domain with an amino acid linker; for example a
Gly-Ser-Gly linker. In some embodiments, at least one H-NOX domain
comprises a tag. In some embodiments, at least one H-NOX domain
comprises a His.sub.6 tag. In some embodiments, the His.sub.6 tag
is linked to the foldon domain with an amino acid linker; for
example an Arg-Gly-Ser linker. In some embodiments, all of the
H-NOX domains comprise a His.sub.6 tag. In some embodiments, the
trimeric H-NOX protein comprises the amino acid sequence set forth
in SEQ ID NO:6 or SEQ ID NO:8.
[0117] The exemplary H-NOX domain from T. tengcongensis is
approximately 26.7 kDal. In some embodiments, the polymeric H-NOX
protein has an atomic mass greater than any of about 50 kDal, 75
kDal, 100 kDal, 125kDal, to about 150 kDal.
[0118] The invention provides polymeric H-NOX proteins that show
greater accumulation in one or more tissues in an individual
compared to a corresponding monomeric H-NOX protein comprising a
single H-NOX domain following administration of the H-NOX protein
to the individual. A corresponding H-NOX protein refers to a
monomeric form of the H-NOX protein comprising at least one of the
H-NOX domains of the polymeric H-NOX protein. Tissues of
preferential polymeric H-NOX accumulation include, but are not
limited to tumors and tissue with damaged vasculature. In some
embodiments the polymeric H-NOX protein persists in a mammal for at
least about 1, 2, 3, 4, 6, 12 or 24 hours following administration
of the H-NOX protein to the individual. In some embodiments the
polymeric H-NOX protein persists in a mammal for about 1-2, 2-3,
3-4, 4-6, 6-12 or 12-24 hours following administration of the H-NOX
protein to the individual In some embodiments, less than about 10%
of the polymeric H-NOX is cleared from mammal by the kidneys within
less than any of about 1 hour, 2 hours or 3 hours following
administration of the H-NOX protein to the individual.
Sources of H-NOX Proteins and H-NOX Domains
[0119] H-NOX proteins and H-NOX domains from any genus or species
can be used in the compositions, kits, and methods described
herein. In various embodiments, the H-NOX protein or the H-NOX
domains of a polymeric H-NOX protein is a protein or domain from a
mammal (e.g., a primate (e.g., human, monkey, gorilla, ape, lemur,
etc), a bovine, an equine, a porcine, a canine, or a feline), an
insect, a yeast, or a bacteria or is derived from such a protein.
Exemplary mammalian H-NOX proteins include wild-type human and rat
soluble guanylate cyclase (such as the 31 subunit). Non-limiting
examples of H-NOX proteins include wild-type mammalian H-NOX
proteins, e.g. H. sapiens, M. musculus, C. familiaris, B. Taurus,
C. lupus and R. norvegicus and examples of prokaryotic wild-type
H-NOX proteins include T. tengcongensis, V. cholera, V. fischerii,
N. punctiforme, D. desulfuricans, L. pneumophila 1, L. pneumophila
2, and C. acetobutvlicum. Examples of H-NOX proteins including
their NCBI accession numbers may be found in U.S. Pat. Nos.
8,404,631 and 8,404,632, WO 2007/139791 and WO 2007/139767; the
contents of each is incorporated herein by reference in its
entirety.
[0120] Additional H-NOX proteins, H-NOX domains of polymeric H-NOX
proteins, and nucleic acids, which may be suitable for use in the
pharmaceutical compositions and methods described herein, can be
identified using standard methods. For example, standard sequence
alignment and/or structure prediction programs can be used to
identify additional H-NOX proteins and nucleic acids based on the
similarity of their primary and/or predicted protein secondary
structure with that of known H-NOX proteins and nucleic acids. For
example, the Pfam database uses defined alignment algorithms and
Hidden Markov Models (such as Pfam 21.0) to categorize proteins
into families, such as the H-NOX protein family (Pfam--A database
of protein domain family alignments and Hidden Markov Models,
Copyright (C) 1996-2006 The Pfam Consortium; GNU LGPL Free Software
Foundation, Inc., 59 Temple Place--Suite 330, Boston, Mass.
02111-1307, USA). Standard databases such as the swissprot-trembl
database (world-wide web at "expasy.org", Swiss Institute of
Bioinformatics Swiss-Prot group CMU--1 rue Michel Servet CH-1211
Geneva 4, Switzerland) can also be used to identify members of the
H-NOX protein family. The secondary and/or tertiary structure of an
H-NOX protein can be predicted using the default settings of
standard structure prediction programs, such as PredictProtein (630
West, 168 Street, BB217, New York, N.Y. 10032, USA). Alternatively,
the actual secondary and/or tertiary structure of an H-NOX protein
can be determined using standard methods.
[0121] In some embodiments, the H-NOX domain has the same amino
acid in the corresponding position as any of following distal
pocket residues in T. tengcongensis H-NOX: Thr4, Ile5, Thr8, Trp9,
Trp67, Asn74, Ile75, Phe78, Phe82, Tyr140, Leu144, or any
combination of two or more of the foregoing. In some embodiments,
the H-NOX domain has a proline or an arginine in a position
corresponding to that of Pro115 or Arg135 of T. tengcongensis
H-NOX, respectively, based on sequence alignment of their amino
acid sequences. In some embodiments, the H-NOX domain has a
histidine that corresponds to His105 of R. norvegicus .beta.1
H-NOX. In some embodiments, the H-NOX domain has or is predicted to
have a secondary structure that includes six alpha-helices,
followed by two beta-strands, followed by one alpha-helix, followed
by two beta-strands. This secondary structure has been reported for
H-NOX proteins.
[0122] If desired, a newly identified H-NOX protein or H-NOX domain
can be tested to determine whether it binds heme using standard
methods. The ability of an H-NOX domain to function as an O.sub.2
carrier can be tested by determining whether the H-NOX domain binds
O.sub.2 using standard methods, such as those described herein. If
desired, one or more of the mutations described herein can be
introduced into the H-NOX domain to optimize its characteristics as
an O.sub.2 carrier. For example, one or more mutations can be
introduced to alter its O.sub.2 dissociation constant, k.sub.off
for oxygen, rate of heme autoxidation, NO reactivity, NO stability
or any combination of two or more of the foregoing. Standard
techniques such as those described herein can be used to measure
these parameters.
Mutant H-NOX Proteins
[0123] As discussed further herein, an H-NOX protein or an H-NOX
domain of a polymeric H-NOX protein may contain one or more
mutations, such as a mutation that alters the O.sub.2 dissociation
constant, the k.sub.off for oxygen, the rate of heme autoxidation,
the NO reactivity, the NO stability, or any combination of two or
more of the foregoing compared to that of the corresponding
wild-type protein. In some embodiments, the invention provides a
polymeric H-NOX protein comprising one or more H-NOX domains that
may contain one or more mutations, such as a mutation that alters
the O.sub.2 dissociation constant, the k.sub.off for oxygen, the
rate of heme autoxidation, the NO reactivity, the NO stability, or
any combination of two or more of the foregoing compared to that of
the corresponding wild-type protein. Panels of engineered H-NOX
domains may be generated by random mutagenesis followed by
empirical screening for requisite or desired dissociation
constants, dissociation rates, NO-reactivity, stability,
physio-compatibility, or any combination of two or more of the
foregoing in view of the teaching provided herein using techniques
as described herein and, additionally, as known by the skilled
artisan. Alternatively, mutagenesis can be selectively targeted to
particular regions or residues such as distal pocket residues
apparent from the experimentally determined or predicted
three-dimensional structure of an H-NOX protein (see, for example,
Boon, E. M. et al. (2005). Nature Chemical Biology 1:53-59, which
is hereby incorporated by reference in its entirety, particularly
with respect to the sequences of wild-type and mutant H-NOX
proteins) or evolutionarily conserved residues identified from
sequence alignments (see, for example, Boon E. M. et al. (2005).
Nature Chemical Biology 1:53-59, which is hereby incorporated by
reference in its entirety, particularly with respect to the
sequences of wild-type and mutant H-NOX proteins).
[0124] In some embodiments of the invention, the mutant H-NOX
protein or mutant H-NOX domain of a polymeric H-NOX protein has a
sequence that differs from that of all H-NOX proteins or domains
occurring in nature. In various embodiments, the amino acid
sequence of the mutant protein is at least about any of 10, 15, 20,
25, 30, 40, 50, 60, 70, 80, 90, 95, 97, 98, 99, or 99.5% identical
to that of the corresponding region of an H-NOX protein occurring
in nature. In various embodiments, the amino acid sequence of the
mutant protein is about 10-20%, 20-30%, 30-40%, 40-50%, 50-60%,
60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or 99.5% identical to that
of the corresponding region of an H-NOX protein occurring in
nature. In some embodiments, the mutant protein is a protein
fragment that contains at least about any of 25, 50, 75, 100, 150,
200, 300, or 400 contiguous amino acids from a full-length protein.
In some embodiments, the mutant protein is a protein fragment that
contains 25-50, 50-75, 75-100, 100-150, 150-200, 200-300, or
300-400 contiguous amino acids from a full-length protein. Sequence
identity can be measured, for example, using sequence analysis
software with the default parameters specified therein (e.g.,
Sequence Analysis Software Package of the Genetics Computer Group,
University of Wisconsin Biotechnology Center, 1710 University
Avenue, Madison, Wis. 53705). This software program matches similar
sequences by assigning degrees of homology to various amino acids
replacements, deletions, and other modifications.
[0125] In some embodiments of the invention, the mutant H-NOX
protein or mutant H-NOX domain of a polymeric H-NOX protein
comprises the insertion of one or more amino acids (e.g., the
insertion of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). In some
embodiments of the invention, the mutant H-NOX protein or mutant
H-NOX domain comprises the deletion of one or more amino acids
(e.g., a deletion of N-terminal, C-terminal, and/or internal
residues, such as the deletion of at least about any of 5, 10, 15,
25, 50, 75, 100, 150, 200, 300, or more amino acids or a deletion
of 5-10, 10-15, 15-25, 25-50, 50-75, 75-100, 100-150, 150-200,
200-300, or 300-400 amino acids). In some embodiments of the
invention, the mutant H-NOX protein or mutant H-NOX domain
comprises the replacement of one or more amino acids (e.g., the
replacement of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), or
combinations of two or more of the foregoing. In some embodiments,
a mutant protein has at least one amino acid alteration compared to
a protein occurring in nature. In some embodiments, a mutant
nucleic acid sequence encodes a protein that has at least one amino
acid alteration compared to a protein occurring in nature. In some
embodiments, the nucleic acid is not a degenerate version of a
nucleic acid occurring in nature that encodes a protein with an
amino acid sequence identical to a protein occurring in nature.
[0126] In some embodiments the mutation in the H-NOX protein or
H-NOX domain of a polymeric H-NOX protein is an evolutionary
conserved mutations (also denoted class I mutations). Examples of
class I mutations are listed in Table 1A. In Table 1A, mutations
are numbered/annotated according to the sequence of human .beta.1
H-NOX, but are analogous for all H-NOX sequences. Thus, the
corresponding position in any other H-NOX protein can be mutated to
the indicated residue. For example, Phe4 of human .beta.1 H-NOX can
be mutated to a tyrosine since other H-NOX proteins have a tyrosine
in this position. The corresponding phenylalanine residue can be
mutated to a tyrosine in any other H-NOX protein. In particular
embodiments, the one or more mutations are confined to
evolutionarily conserved residues. In some embodiments, the one or
more mutations may include at least one evolutionarily conserved
mutation and at least one non-evolutionarily conserved mutation. If
desired, these mutant H-NOX proteins are subjected to empirical
screening for NO/O.sub.2 dissociation constants, NO-reactivity,
stability, and physio-compatibility in view of the teaching
provided herein.
TABLE-US-00001 TABLE 1A Exemplary Class I H-NOX mutations targeting
evolutionary conserved residues F4Y Q30G I145Y F4L E33P I145H H7G
N61G K151E A8E C78H I157F L9W A109F E183F
[0127] In some embodiments, the mutation is a distal pocket
mutation, such as mutation of a residue in alpha-helix A, D, E, or
G (Pellicena, P. et al. (Aug. 31, 2004). Proc Natl. Acad Sci USA
101(35):12854-12859). Exemplary distal pocket mutations (also
denoted class II mutations) are listed in Table 1B. In Table 1B,
mutations are numbered/annotated according to the sequence of human
.beta.1 H-NOX, but are analogous for all H-NOX sequences. Because
several substitutions provide viable mutations at each recited
residue, the residue at each indicated position can be changed to
any other naturally or non-naturally-occurring amino acid (denoted
"X"). Such mutations can produce H-NOX proteins with a variety of
desired affinity, stability, and reactivity characteristics.
TABLE-US-00002 TABLE 1B Exemplary Class II H-NOX mutations
targeting distal pocket residues V8X M73X I145X L9X F77X I149X F70X
C78X
[0128] In particular embodiments, the mutation is a heme distal
pocket mutation. As described herein, a crucial molecular
determinant that prevents O.sub.2 binding in NO-binding members of
the H-NOX family is the lack of a H-bond donor in the distal pocket
of the heme. Accordingly, in some embodiments, the mutation alters
H-bonding between the H-NOX domain and the ligand within the distal
pocket. In some embodiments, the mutation disrupts an H-bond donor
of the distal pocket and/or imparts reduced O.sub.2 ligand-binding
relative to the corresponding wild-type H-NOX domain. Exemplary
distal pocket residues include Thr4, Ile5, Thr8, Trp9, Trp67,
Asn74, Ile75, Phe78, Phe82, Tyr140, and Leu144 of T. tengcongensis
H-NOX and the corresponding residues in any other H-NOX protein. In
some embodiments, the H-NOX protein or H-NOX domain of a polymeric
H-NOX protein comprises one or more distal pocket mutations. In
some embodiments, the H-NOX protein or H-NOX domain of a polymeric
H-NOX protein comprises one, two, three, four, five, six, seven,
eight, nine, ten or more than ten distal pocket mutations. In some
embodiments, the distal pocket mutation corresponds to a L144F
mutation of T. tengcongensis H-NOX. In some embodiments, the distal
pocket mutation is a L144F mutation of T. tengcongensis H-NOX. In
some embodiments, H-NOX protein or the H-NOX domain of a polymeric
H-NOX protein comprises two distal pocket mutations. In some
embodiments, the H-NOX protein or H-NOX domain of a polymeric H-NOX
protein corresponds to a W9F/L144F mutation of T. tengcongensis
H-NOX. In some embodiments, the H-NOX protein or H-NOX domain of a
polymeric H-NOX protein is a W9F/L144F mutation of T. tengcongensis
H-NOX.
[0129] Residues that are not in the distal pocket can also affect
the three-dimensional structure of the heme group; this structure
in turn affects the binding of O.sub.2 and NO to iron in the heme
group. Accordingly, in some embodiments, the H-NOX protein or H-NOX
domain of a polymeric H-NOX protein has one or more mutations
outside of the distal pocket. Examples of residues that can be
mutated but are not in the distal pocket include Pro115 and Arg135
of T. tengcongensis H-NOX. In some embodiments, the mutation is in
the proximal pocket which includes His105 as a residue that ligates
to the heme iron.
[0130] In some embodiments when two or more mutations are present;
at least one mutation is in the distal pocket, and at least one
mutation is outside of the distal pocket (e.g., a mutation in the
proximal pocket). In some embodiments, all the mutations are in the
distal pocket.
[0131] To reduce the immunogenicity of H-NOX protein or H-NOX
domains derived from sources other than humans, amino acids in an
H-NOX protein or H-NOX domain can be mutated to the corresponding
amino acids in a human H-NOX. For example, one or more amino acids
on the surface of the tertiary structure of a non-human H-NOX
protein or H-NOX domain can be mutated to the corresponding amino
acid in a human H-NOX protein or H-NOX domain. In some variations,
mutation of one or more surface amino acids may be combined with
mutation of two or more distal pocket residues, mutation of one or
more residues outside of the distal pocket (e.g., a mutation in the
proximal pocket), or combinations of two or more of the
foregoing.
[0132] The invention also relates to any combination of mutation
described herein, such as double, triple, or higher multiple
mutations. For example, combinations of any of the mutations
described herein can be made in the same H-NOX protein. Note that
mutations in equivalent positions in other mammalian or
non-mammalian H-NOX proteins are also encompassed by this
invention. Exemplary mutant H-NOX proteins or mutant H-NOX domains
comprise one or more mutations that impart altered O.sub.2 or NO
ligand-binding relative to the corresponding wild-type H-NOX domain
and are operative as a physiologically compatible mammalian O.sub.2
blood gas carrier.
[0133] The residue number for a mutation indicates the position in
the sequence of the particular H-NOX protein being described. For
example, T. tengcongensis I5A refers to the replacement of
isoleucine by alanine at the fifth position in T. tengcongensis
H-NOX. The same isoleucine to alanine mutation can be made in the
corresponding residue in any other H-NOX protein or H-NOX domain
(this residue may or may not be the fifth residue in the sequence
of other H-NOX proteins). Since the amino acid sequences of
mammalian .beta.1 H-NOX domains differ by at most two amino acids,
mutations that produce desirable mutant H-NOX proteins or H-NOX
domains when introduced into wild-type rat .beta.1 H-NOX proteins
are also expected to produce desirable mutant H-NOX proteins or
H-NOX domains when introduced into wild-type .beta.1 H-NOX proteins
or H-NOX domains from other mammals, such as humans.
[0134] In some embodiments, the H-NOX protein is a trimer
comprising three T. tengcongensis L144F H-NOX domains and three
foldon domains. In some embodiments, the H-NOX protein is a trimer
comprising three T. tengcongensis W9F/L144F H-NOX domains and three
foldon domains. In some embodiments, the H-NOX protein is a trimer
comprising three T. tengcongensis wildtype H-NOX domains and three
foldon domains.
Modifications to H-NOX Proteins
[0135] Any of the wild-type or mutant H-NOX proteins, including
polymeric H-NOX proteins, can be modified and/or formulated using
standard methods to enhance therapeutic or industrial applications.
For example, and particularly as applied to heterologous engineered
H-NOX proteins, a variety of methods are known in the art for
insulating such agents from immune surveillance, including
crosslinking, PEGylation, carbohydrate decoration, etc. (e.g.,
Rohlfs, R. J. et al. (May 15, 1998). J. Biol. Chem.
273(20):12128-12134; Migita, R. et al. (June 1997). J. Appl.
Physiol. 82(6):1995-2002; Vandegriff, K. D. et al. (Aug. 15, 2004).
Biochem J. 382(Pt 1):183-189, which are each hereby incorporated by
reference in their entireties, particularly with respect to the
modification of proteins) as well as other techniques known to the
skilled artisan. Fusing an H-NOX protein, including a polymeric
H-NOX protein, with a human protein such as human serum albumin can
increase the serum half-life, viscosity, and colloidal oncotic
pressure. In some embodiments, an H-NOX protein is modified during
or after its synthesis to decrease its immunogenicity and/or to
increase its plasma retention time. H-NOX proteins can also be
encapsulated (such as encapsulation within liposomes or
nanoparticles).
[0136] In some embodiments, the H-NOX protein comprises one of more
tags; e.g. to assist in purification of the H-NOX protein. Examples
of tags include, but are not limited to His.sub.6, FLAG, GST, and
MBP. In some embodiments, the H-NOX protein comprises one of more
His.sub.6 tags. The one or more His.sub.6 tags may be removed prior
to use of the polymeric H-NOX protein; e.g. by treatment with an
exopeptidase. In some embodiments, the H-NOX protein is a trimer
comprising three T. tengcongensis L144F H-NOX domains, three foldon
domains, and three His.sub.6 tags. In some embodiments, the H-NOX
protein is a trimer comprising three T. tengcongensis W9F/L144F
H-NOX domains, three foldon domains, and three His.sub.6 tags. In
some embodiments, the H-NOX protein is a trimer comprising three T.
tengcongensis wildtype H-NOX domains, three foldon domains, and
three His.sub.6 tags.
[0137] In some embodiments, the H-NOX protein comprises one or more
polyethylene glycol (PEG) molecules (i.e., is PEGylated). In some
embodiments, the H-NOX protein is a trimer comprising three T.
tengcongensis L144F H-NOX domains, three foldon domains, and one or
more polyethylene glycol molecules (PEGylated trimer Tt H-NOX
L144F). In some embodiments, the H-NOX protein is a trimer
comprising three T. tengcongensis W9F/L144F H-NOX domains, three
foldon domains, and one or more polyethylene glycol molecules. In
some embodiments, the H-NOX protein is a trimer comprising three T.
tengcongensis wildtype H-NOX domains, three foldon domains, and one
or more polyethylene glycol molecules. In some embodiments, the
molecular weight of the PEG is between about 1 kDa and about 50
kDa. In some embodiments, the molecular weight of the PED is
between about any of 1 kDa and 50 kDa, 1 kDa and 40 kDa, 1 kDa and
30 kDa, 1 kDa and 25 kDa, 1 kDa and 20 kDa, 1 kDa and 15 kDa, 1 kDa
and 10 kDa, 1 kDa and 5 kDa, 5 kDa and 50 kDa, 5 kDa and 40 kDa, 5
kDa and 30 kDa, 5 kDa and 25 kDa, 5 kDa and 20 kDa, 5 kDa and 15
kDa, 5 kDa and 10 kDa, 10 kDa and 50 kDa, 10 kDa and 40 kDa, 10 kDa
and 30 kDa, 10 kDa and 25 kDa, 10 kDa and 20 kDa, 10 kDa and 15
kDa, 15 kDa and 50 kDa, 15 kDa and 40 kDa, 15 kDa and 35 kDa, 15
kDa and 30 kDa, 15 kDa and 25 kDa, 15 kDa and 20 kDa, 20 kDa and 50
kDa, 20 kDa and 40 kDa, 20 kDa and 30 kDa, 20 kDa and 25 kDa, 25
kDa and 50 kDa, 25 kDa and 40 kDa, 25 kDa and 30 kDa, 30 kDa and 50
kDa, 30 kDa and 40 kDa, or 40 kDa and 50 kDa. In some embodiments,
the H-NOX protein comprises any one of more than about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 PEG molecules
per H-NOX monomer or any number therebetween. In some embodiments,
the H-NOX protein comprises an average of 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 PEG molecules per H-NOX
monomer or any number therebetween.
Polymerization Domains
[0138] In some aspects, the invention provides polymeric H-NOX
proteins comprising two or more H-NOX domains and one or more
polymerization domains. Polymerization domains are used to link two
or more H-NOX domains to form a polymeric H-NOX protein. One or
more polymerization domains may be used to produce dimers, trimers,
tetramers, pentamers, etc. of H-NOX proteins. Polymerization
domains are known in the art, such as: the foldon of T4
bacteriophage fibritin, Arc, POZ, coiled coil domains (including
GCN4, leucine zippers, Velcro), uteroglobin, collagen, 3-stranded
coiled colis (matrilin-1), thrombosporins, TRPV1-C, P53, Mnt,
avadin, streptavidin, Bcr-Abl, COMP, verotoxin subunit B, CamKII,
RCK, and domains from N ethylmaleimide-sensitive fusion protein,
STM3548, KaiC, TyrR, Hcp1, CcmK4, GP41, anthrax protective antigen,
aerolysin, a-hemolysin, C4b-binding protein, Mi-CK, arylsurfatase
A, and viral capsid proteins. The polymerization domains may be
covalently or non-covalently linked to the H-NOX domains. In some
embodiments, a polymerization domain is linked to an H-NOX domain
to form a monomer subunit such that the polymerization domains from
a plurality of monomer subunits associate to form a polymeric H-NOX
domain. In some embodiments, the C-terminus of an H-NOX domain is
linked to the N-terminus of a polymerization domain. In other
embodiments, the N-terminus of an H-NOX domain is linked to the
N-terminus of a polymerization domain. In yet other embodiments,
the C-terminus of an H-NOX domain is linked to the C-terminus of a
polymerization domain. In some embodiments, the N-terminus of an
H-NOX domain is linked to the C-terminus of a polymerization
domain.
[0139] Linkers may be used to join a polymerization domain to an
H-NOX domain; for example, for example, amino acid linkers. In some
embodiments, a linker comprising any one of one, two, three, four,
five, six, seven, eight, nine, ten or more than ten amino acids may
be placed between the polymerization domain and the H-NOX domain.
Exemplary linkers include but are not limited to Gly-Ser-Gly and
Arg-Gly-Ser linkers.
Bacteriophage T4 Fibritin Trimerization Domain
[0140] An exemplary polymerization domain is the foldon domain of
bacteriophage T4. The wac gene from the bacteriophage T4 encodes
the fibritin protein, a 486 amino acid protein with a C-terminal
trimerization domain (residues 457-483) (Efimov, V. P. et al.
(1994) J Mol Biol 242:470-486). The domain is able to trimerize
fibritin both in vitro and in vivo (Boudko, S. P. et al. (2002) Eur
J Biochem 269:833-841; Letarov, A. V., et al., (1999) Biochemistry
(Mosc) 64:817-823; Tao, Y., et al., (1997) Structure 5:789-798).
The isolated 27 residue trimerization domain, often referred to as
the "foldon domain," has been used to construct chimeric trimers in
a number of different proteins (including HIV envelope
glycoproteins (Yang, X. et al., (2002) J Virol 76:4634-4642),
adenoviral adhesins (Papanikolopoulou, K., et al., (2004) J Biol
Chem 279:8991-8998; Papanikolopoulou, K. et al. (2004) J Mol Biol
342:219-227), collagen (Zhang, C., et al. (2009) Biotechnol Prog
25:1660-1668), phage P22 gp26 (Bhardwaj, A., et al. (2008) Protein
Sci 17:1475-1485), and rabies virus glycoprotein (Sissoeff, L., et
al. (2005) J Gen Virol 86:2543-2552). An exemplary sequence of the
foldon domain is shown in FIG. 1 and provided by SEQ ID NO:4.
[0141] The isolated foldon domain folds into a single 0-hairpin
structure and trimerizes into a .beta.-propeller structure
involving three hairpins (Guthe, S. et al. (2004) J Mol Biol
337:905-915). The structure of the foldon domain alone has been
determined by NMR (Guthe, S. et al. (2004) J Mol Biol 337:905-915)
and the structures of several proteins trimerized with the foldon
domain have been solved by X-ray crystallography (Papanikolopoulou,
K., et al., (2004) J Biol Chem 279:8991-8998; Stetefeld, J. et al.
(2003) Structure 11:339-346; Yokoi, N. et al. (2010) Small
6:1873-1879). The domain folds and trimerizes rapidly reducing the
opportunity for misfolding intermediates or off-pathway
oligomerization products (Guthe, S. et al. (2004) J Mol Biol
337:905-915). The foldon domain is very stable, able to maintain
tertiary structure and oligomerization in >10% SDS, 6.0 M
guanidine hydrochloride, or 80.degree. C. (Bhardwaj, A., et al.
(2008) Protein Sci 17:1475-1485; Bhardwaj, A., et al. (2007) J Mol
Biol 371:374-387) and can improve the stability of sequences fused
to the foldon domain (Du, C. et al. (2008) Appl Microbiol
Biotechnol 79:195-202.
[0142] In some embodiments, the C-terminus of an H-NOX domain is
linked to the N-terminus of a foldon domain. In other embodiments,
the N-terminus of an H-NOX domain is linked to the N-terminus of a
foldon domain. In yet other embodiments, the C-terminus of an H-NOX
domain is linked to the C-terminus of a foldon domain. In some
embodiments, the N-terminus of an H-NOX domain is linked to the
C-terminus of a foldon domain.
[0143] In some embodiments, linkers are be used to join a foldon
domain to an H-NOX domain. In some embodiments, a linker comprising
any one of one, two, three, four, five, six, seven, eight, nine,
ten or more than ten amino acids may be placed between the
polymerization domain and the H-NOX domain. Exemplary linkers
include but are not limited to Gly-Ser-Gly and Arg-Gly-Ser linkers.
In some embodiments, the invention provides a trimeric H-NOX
protein comprising from N-terminus to C-terminus: a T.
tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker, and a
foldon domain. In some embodiments, the invention provides a
trimeric H-NOX protein comprising from N-terminus to C-terminus: a
T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker, a
foldon domain, an Arg-Gly-Ser amino acid linker, and a His.sub.6
tag. In some embodiments, the T. tengcongensis H-NOX domain
comprises an L144F mutation. In some embodiments, the T.
tengcongensis H-NOX domain comprises a W9F mutation and a L144F
mutation. In some embodiments, the T. tengcongensis H-NOX domain is
a wild-type H-NOX domain.
Monomeric H-NOX Domain Subunits
[0144] In one aspect, the invention provides recombinant monomeric
H-NOX proteins (i.e. monomeric H-NOX subunits of polymeric H-NOX
proteins) that can associate to form polymeric H-NOX proteins. In
some embodiments, the invention provides recombinant H-NOX proteins
comprising an H-NOX domain as described herein and a polymerization
domain. The H-NOX domain and the polymerization domain may be
covalently linked or noncovalently linked. In some embodiments, the
C-terminus of an H-NOX domain of the recombinant monomeric H-NOX
protein is linked to the N-terminus of a polymerization domain. In
other embodiments, the N-terminus of an H-NOX domain of the
recombinant monomeric H-NOX protein is linked to the N-terminus of
a polymerization domain. In yet other embodiments, the C-terminus
of an H-NOX domain of the recombinant monomeric H-NOX protein is
linked to the C-terminus of a polymerization domain. In some
embodiments, the N-terminus of an H-NOX domain of the recombinant
monomeric H-NOX protein is linked to the C-terminus of a
polymerization domain. In some embodiments, the recombinant
monomeric H-NOX protein does not comprise a guanylyl cyclase
domain.
[0145] In some embodiments, the monomeric H-NOX protein comprises a
wild-type H-NOX domain. In some embodiments of the invention, the
monomeric H-NOX protein comprises one of more mutations in the
H-NOX domain. In some embodiments, the one or more mutations alter
the O.sub.2 dissociation constant, the k.sub.off for oxygen, the
rate of heme autooxidation, the NO reactivity, the NO stability or
any combination of two or more of the foregoing compared to that of
the corresponding wild-type H-NOX domain. In some embodiments, the
mutation is a distal pocket mutation. In some embodiments, the
mutation comprises a mutation that is not in the distal pocket. In
some embodiments, the distal pocket mutation corresponds to a L144
mutation of T. tengcongensis (e.g. a L144F mutation). In some
embodiments, the recombinant monomeric H-NOX protein comprises two
distal pocket mutations corresponding to a W9 and a L144 mutation
of T. tengcongensis (e.g. a W9F/L144F mutation).
[0146] In some aspects, the invention provides recombinant
monomeric H-NOX proteins that associate to form trimeric H-NOX
proteins. In some embodiments, the recombinant H-NOX protein
comprises an H-NOX domain and a trimerization domain. In some
embodiments, the trimerization domain is a foldon domain as
discussed herein. In some embodiments, the H-NOX domain is a T.
tengcongensis H-NOX domain. In some embodiments the C-terminus of
the T. tengcongensis H-NOX domain is covalently linked to the
N-terminus of the foldon domain. In some embodiments the C-terminus
of the T. tengcongensis H-NOX domain is covalently linked to the
C-terminus of the foldon domain. In some embodiments, the T.
tengcongensis domain is an L144F H-NOX domain. In some embodiments,
the T. tengcongensis domain is a W9F/L144F H-NOX domain. In some
embodiments, the T. tengcongensis domain is a wild-type H-NOX
domain.
[0147] In some embodiments, the H-NOX domain is covalently linked
to the polymerization domain using an amino acid linker sequence.
In some embodiments, the amino acid linker sequence is one, two,
three, four, five, six, seven, eight, nine, ten or more than ten
amino acids in length. Exemplary amino acid linker sequences
include but are not limited to a Gly-Ser-Gly sequence and an
Arg-Gly-Ser sequence. In some embodiments, the polymeric H-NOX
protein is a trimeric H-NOX protein comprising three H-NOX domains
and three trimerization sequences wherein the H-NOX domain is
covalently linked to the trimerization domain via an amino acid
linker sequence. In some embodiments, the monomeric H-NOX protein
comprises the following from the N-terminus to the C-terminus: an
L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid
linker sequence, and a foldon domain. In some embodiments, the
monomeric H-NOX protein comprises the following from the N-terminus
to the C-terminus: a W9F/L144F T. tengcongensis H-NOX domain, a
Gly-Ser-Gly amino acid linker sequence, and a foldon domain. In
some embodiments, the monomeric H-NOX protein comprises the
following from the N-terminus to the C-terminus: a wild-type T.
tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker
sequence, and a foldon domain.
[0148] In some embodiments, the recombinant monomeric H-NOX protein
comprises a tag; e.g., a His.sub.6, a FLAG, a GST, or an MBP tag.
In some embodiments, the recombinant monomeric H-NOX protein
comprises a His.sub.6 tag. In some embodiments, the recombinant
monomeric H-NOX protein does not comprise a tag. In some
embodiments, the tag (e.g. a His.sub.6 tag) is covalently linked to
the polymerization domain using an amino acid spacer sequence. In
some embodiments, the amino acid linker sequence is one, two,
three, four, five, six, seven, eight, nine, ten or more than ten
amino acids in length. Exemplary amino acid linker sequences
include but are not limited to a Gly-Ser-Gly sequence and an
Arg-Gly-Ser sequence. In some embodiments, the polymeric H-NOX
protein is a trimeric H-NOX protein comprising three H-NOX domains,
three trimerization sequences, and three His.sub.6 tags, wherein
the H-NOX domain is covalently linked to the trimerization domain
via an amino acid linker sequence and the trimerization domain is
covalently linked to the His.sub.6 tag via an amino acid linker
sequence. In some embodiments, the monomeric H-NOX protein
comprises the following from the N-terminus to the C-terminus: an
L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid
linker sequence, a foldon domain, an Arg-Gly-Ser linker sequence,
and a His.sub.6 tag. In some embodiments, the monomeric H-NOX
protein comprises the following from the N-terminus to the
C-terminus: a W9F/L144F T. tengcongensis H-NOX domain, a
Gly-Ser-Gly amino acid linker sequence, a foldon domain, an
Arg-Gly-Ser linker sequence, and a His.sub.6 tag. In some
embodiments, the monomeric H-NOX protein comprises the following
from the N-terminus to the C-terminus: a wild-type T. tengcongensis
H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, a foldon
domain, an Arg-Gly-Ser linker sequence, and a His.sub.6 tag.
[0149] In some embodiments the recombinant monomeric H-NOX protein
comprises the amino acid sequence of SEQ ID NO:6 or SEQ ID
NO:8.
Characteristics of Wild-Type and Mutant H-NOX Proteins
[0150] The present invention provides the use of O.sub.2 carrier
polypeptides for use in enhancing tumor immunogenicity; for
example, by inhibiting the immune suppressive activities associated
with tumor hypoxia. A non-limiting exemplary family of O.sub.2
carrier polypeptides is the H-NOX family of O.sub.2 carrier
polypeptides. As described herein, a large number of diverse H-NOX
mutant proteins, including polymeric H-NOX proteins, providing
ranges of NO and O.sub.2 dissociation constants, O.sub.2 k.sub.off,
NO reactivity, and stability have been generated. To provide
operative blood gas carriers, the H-NOX proteins may be used to
functionally replace or supplement endogenous O.sub.2 carriers,
such as hemoglobin. In some embodiments, H-NOX proteins such as
polymeric H-NOX proteins, are used to deliver O.sub.2 to hypoxic
tumor tissue (e.g. a glioblastoma) as an adjuvant to radiation
therapy or chemotherapy. Accordingly, in some embodiments, an H-NOX
protein has a similar or improved O.sub.2 association rate, O.sub.2
dissociation rate, dissociation constant for O.sub.2 binding, NO
stability, NO reactivity, autoxidation rate, plasma retention time,
or any combination of two or more of the foregoing compared to an
endogenous O.sub.2 carrier, such as hemoglobin. In some
embodiments, the H-NOX protein is a polymeric H-NOX protein. In
some embodiments, the polymeric H-NOX protein is a trimeric H-NOX
protein comprising three monomers, each monomer comprising a T.
tengcongensis L144F H-NOX domain and a foldon domain. In some
embodiments, the polymeric H-NOX protein is a trimeric H-NOX
protein comprising three monomers, each monomer comprising a T.
tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some
embodiments, the polymeric H-NOX protein is a trimeric H-NOX
protein comprising three monomers, each monomer comprising a T.
tengcongensis L144F H-NOX domain and a foldon domain.
[0151] In various embodiments, the k.sub.off for O.sub.2 for an
H-NOX protein, including a polymeric H-NOX protein, is between
about 0.01 to about 200 s.sup.-1 at 20.degree. C., such as about
0.1 to about 200 s.sup.-1, about 0.1 to 100 s.sup.-, about 1.0 to
about 16.0 s.sup.-1, about 1.35 to about 23.4 s.sup.-, about 1.34
to about 18 s.sup.-1, about 1.35 to about 14.5 s.sup.-1, about 0.21
to about 23.4 s.sup.-1, about 1.35 to about 2.9 s.sup.-1 about 2 to
about 3 s.sup.-1, about 5 to about 15 s.sup.-1, or about 0.1 to
about 1 s.sup.-1. In some embodiments, the H-NOX protein has a
k.sub.off for oxygen that is less than or equal to about 0.65
s.sup.-1 at 20.degree. C. (such as between about 0.21 s.sup.-1 to
about 0.65 s.sup.-1 at 20.degree. C.).
[0152] In various embodiments, the k.sub.on for O.sub.2 for an
H-NOX protein, including a polymeric H-NOX protein, is between
about 0.14 to about 60 .mu.M.sup.-1s.sup.-1 at 20.degree. C., such
as about 6 to about 60 .mu.M.sup.-1s.sup.-1, about 6 to 12
.mu.M.sup.-1s.sup.-1, about 15 to about 60 .mu.M.sup.-1s.sup.-1,
about 5 to about 18 .mu.M.sup.-1s.sup.-1, or about 6 to about 15
.mu.M.sup.-1s.sup.-1.
[0153] In various embodiments, the kinetic or calculated K.sub.D
for O.sub.2 binding by an H-NOX protein, including a polymeric
H-NOX protein, is between about 1 nM to 1 mM, about 1 .mu.M to
about 10 .mu.M, or about 10 .mu.M to about 50 .mu.M. In some
embodiments the calculated K.sub.D for O.sub.2 binding is any one
of about 2 nM to about 2 .mu.M, about 2p M to about 1 mM, about 100
nM to about 1 .mu.M, about 9 .mu.M to about 50 .mu.M, about 100
.mu.M to about 1 mM, about 50 nM to about 10 .mu.M, about 2 nM to
about 50 .mu.M, about 100 nM to about 1.9 .mu.M, about 150 nM to
about 1 .mu.M, or about 100 nM to about 255 nM, about 20 nM to
about 2 .mu.M, 20 nM to about 75 nM, about 1 .mu.M to about 2
.mu.M, about 2 .mu.M to about 10 .mu.M, about 2 .mu.M to about 9
.mu.M, or about 100 nM to 500 nM at 20.degree. C. In some
embodiments, the kinetic or calculated K.sub.D for O.sub.2 binding
is less than about any of 100 nM, 80 nM, 50 nM, 30 nM, 25 nM, 20
nM, or 10 nM at 20.degree. C.
[0154] In various embodiments, the kinetic or calculated K.sub.D
for O.sub.2 binding by an H-NOX protein, including a polymeric
H-NOX protein, is within about 0.01 to about 100-fold of that of
hemoglobin under the same conditions (such as at 20.degree. C.),
such as between about 0.1 to about 10-fold or between about 0.5 to
about 2-fold of that of hemoglobin under the same conditions (such
as at 20.degree. C.). In various embodiments, the kinetic or
calculated K.sub.D for NO binding by an H-NOX protein is within
about 0.01 to about 100-fold of that of hemoglobin under the same
conditions (such as at 20.degree. C.), such as between about 0.1 to
about 10-fold or between about 0.5 to about 2-fold of that of
hemoglobin under the same conditions (such as at 20.degree.
C.).
[0155] In some embodiments, less than about any of 50, 40, 30, 10,
or 5% of an H-NOX protein, including a polymeric H-NOX protein, is
oxidized after incubation for about any of 1, 2, 4, 6, 8, 10, 15,
or 20 hours at 20.degree. C.
[0156] In various embodiments, the NO reactivity of an H-NOX
protein, including a polymeric H-NOX protein, is less than about
700 s.sup.-1 at 20.degree. C., such as less than about 600
s.sup.-1, 500 s.sup.-1, 400 s.sup.-1, 300 s.sup.-1, 200 s.sup.-1,
100 s.sup.-1, 75 s.sup.-1, 50 s.sup.-1, 25 s.sup.-1, 20 s.sup.-1,
10 s.sup.-1, 50 s.sup.-1, 3 s.sup.-1, 2 s.sup.-1, 1.8 s.sup.-1, 1.5
s.sup.-1, 1.2 s.sup.-1, 1.0 s.sup.-1, 0.8 s.sup.-1, 0.7 s.sup.-1,
or 0.6 s.sup.-1 at 20.degree. C. In various embodiments, the NO
reactivity of an H-NOX protein is between about 0.1 to about 600
s.sup.-1 at 20.degree. C., such as between about 0.5 to about 400
s.sup.-1, about 0.5 to about 100 s.sup.-1, about 0.5 to about 50
s.sup.-1, about 0.5 to about 10 s.sup.-1, about 1 to about 5
s.sup.-1, or about 0.5 to about 2.1 s.sup.-1 at 20.degree. C. In
various embodiments, the reactivity of an H-NOX protein is at least
about 10, 100, 1,000, or 10,000 fold lower than that of hemoglobin
under the same conditions, such as at 20.degree. C.
[0157] In various embodiments, the rate of heme autoxidation of an
H-NOX protein, including a polymeric H-NOX protein, is less than
about 1.0 h-tat 37.degree. C., such as less than about any of 0.9
h.sup.-1, 0.8 h.sup.-1, 0.7 h.sup.-1, 0.6 h.sup.-1, 0.5 h.sup.-1,
0.4 h.sup.-1, 0.3 h.sup.-1, 0.2 h.sup.-1, 0.1 h.sup.-1, or 0.05
h.sup.-1 at 37 C. In various embodiments, the rate of heme
autoxidation of an H-NOX protein is between about 0.006 to about
5.0 h.sup.-1 at 37.degree. C., such as about 0.006 to about 1.0
h.sup.-1, 0.006 to about 0.9 h.sup.-1, or about 0.06 to about 0.5
h.sup.-1 at 37.degree. C.
[0158] In various embodiments, a mutant H-NOX protein, including a
polymeric H-NOX protein, has (a) an O.sub.2 or NO dissociation
constant, association rate (k.sub.on for O.sub.2 or NO), or
dissociation rate (k.sub.off for O.sub.2 or NO) within 2 orders of
magnitude of that of hemoglobin, (b) has an NO affinity weaker
(e.g., at least about 10-fold, 100-fold, or 1000-fold weaker) than
that of sGC .beta.1, respectively, (c) an NO reactivity with bound
02 at least 1000-fold less than hemoglobin, (d) an in vivo plasma
retention time at least 2, 10, 100, or 1000-fold higher than that
of hemoglobin, or (e) any combination of two or more of the
foregoing.
[0159] Exemplary suitable O.sub.2 carriers provide dissociation
constants within two orders of magnitude of that of hemoglobin,
i.e. between about 0.01 and 100-fold, such as between about 0.1 and
10-fold, or between about 0.5 and 2-fold of that of hemoglobin. A
variety of established techniques may be used to quantify
dissociation constants, such as the techniques described herein
(Boon, E. M. et al. (2005). Nature Chem. Biol. 1:53-59; Boon, E. M.
et al. (October 2005). Curr. Opin. Chem. Biol. 9(5):441-446; Boon.
E. M. et al. (2005). J. Inorg. Biochem. 99(4):892-902), Vandegriff,
K. D. et al. (Aug. 15, 2004). Biochem J. 382(Pt 1):183-189, which
are each hereby incorporated by reference in their entireties,
particularly with respect to the measurement of dissociation
constants), as well as those known to the skilled artisan.
Exemplary O.sub.2 carriers provide low or minimized NO reactivity
of the H-NOX protein with bound O.sub.2, such as an NO reactivity
lower than that of hemoglobin. In some embodiments, the NO
reactivity is much lower, such as at least about 10, 100, 1,000, or
10,000-fold lower than that of hemoglobin. A variety of established
techniques may be used to quantify NO reactivity (Boon, E. M. et
al. (2005). Nature Chem. Biol. 1:53-59; Boon, E. M. et al. (October
2005). Curr. Opin. Chem. Biol. 9(5):441-446; Boon, E. M. et al.
(2005). J. Inorg. Biochem. 99(4):892-902), Vandegriff, K. D. et al.
(Aug. 15, 2004). Biochem J. 382(Pt 1):183-189, which are each
hereby incorporated by reference in their entireties, particularly
with respect to the measurement of NO reactivity) as well as those
known to the skilled artisan. Because wild-type T. tengcongensis
H-NOX has such a low NO reactivity, other wild-type H-NOX proteins
and mutant H-NOX proteins may have a similar low NO reactivity. For
example, T. tengcongensis H-NOX Y140H has an NO reactivity similar
to that of wild-type T. tengcongensis H-NOX.
[0160] In addition, suitable O.sub.2 carriers provide high or
maximized stability, particularly in vivo stability. A variety of
stability metrics may be used, such as oxidative stability (e.g.,
stability to autoxidation or oxidation by NO), temperature
stability, and in vivo stability. A variety of established
techniques may be used to quantify stability, such as the
techniques described herein (Boon, E. M. et al. (2005). Nature
Chem. Biol. 1:53-59; Boon, E. M. et al. (October 2005). Curr. Opin.
Chem. Biol. 9(5):441-446; Boon, E. M. et al. (2005). J. Inorg.
Biochem. 99(4):892-902), as well as those known to the skilled
artisan. For in vivo stability in plasma, blood, or tissue,
exemplary metrics of stability include retention time, rate of
clearance, and half-life. H-NOX proteins from thermophilic
organisms are expected to be stable at high temperatures. In
various embodiments, the plasma retention times are at least about
2-, 10-, 100-, or 1000-fold greater than that of hemoglobin (e.g.
Bobofchak, K. M. et al. (August 2003). Am. J. Physiol. Heart Circ.
Physiol. 285(2):H549-H561). As will be appreciated by the skilled
artisan, hemoglobin-based blood substitutes are limited by the
rapid clearance of cell-free hemoglobin from plasma due the
presence of receptors for hemoglobin that remove cell-free
hemoglobin from plasma. Since there are no receptors for H-NOX
proteins in plasma, wild-type and mutant H-NOX proteins are
expected to have a longer plasma retention time than that of
hemoglobin. If desired, the plasma retention time can be increased
by PEGylating or crosslinking an H-NOX protein or fusing an H-NOX
protein with another protein using standard methods (such as those
described herein and those known to the skilled artisan).
[0161] In various embodiments, the H-NOX protein, including a
polymeric H-NOX protein, has an O.sub.2 dissociation constant
between about 1 nM to about 1 mM at 20.degree. C. and a NO
reactivity at least about 10-fold lower than that of hemoglobin
under the same conditions, such as at 20.degree. C. In some
embodiments, the H-NOX protein has an O.sub.2 dissociation constant
between about 1 nM to about 1 mM at 20.degree. C. and a NO
reactivity less than about 700 s.sup.-1 at 20.degree. C. (e.g.,
less than about 600 s.sup.-1, 500 s.sup.-1, 100 s.sup.-1, 20
s.sup.-1, or 1.8 s.sup.-1 at 20.degree. C.). In some embodiments,
the H-NOX protein has an O.sub.2 dissociation constant within 2
orders of magnitude of that of hemoglobin and a NO reactivity at
least about 10-fold lower than that of hemoglobin under the same
conditions, such as at 20.degree. C. In some embodiments, the H-NOX
protein has a k.sub.off for oxygen between about 0.01 to about 200
s.sup.-1 at 20.degree. C. and an NO reactivity at least about
10-fold lower than that of hemoglobin under the same conditions,
such as at 20.degree. C. In some embodiments, the H-NOX protein has
a k.sub.off for oxygen that is less than about 0.65 s.sup.-1 at
20.degree. C. (such as between about 0.21 s.sup.-1 to about 0.64
s.sup.-1 at 20.degree. C.) and a NO reactivity at least about
10-fold lower than that of hemoglobin under the same conditions,
such as at 20.degree. C. In some embodiments of the invention, the
O.sub.2 dissociation constant of the H-NOX protein is between about
1 nM to about 1 .mu.M (1000 nM), about 1 .mu.M to about 10 .mu.M,
or about 10 .mu.M to about 50 .mu.M. In particular embodiments, the
O.sub.2 dissociation constant of the H-NOX protein is between about
2 nM to about 50 .mu.M, about 50 nM to about 10 .mu.M, about 100 nM
to about 1.9 .mu.M, about 150 nM to about 1 .mu.M, or about 100 nM
to about 255 nM at 20.degree. C. In various embodiments, the
O.sub.2 dissociation constant of the H-NOX protein is less than
about 80 nM at 20.degree. C., such as between about 20 nM to about
75 nM at 20.degree. C. In some embodiments, the NO reactivity of
the H-NOX protein is at least about 100-fold lower or about 1,000
fold lower than that of hemoglobin, under the same conditions, such
as at 20.degree. C. In some embodiments, the NO reactivity of the
H-NOX protein is less than about 700 s.sup.-1 at 20.degree. C.,
such as less than about 600 s.sup.-1, 500 s.sup.-1, 400 s.sup.-1,
300 s.sup.-1, 200 s.sup.-1, 100 s.sup.-1, 75 s.sup.-1, 50 s.sup.-1,
25 s.sup.-1, 20 s.sup.-1, 10 s.sup.-1, 50 s.sup.-1, 3 s.sup.-1, 2
s.sup.-1, 1.8 s.sup.-1, 1.5 s.sup.-1, 1.2 s.sup.-1, 1.0 s.sup.-1,
0.8 s.sup.-1, 0.7 s.sup.-1, or 0.6 s.sup.-1 at 20.degree. C. In
some embodiments, the k.sub.off for oxygen of the H-NOX protein is
between 0.01 to 200 s.sup.-1 at 20.degree. C., such as about 0.1 to
about 200 s.sup.-1, about 0.1 to 100 s.sup.-1, about 1.35 to about
23.4 s.sup.-1, about 1.34 to about 18 s.sup.-1, about 1.35 to about
14.5 s.sup.-1, about 0.21 to about 23.4 s.sup.-1, about 2 to about
3 s.sup.-1, about 5 to about 15 s.sup.-1, or about 0.1 to about 1
s.sup.-1. In some embodiments, the O.sub.2 dissociation constant of
the H-NOX protein is between about 100 nM to about 1.9 .mu.M at
20.degree. C., and the k.sub.off for oxygen of the H-NOX protein is
between about 1.35 s.sup.-1 to about 14.5 s.sup.-1 at 20.degree. C.
In some embodiments, the rate of heme autoxidation of the H-NOX
protein is less than about 1 h.sup.-1 at 37.degree. C., such as
less than about any of 0.9 h.sup.-1, 0.8 h.sup.-1, 0.7 h.sup.-1,
0.6 h.sup.-1, 0.5 h.sup.-1, 0.4 h.sup.-1, 0.3 h.sup.-1, 0.2
h.sup.-1, or 0.1 h.sup.-1. In some embodiments, the k.sub.off for
oxygen of the H-NOX protein is between about 1.35 s.sup.-1 to about
14.5 s.sup.-1 at 20.degree. C., and the rate of heme autoxidation
of the H-NOX protein is less than about 1 h.sup.-1 at 37.degree. C.
In some embodiments, the k.sub.off for oxygen of the H-NOX protein
is between about 1.35 s.sup.-1 to about 14.5 s.sup.-1 at 20.degree.
C., and the NO reactivity of the H-NOX protein is less than about
700 s.sup.-1 at 20.degree. C. (e.g., less than about 600 s.sup.-1,
500 s.sup.-1, 100 s.sup.-1, 20 s.sup.-1, or 1.8 s.sup.-1 at
20.degree. C.). In some embodiments, the rate of heme autoxidation
of the H-NOX protein is less than about 1 h.sup.-1 at 37.degree.
C., and the NO reactivity of the H-NOX protein is less than about
700 s.sup.-1 at 20.degree. C. (e.g., less than about 600 s.sup.-1,
500 s.sup.-1, 100 s.sup.-1, 20 s.sup.-1, or 1.8 s.sup.-1 at
20.degree. C.).
[0162] In some embodiments, the viscosity of the H-NOX protein
solution, including a polymeric H-NOX protein solution, is between
1 and 4 centipoise (cP). In some embodiments, the colloid oncotic
pressure of the H-NOX protein solution is between 20 and 50 mm
Hg.
Measurement of O.sub.2 and/or NO Binding
[0163] One skilled in the art can readily determine the oxygen and
nitric oxide binding characteristics of any H-NOX protein including
a polymeric H-NOX protein such as a trimeric H-NOX protein by
methods known in the art and by the non-limiting exemplary methods
described below.
Kinetic K.sub.D: Ratio of k.sub.off to k.sub.on
[0164] The kinetic K.sub.D value is determined for wild-type and
mutant H-NOX proteins, including polymeric H-NOS proteins,
essentially as described by Boon, E. M. et al. (2005). Nature
Chemical Biology 1:53-59, which is hereby incorporated by reference
in its entirety, particularly with respect to the measurement of
O.sub.2 association rates, O.sub.2 dissociation rates, dissociation
constants for O.sub.2 binding, autoxidation rates, and NO
dissociation rates.
k.sub.on (O.sub.2 Association Rate)
[0165] O.sub.2 association to the heme is measured using flash
photolysis at 20.degree. C. It is not possible to flash off the
Fe.sup.II-O.sub.2 complex as a result of the very fast geminate
recombination kinetics; thus, the Fen-CO complex is subjected to
flash photolysis with laser light at 560 nm (Hewlett-Packard, Palo
Alto, Calif.), producing the 5-coordinate Fe.sup.II intermediate,
to which the binding of molecular O.sub.2 is followed at various
wavelengths. Protein samples are made by anaerobic reduction with
10 mM dithionite, followed by desalting on a PD-10 column
(Millipore, Inc., Billerica, Mass.). The samples are then diluted
to 20 .mu.M heme in 50 mM TEA, 50 mM NaCl, pH 7.5 buffer in a
controlled-atmosphere quartz cuvette, with a size of 100 .mu.L to 1
mL and a path-length of 1-cm. CO gas is flowed over the headspace
of this cuvette for 10 minutes to form the Fe.sup.II-CO complex,
the formation of which is verified by UV-visible spectroscopy
(Soret maximum 423 nm). This sample is then either used to measure
CO-rebinding kinetics after flash photolysis while still under 1
atmosphere of CO gas, or it is opened and stirred in air for 30
minutes to fully oxygenate the buffer before flash photolysis to
watch O.sub.2-rebinding events. O.sub.2 association to the heme is
monitored at multiple wavelengths versus time. These traces are fit
with a single exponential using Igor Pro software (Wavemetrics,
Inc., Oswego, Oreg.; latest 2005 version). This rate is independent
of observation wavelength but dependent on O.sub.2 concentration.
UV-visible spectroscopy is used throughout to confirm all the
complexes and intermediates (Cary 3K, Varian, Inc. Palo Alto,
Calif.). Transient absorption data are collected using instruments
described in Dmochowski, I. J. et al. (Aug. 31, 2000). J Inorg
Biochem. 81(3):221-228, which is hereby incorporated by reference
in its entirety, particularly with respect to instrumentation. The
instrument has a response time of 20 ns, and the data are digitized
at 200 megasamples s.sup.-1.
k.sub.off (O.sub.2 Dissociation Rate)
[0166] To measure the k.sub.off, Fe.sup.II-O.sub.2 complexes of
protein (5 .mu.M heme), are diluted in anaerobic 50 mM TEA, 50 mM
NaCl, pH 7.5 buffer, and are rapidly mixed with an equal volume of
the same buffer (anaerobic) containing various concentrations of
dithionite and/or saturating CO gas. Data are acquired on a HI-TECH
Scientific SF-61 stopped-flow spectrophotometer equipped with a
Neslab RTE-100 constant-temperature bath set to 20.degree. C. (TGK
Scientific LTD., Bradford On Avon, United Kingdom). The
dissociation of O.sub.2 from the heme is monitored as an increase
in the absorbance at 437 nm, a maximum in the
Fe.sup.II--Fe.sup.II--O.sub.2 difference spectrum, or 425 nm, a
maximum in the Fe.sup.II--Fe.sup.II--CO difference spectrum. The
final traces are fit to a single exponential using the software
that is part of the instrument. Each experiment is done a minimum
of six times, and the resulting rates are averaged. The
dissociation rates measured are independent of dithionite
concentration and independent of saturating CO as a trap for the
reduced species, both with and without 10 mM dithionite
present.
Kinetic K.sub.D
[0167] The kinetic K.sub.D is determined by calculating the ratio
of k.sub.off to k.sub.on using the measurements of k.sub.off and
k.sub.on described above.
[0168] Calculated K.sub.D
[0169] To measure the calculated K.sub.D, the values for the
k.sub.off and kinetic K.sub.D that are obtained as described above
are graphed. A linear relationship between k.sub.off and kinetic
K.sub.D is defined by the equation (y=mx+b). k.sub.off values were
then interpolated along the line to derive the calculated K.sub.D
using Excel: MAC 2004 (Microsoft, Redmond, Wash.). In the absence
of a measured k.sub.on, this interpolation provides a way to relate
k.sub.off to K.sub.D.
Rate of Autoxidation
[0170] To measure the rate of autoxidation, the protein samples are
anaerobically reduced, then diluted to 5 .mu.M heme in aerobic 50
mM TEA, 50 mM NaCl, pH 7.5 buffer. These samples are then incubated
in a Cary 3E spectrophotometer equipped with a Neslab RTE-100
constant-temperature bath set to 37.degree. C. and scanned
periodically (Cary 3E, Varian, Inc., Palo Alto, Calif.). The rate
of autoxidation is determined from the difference between the
maximum and minimum in the Fe.sup.III-Fe.sup.II difference spectrum
plotted versus time and fit with a single exponential using Excel:
MAC 2004 (Microsoft, Redmond, Wash.).
Rate of Reaction with NO
[0171] NO reactivity is measured using purified proteins (H-NOX,
polymeric H-NOX, Homo sapiens hemoglobin (Hs Hb) etc.) prepared at
2 .mu.M in buffer A and NO prepared at 200 .mu.M in Buffer A
(Buffer A: 50 mM Hepes, pH 7.5, 50 mM NaCl). Data are acquired on a
HI-TECH Scientific SF-61 stopped-flow spectrophotometer equipped
with a Neslab RTE-100 constant-temperature bath set to 20.degree.
C. (TGK Scientific LTD., Bradford On Avon, United Kingdom). The
protein is rapidly mixed with NO in a 1:1 ratio with an integration
time of 0.00125 sec. The wavelengths of maximum change are fit to a
single exponential using the software that is part of the
spectrometer, essentially measuring the rate-limiting step of
oxidation by NO. The end products of the reaction are ferric-NO for
the HNOX proteins and ferric-aquo for Hs Hb.
p50 Measurements
[0172] If desired, the p50 value for mutant or wild-type H-NOX
proteins can be measured as described by Guarnone, R. et al.
(September/October 1995). Haematologica 80(5):426-430, which is
hereby incorporated by reference in its entirety, particularly with
respect to the measurement of p50 values. The p50 value is
determined using a HemOx analyzer. The measurement chamber starts
at 0% oxygen and slowly is raised, incrementally, towards 100%
oxygen. An oxygen probe in the chamber measures the oxygen
saturation %. A second probe (UV-Vis light) measures two
wavelengths of absorption, tuned to the alpha and beta peaks of the
hemoprotein's (e.g., a protein such as H-NOX complexed with heme)
UV-Vis spectra. These absorption peaks increase linearly as
hemoprotein binds oxygen. The percent change from unbound to 100%
bound is then plotted against the % oxygen values to generate a
curve. The p50 is the point on the curve where 50% of the
hemoprotein is bound to oxygen.
[0173] Specifically, the Hemox-Analyzer (TCS Scientific
Corporation, New Hope, Pa.) determines the oxyhemoprotein
dissociation curve (ODC) by exposing 50 .mu.L of blood or
hemoprotein to an increasing partial pressure of oxygen and
deoxygenating it with nitrogen gas. A Clark oxygen electrode
detects the change in oxygen tension, which is recorded on the
x-axis of an x-y recorder. The resulting increase in oxyhemoprotein
fraction is simultaneously monitored by dual-wavelength
spectrophotometry at 560 nm and 576 nm and displayed on the y-axis.
Blood samples are taken from the antemedial vein, anticoagulated
with heparin, and kept at 4.degree. C. on wet ice until the assay.
Fifty .mu.L of whole blood are diluted in 5 .mu.L of
Hemox-solution, a manufacturer-provided buffer that keeps the pH of
the solution at a value of 7.4-0.01. The sample-buffer is drawn
into a cuvette that is part of the Hemox-Analyzer and the
temperature of the mixture is equilibrated and brought to
37.degree. C.; the sample is then oxygenated to 100% with air.
After adjustment of the pO.sub.2 value the sample is deoxygenated
with nitrogen; during the deoxygenation process the curve is
recorded on graph paper. The P50 value is extrapolated on the
x-axis as the point at which O.sub.2 saturation is 50% using the
software that is part of the Hemox-Analyzer. The time required for
a complete recording is approximately 30 minutes.
H-NOX Nucleic Acids
[0174] The invention also features nucleic acids encoding any of
the mutant H-NOX proteins, polymeric H-NOX, or recombinant monomer
H-NOX protein subunits as described herein.
[0175] In particular embodiments, the nucleic acid includes a
segment of or the entire nucleic acid sequence of any of nucleic
acids encoding an H-NOX protein or an H-NOX domain. In some
embodiments, the nucleic acid includes at least about 50, 100, 150,
200, 300, 400, 500, 600, 700, 800, or more contiguous nucleotides
from a H-NOX nucleic acid and contains one or more mutations (e.g.,
1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations) compared to the H-NOX
nucleic acid from which it was derived. In various embodiments, a
mutant H-NOX nucleic acid contains less than about 20, 15, 12, 10,
9, 8, 7, 6, 5, 4, 3, or 2 mutations compared to the H-NOX nucleic
acid from which it was derived. The invention also features
degenerate variants of any nucleic acid encoding a mutant H-NOX
protein.
[0176] In some embodiments, the nucleic acid includes nucleic acids
encoding two or more H-NOX domains. In some embodiments, the
nucleic acids including two or more H-NOX domains are linked such
that a polymeric H-NOX protein is expressed from the nucleic acid.
In further embodiments, the nucleic acid includes nucleic acids
encoding one or more polymerization domains. In some embodiments,
the nucleic acids including the two or more H-NOX domains and the
one or more polymerization domains are linked such that a polymeric
H-NOX protein is expressed from the nucleic acid.
[0177] In some embodiments, the nucleic acid includes a segment or
the entire nucleic acid sequence of any nucleic acid encoding a
polymerization domain. In some embodiments the nucleic acid
comprises a nucleic acid encoding an H-NOX domain and a
polymerization domain. In some embodiments, the nucleic acid
encoding an H-NOX domain and the nucleic acid encoding a
polymerization domain a linked such that the produced polypeptide
is a fusion protein comprising an H-NOX domain and a polymerization
domain.
[0178] In some embodiments, the nucleic acid comprises nucleic acid
encoding one or more His.sub.6 tags. In some embodiments the
nucleic acid further comprised nucleic acids encoding linker
sequences positioned between nucleic acids encoding the H-NOX
domain, the polymerization domain and/or a His.sub.6 tag.
[0179] In some embodiments, the invention provides a nucleic acid
encoding an H-NOX domain and a foldon domain. In some embodiments,
the H-NOX domain is a T. thermoanaerobacter H-NOX domain. In some
embodiments, the H-NOX domain is a wild-type T. thermoanaerobacter
H-NOX domain. In some embodiments, the H-NOX domain is a T.
thermoanaerobacter L144F H-NOX domain. In some embodiments, the
H-NOX domain is a T. thermoanaerobacter W9F/L144F H-NOX domain.
[0180] In some embodiments, the invention provides nucleic acids
encoding the following 5' to 3': a L144F T. tengcongensis H-NOX
domain, a Gly-Ser-Gly amino acid linker sequence, and a foldon
domain. In some embodiments, the invention provides nucleic acids
encoding the following 5' to 3': a W9F/L144F T. tengcongensis H-NOX
domain, a Gly-Ser-Gly amino acid linker sequence, and a foldon
domain. In some embodiments, the invention provides nucleic acids
encoding the following 5' to 3': a wild-type T. tengcongensis H-NOX
domain, a Gly-Ser-Gly amino acid linker sequence, and a foldon
domain.
[0181] In some embodiments, the invention provides nucleic acids
encoding the following 5' to 3': a L144F T. tengcongensis H-NOX
domain, a Gly-Ser-Gly amino acid linker sequence, a foldon domain,
an Arg-Gly-Ser linker sequence, and a His.sub.6 tag. In some
embodiments, the invention provides nucleic acids encoding the
following 5' to 3': a W9F/L144F T. tengcongensis H-NOX domain, a
Gly-Ser-Gly amino acid linker sequence, a foldon domain, an
Arg-Gly-Ser linker sequence, and a His.sub.6 tag. In some
embodiments, the invention provides nucleic acids encoding the
following 5' to 3': a wild-type T. tengcongensis H-NOX domain, a
Gly-Ser-Gly amino acid linker sequence, a foldon domain, an
Arg-Gly-Ser linker sequence, and a His.sub.6 tag.
[0182] In some embodiments, the nucleic acid comprises the nucleic
acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:7.
[0183] The invention also includes a cell or population of cells
containing at least one nucleic acid encoding a mutant H-NOX
protein described herein. Exemplary cells include insect, plant,
yeast, bacterial, and mammalian cells. These cells are useful for
the production of mutant H-NOX proteins using standard methods,
such as those described herein.
[0184] In some embodiments, the invention provides a cell
comprising a nucleic acid encoding an H-NOX domain and a foldon
domain. In some embodiments, the H-NOX domain is a T.
thermoanaerobacter H-NOX domain. In some embodiments, the H-NOX
domain is a wild-type T. thermoanaerobacter H-NOX domain. In some
embodiments, the H-NOX domain is a T. thermoanaerobacter L144F
H-NOX domain. In some embodiments, the H-NOX domain is a T.
thermoanaerobacter W9F/L144F H-NOX domain. In some embodiments, the
invention provides a cell comprising a nucleic acid comprising the
nucleic acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:7.
Formulations of H-NOX Proteins
[0185] The present invention provides formulations of O.sub.2
carrier polypeptides for use in enhancing tumor immunogenicity; for
example, by inhibiting the immune suppressive activities associated
with tumor hypoxia. A non-limiting exemplary family of O.sub.2
carrier polypeptides is the H-NOX family of O.sub.2 carrier
polypeptides. Any wild-type or mutant H-NOX protein, including
polymeric H-NOX proteins, described herein may be used for the
formulation of pharmaceutical or non-pharmaceutical compositions.
In some embodiments, the formulations comprise a monomeric H-NOX
protein comprising an H-NOX domain and a polymerization domain such
that the monomeric H-NOX proteins associate in vitro or in vivo to
produce a polymeric H-NOX protein. As discussed further below,
these formulations are useful in a variety of therapeutic and
industrial applications.
[0186] In some embodiments, the pharmaceutical composition includes
one or more wild-type or mutant H-NOX proteins described herein
including polymeric H-NOX proteins and a pharmaceutically
acceptable carrier or excipient. Examples of pharmaceutically
acceptable carriers or excipients include, but are not limited to,
any of the standard pharmaceutical carriers or excipients such as
phosphate buffered saline solutions, water, emulsions such as
oil/water emulsion, and various types of wetting agents. Exemplary
diluents for aerosol or parenteral administration are phosphate
buffered saline or normal (0.9%) saline. Compositions comprising
such carriers are formulated by well-known conventional methods
(see, for example, Remington's Pharmaceutical Sciences, 18th
edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990;
and Remington, The Science and Practice of Pharmacy 20th Ed. Mack
Publishing, 2000, which are each hereby incorporated by reference
in their entireties, particularly with respect to formulations). In
some embodiments, the formulations are sterile. In some
embodiments, the formulations are essentially free of
endotoxin.
[0187] While any suitable carrier known to those of ordinary skill
in the art may be employed in the pharmaceutical compositions of
this invention, the type of carrier will vary depending on the mode
of administration. Compositions can be formulated for any
appropriate manner of administration, including, for example,
intravenous, intra-arterial, intravesicular, intratumoral,
inhalation, intraperitoneal, intrapulmonary, intramuscular,
subcutaneous, intra-tracheal, transmucosal, intraocular,
intrathecal, or transdermal administration. For parenteral
administration, such as subcutaneous injection, the carrier may
include, e.g., water, saline, alcohol, a fat, a wax, or a buffer.
For oral administration, any of the above carriers or a solid
carrier, such as mannitol, lactose, starch, magnesium stearate,
sodium saccharine, talcum, cellulose, glucose, sucrose, or
magnesium carbonate, may be employed. Biodegradable microspheres
(e.g., polylactate polyglycolate) may also be used as carriers.
[0188] In some embodiments, the pharmaceutical or
non-pharmaceutical compositions include a buffer (e.g., neutral
buffered saline, phosphate buffered saline, etc), a carbohydrate
(e.g., glucose, mannose, sucrose, dextran, etc.), an antioxidant, a
chelating agent (e.g., EDTA, glutathione, etc.), a preservative,
another compound useful for binding and/or transporting oxygen, an
inactive ingredient (e.g., a stabilizer, filler, etc.), or
combinations of two or more of the foregoing. In some embodiments,
the composition is formulated as a lyophilizate. H-NOX proteins may
also be encapsulated within liposomes or nanoparticles using well
known technology. Other exemplary formulations that can be used for
H-NOX proteins are described by, e.g., U.S. Pat. Nos. 6,974,795,
and 6,432,918, which are each hereby incorporated by reference in
their entireties, particularly with respect to formulations of
proteins.
[0189] The compositions described herein may be administered as
part of a sustained release formulation (e.g., a formulation such
as a capsule or sponge that produces a slow release of compound
following administration). Such formulations may generally be
prepared using well known technology and administered by, for
example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Sustained-release
formulations may contain an H-NOX protein dispersed in a carrier
matrix and/or contained within a reservoir surrounded by a rate
controlling membrane. Carriers for use within such formulations are
biocompatible, and may also be biodegradable. In some embodiments,
the formulation provides a relatively constant level of H-NOX
protein release. The amount of H-NOX protein contained within a
sustained release formulation depends upon the site of
implantation, the rate and expected duration of release, and the
nature of the condition to be treated or prevented.
[0190] In some embodiments, the pharmaceutical composition contains
an effective amount of a wild-type or mutant H-NOX protein. In some
embodiments, the pharmaceutical composition contains an effective
amount of a polymeric H-NOX protein comprising two or more
wild-type or mutant H-NOX domains. In some embodiments, the
pharmaceutical composition contains an effective amount of a
recombinant monomeric H-NOX protein comprising a wild-type or
mutant H-NOX domain and a polymerization domain as described
herein. In some embodiments, the formulation comprises a trimeric
H-NOX protein comprising three monomers, each monomer comprising a
T. tengcongensis L144F H-NOX domain and a foldon domain. In some
embodiments, the formulation comprises a trimeric H-NOX protein
comprising three monomers, each monomer comprising a T.
tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some
embodiments, the formulation comprises a trimeric H-NOX protein
comprising three monomers, each monomer comprising a T.
tengcongensis L144F H-NOX domain and a foldon domain. In some
embodiments, the formulation comprises a PEGylated trimeric H-NOX
protein comprising three monomers, each monomer comprising a T.
tengcongensis L144F H-NOX domain and a foldon domain. In some
embodiments, the pharmaceutical composition comprises an O.sub.2
carrier polypeptide (e.g., an H-NOX protein) in an amount effective
to modulate tumor immunity (e.g., enhance an immune response to the
tumor).
[0191] In some embodiments, an effective amount of an H-NOX protein
for administration to a human is between a few grams to over about
350 grams. Other exemplary doses of an H-NOX protein include about
any of 4.4., 5, 10, or 13 G/DL (where G/DL is the concentration of
the H-NOX protein solution prior to infusion into the circulation)
at an appropriate infusion rate, such as about 0.5 ml/min (see, for
example, Winslow, R. Chapter 12 In Blood Substitutes). It will be
appreciated that the unit content of active ingredients contained
in an individual dose of each dosage form need not in itself
constitute an effective amount since the necessary effective amount
could be reached by the combined effect of a plurality of
administrations. The selection of the amount of an H-NOX protein to
include in a pharmaceutical composition depends upon the dosage
form utilized, the condition being treated, and the particular
purpose to be achieved according to the determination of the
ordinarily skilled artisan in the field.
[0192] Exemplary compositions include genetically engineered,
recombinant H-NOX proteins, which may be isolated or purified,
comprising one or more mutations that collectively impart altered
O.sub.2 or NO ligand-binding relative to the corresponding
wild-type H-NOX protein, and operative as a physiologically
compatible mammalian blood gas carrier. For example, mutant H-NOX
proteins as described herein. In some embodiments, the H-NOX
protein is a polymeric H-NOX protein. In some embodiments, the
H-NOX protein is a recombinant monomeric H-NOX protein comprising a
wild-type or mutant H-NOX domain and a polymerization domain as
described herein. In some embodiments, the composition comprises a
trimeric H-NOX protein comprising three monomers, each monomer
comprising a T. tengcongensis L144F H-NOX domain and a foldon
domain. In some embodiments, the composition comprises a trimeric
H-NOX protein comprising three monomers, each monomer comprising a
T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In
some embodiments, the composition comprises a trimeric H-NOX
protein comprising three monomers, each monomer comprising a T.
tengcongensis L144F H-NOX domain and a foldon domain. In some
embodiments, the composition comprises a PEGylated trimeric H-NOX
protein comprising three monomers, each monomer comprising a T.
tengcongensis L144F H-NOX domain and a foldon domain.
[0193] To reduce or prevent an immune response in human subjects
who are administered a pharmaceutical composition, human H-NOX
proteins or domains (either wild-type human proteins or human
proteins into which one or more mutations have been introduced) or
other non-antigenic H-NOX proteins or domains (e.g., mammalian
H-NOX proteins) can be used. To reduce or eliminate the
immunogenicity of H-NOX proteins derived from sources other than
humans, amino acids in an H-NOX protein or H-NOX domain can be
mutated to the corresponding amino acids in a human H-NOX. For
example, one or more amino acids on the surface of the tertiary
structure of a non-human H-NOX protein can be mutated to the
corresponding amino acid in a human H-NOX protein.
Methods to Modulate Tumor Immunity
[0194] In some aspects, the invention provides methods modulate to
tumor immunity and thus can be used in anticancer treatments.
Hypoxic tumor microenvironments suppress the host's immune
anti-tumor defenses by modulating multiple signaling pathways (FIG.
1) including, but not limited to, hypoxia inducible factor (HIF-1)
signaling (Codo et al., 2014 Oncotarget, 5(17), 7651-7662; Lee,
Mace, & Repasky, 2010 Int J Hyperthermia, 26(3), 232-246; Wei
et al., 2011 PLoS One, 6(1), e16195), miRNA epigenetic regulation
of antitumor T cells, MHC1 expression on tumor cells, and
recruitment of tumor associated macrophages and myeloid-derived
suppressor cells (MDSC). Hypoxic activation of the HIF-1 pathway
has been shown to activate adenosinergic A2 and PD-L1 pathways
which in turn inhibit recruitment and activation of helper and
killer T-cells and NK cells (Noman et al., 2014 J Exp Med, 211(5),
781-790; Ohta et al., 2006 Proc Natl Acad Sci USA, 103(35),
13132-13137). Hypoxic activation of the HIF-1 pathway may also lead
to the recruitment and activation of inhibitory regulatory T cells
(Treg), tumor associated macrophages (TAM) and other
myeloid-derived suppressor cells (MDSC) (Chaturvedi et al., 2014
Proc Natl Acad Sci USA, 111(20), E2120-2129; Corzo et al., 2010 J
Exp Med, 207(11), 2439-2453; Wei et al., 2011). HIF-1 pathway
activation may also directly inhibit the ability of tumor cells to
be recognized by immune system by increasing tumor shedding of MHC1
receptors (Siemens et al., 2008 Cancer Res, 68(12), 4746-4753).
[0195] In some aspects, the invention provides methods for
modulating tumor immunity in an individual with a tumor comprising
administering to the individual an effective amount of an O.sub.2
carrier polypeptide (e.g., an H-NOX protein). In some embodiments,
the modulating of tumor immunity comprises enhancing an immune
response to the tumor. In some embodiments, the invention provides
methods for increasing leucocyte infiltration to a tumor in an
individual comprising administering to the individual an effective
amount of an O.sub.2 carrier polypeptide. In some embodiments, the
invention provides methods for increasing lymphocyte infiltration
to a tumor in an individual comprising administering to the
individual an effective amount of an O.sub.2 carrier polypeptide.
In some embodiments, the increase in lymphocyte infiltration to the
tumor comprises an increase in infiltration of one or more of CD4
cells, CD8 cells, or NK cells. In some embodiments, the modulating
of tumor immunity comprises increasing antigen processing. In some
embodiments, the modulating of tumor immunity comprises increasing
the presentation capabilities of dendritic cells (DC). In some
embodiments, the modulating of tumor immunity comprises one or more
of increasing lymphocyte infiltration to the tumor, increasing
antigen processing, or increasing DC presentation capability. In
some embodiments, the modulating of tumor immunity comprises
lymphocyte activation. In some embodiments, the modulating of tumor
immunity comprises cytokine secretion. In some embodiments, the
O.sub.2 carrier polypeptide is a trimeric Tt H-NOX L144F
polypeptide. In some embodiments, the O.sub.2 carrier polypeptide
is a PEGylated trimeric Tt H-NOX L144F polypeptide.
[0196] In some embodiments of the invention, the increase in
lymphocyte infiltration to the tumor is accompanied by inhibition
of one or more of Treg cells, tumor associated macrophages or
myeloid derived suppressor cells in the tumor. In some embodiments,
the increase in lymphocyte infiltration to the tumor is accompanied
by an increase in MHC1 expression on the tumor cells.
[0197] In some embodiments, the invention provides methods for
decreasing expression of HIF-1.alpha. in a tumor in an individual
comprising administering to the individual an effective amount of
an O.sub.2 carrier polypeptide (e.g. an H-NOX protein). In some
embodiments, administration of an effective amount of an O.sub.2
carrier polypeptide (e.g., an H-NOX protein) to an individual
results in a decrease in expression of HIF-1.alpha.. In some
embodiments, the expression of HIF-1.alpha. is decreased by more
than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
100% compared to expression of HIF-1.alpha. in the absence of
treatment with an O.sub.2 carrier polypeptide. In some embodiments,
the expression of HIF-1.alpha. is reduced compared to expression of
HIF-1.alpha. in the absence of treatment with an O.sub.2 carrier
protein for more than about any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6
hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, 24 hr, 30 hr, 36, hr 42 hr or
48 hr. In some embodiments, the O.sub.2 carrier polypeptide is a
trimeric Tt H-NOX L144F polypeptide. In some embodiments, the
O.sub.2 carrier polypeptide is a PEGylated trimeric Tt H-NOX L144F
polypeptide.
[0198] In some embodiments, the invention provides methods for
decreasing expression of HIF-1.alpha. in a tumor in an individual
comprising administering to the individual an effective amount of
an O.sub.2 carrier polypeptide (e.g. an H-NOX protein) wherein the
decrease in expression of HIF-1.alpha. is measured as a decrease in
expression of vascular epithelial cell growth factor (VEGF). In
some embodiments, administration of an effective amount of an
O.sub.2 carrier polypeptide (e.g., an H-NOX protein) to an
individual results in a decrease in expression of VEGF. In some
embodiments, the expression of VEGF is decreased by more than about
any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared
to expression of VEGF in the absence of treatment with an O.sub.2
carrier polypeptide. In some embodiments, the expression of VEGF is
reduced compared to expression of VEGF in the absence of treatment
with an O.sub.2 carrier protein for more than about any of 1 hr, 2
hr, 3 hr, 4 hr, 5 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, 24
hr, 30 hr, 36, hr 42 hr or 48 hr. In some embodiments, the O.sub.2
carrier polypeptide is a trimeric Tt H-NOX L144F polypeptide. In
some embodiments, the O.sub.2 carrier polypeptide is a PEGylated
trimeric Tt H-NOX L144F polypeptide.
[0199] In some embodiments, the invention provides methods for
decreasing expression of HIF-1.alpha. in a tumor in an individual
comprising administering to the individual an effective amount of
an O.sub.2 carrier polypeptide (e.g. an H-NOX protein) herein the
decrease in expression of HIF-1.alpha. is measured as a decrease in
expression of glucose transporter type 1 (Glut1). In some
embodiments, administration of an effective amount of an O.sub.2
carrier polypeptide (e.g., an H-NOX protein) to an individual
results in a decrease in expression of Glut1. In some embodiments,
the expression of Glut1 is decreased by more than about any of 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to
expression of Glut1 in the absence of treatment with an O.sub.2
carrier polypeptide. In some embodiments, the expression of Glut1
is reduced compared to expression of Glut1 in the absence of
treatment with an O.sub.2 carrier protein for more than about any
of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr,
20 hr, 24 hr, 30 hr, 36, hr 42 hr or 48 hr. In some embodiments,
the O.sub.2 carrier polypeptide is a trimeric Tt H-NOX L144F
polypeptide. In some embodiments, the O.sub.2 carrier polypeptide
is a PEGylated trimeric Tt H-NOX L144F polypeptide.
[0200] In some embodiments, the invention provides methods for
decreasing expression of PD-L1 in a tumor in an individual
comprising administering to the individual an effective amount of
an O.sub.2 carrier polypeptide (e.g. an H-NOX protein). In some
embodiments, administration of an effective amount of an O.sub.2
carrier polypeptide (e.g., an H-NOX protein) to an individual
results in a decrease in expression of PD-L1. In some embodiments,
the expression of PD-L1 is decreased by more than about any of 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to
expression of PD-L1 in the absence of treatment with an O.sub.2
carrier polypeptide. In some embodiments, administration of an
effective amount of an O.sub.2 carrier polypeptide (e.g., an H-NOX
protein) to an individual results in a decrease in the interaction
of PD-L1 with PD-1. In some embodiments, the interaction of PD-L1
with PD1 is decreased by more than about any of 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90% or 100% compared to interaction of PD-L1
with PD1 in the absence of treatment with an O.sub.2 carrier
polypeptide. In some embodiments, the expression of PD-L1 is
reduced compared to expression of PD-L1 in the absence of treatment
with an O.sub.2 carrier protein for more than about any of 1 hr, 2
hr, 3 hr, 4 hr, 5 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr, 20 hr, 24
hr, 30 hr, 36, hr 42 hr or 48 hr. In some embodiments, the O.sub.2
carrier polypeptide is a trimeric Tt H-NOX L144F polypeptide. In
some embodiments, the O.sub.2 carrier polypeptide is a PEGylated
trimeric Tt H-NOX L144F polypeptide.
[0201] In some embodiments, the invention provides methods for
decreasing expression of A2AR in a tumor in an individual
comprising administering to the individual an effective amount of
an O.sub.2 carrier polypeptide (e.g. an H-NOX protein). In some
embodiments, administration of an effective amount of an O.sub.2
carrier polypeptide (e.g., an H-NOX protein) to an individual
results in a decrease in expression of A2AR. In some embodiments,
the expression of A2AR is decreased by more than about any of 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to
expression of A2AR in the absence of treatment with an O.sub.2
carrier polypeptide. In some embodiments, administration of an
effective amount of an O.sub.2 carrier polypeptide (e.g., an H-NOX
protein) to an individual results in a decrease in the interaction
of A2AR with adenosine. In some embodiments, the interaction of
A2AR with adenosine is decreased by more than about any of 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to
interaction of A2AR with adenosine in the absence of treatment with
an O.sub.2 carrier polypeptide. In some embodiments, the expression
of A2AR is reduced compared to expression of A2AR in the absence of
treatment with an O.sub.2 carrier protein for more than about any
of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 8 hr, 10 hr, 12 hr, 16 hr,
20 hr, 24 hr, 30 hr, 36, hr 42 hr or 48 hr. In some embodiments,
the O.sub.2 carrier polypeptide is a trimeric Tt H-NOX L144F
polypeptide. In some embodiments, the O.sub.2 carrier polypeptide
is a PEGylated trimeric Tt H-NOX L144F polypeptide.
[0202] In some embodiments, the invention provides methods for
decreasing expression of HIF-2.alpha. in a tumor in an individual
comprising administering to the individual an effective amount of
an O.sub.2 carrier polypeptide (e.g. an H-NOX protein). In some
embodiments, administration of an effective amount of an O.sub.2
carrier polypeptide (e.g., an H-NOX protein) to an individual
results in a decrease in expression of HIF-2.alpha.. In some
embodiments, the expression of HIF-2.alpha. is decreased by more
than about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
100% compared to expression of HIF-2.alpha. in the absence of
treatment with an O.sub.2 carrier polypeptide. In some embodiments,
administration of an effective amount of an O.sub.2 carrier
polypeptide (e.g., an H-NOX protein) to an individual results in a
decrease in the expression of HIF-2u. In some embodiments, the
expression of HIF-2u is decreased by more than about any of 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to
expression of HIF-2.alpha. in the absence of treatment with an
O.sub.2 carrier polypeptide. In some embodiments, the expression of
HIF-2.alpha. is reduced compared to expression of HIF-2.alpha. in
the absence of treatment with an O.sub.2 carrier protein for more
than about any of 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 8 hr, 10 hr,
12 hr, 16 hr, 20 hr, 24 hr, 30 hr, 36, hr 42 hr or 48 hr. In some
embodiments, the O.sub.2 carrier polypeptide is a trimeric Tt H-NOX
L144F polypeptide. In some embodiments, the O.sub.2 carrier
polypeptide is a PEGylated trimeric Tt H-NOX L144F polypeptide.
[0203] In some embodiments, the invention provides methods for
modulating tumor immunity (e.g., enhancing an immune response to a
tumor) in an individual by any of the methods described herein.
Examples of tumors include but are not limited to a brain tumor, a
glioblastoma, a bone tumor, a pancreatic tumor, a skin tumor, a
tumor of the head or neck, a melanoma, a lung tumor, a uterine
tumor, an ovarian tumor, a colorectal tumor, an anal tumor, a liver
tumor, a hepatocellular carcinoma, a stomach tumor, a testicular
tumor, an endometrial tumor, a cervical tumor, a vaginal tumor, a
Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an esophageal tumor,
an intestinal tumor, a thyroid tumor, an adrenal tumor, a bladder
tumor, a kidney tumor, a breast tumor, a multiple myeloma tumor, a
sarcoma, or a squamous cell tumor.
[0204] In some embodiments, the invention provides methods for
modulating tumor immunity (e.g., enhancing an immune response to a
tumor) in an individual by any of the methods described herein
thereby providing methods for treating cancer in an individual.
Examples of cancers that may be treated by the methods of the
invention include but are not limited to brain cancer,
glioblastoma, bone cancer, pancreatic cancer, skin cancer, cancer
of the head or neck, melanoma, lung cancer, uterine cancer, ovarian
cancer, colorectal cancer, anal cancer, liver cancer,
hepatocellular carcinoma, stomach cancer, testicular cancer,
endometrial cancer, cervical cancer, Hodgkin's Disease,
non-Hodgkin's lymphoma, esophageal cancer, intestinal cancer,
thyroid cancer, adrenal cancer, bladder cancer, kidney cancer,
breast cancer, multiple myeloma, sarcoma, or squamous cell
cancer.
[0205] In some embodiments, the invention provides methods for
modulating tumor immunity in an individual by any of the methods
described herein. In some embodiments, the individual is a mammal;
for example a human. In some embodiments, the mammal is a pet, a
laboratory research animal, or a farm animal. Non-limiting examples
of pets, research animals or farm animals include dogs, cats,
horses, monkeys, rabbits, rats, mice, guinea pigs, hamsters, pigs
and cows.
[0206] O.sub.2 carrier polypeptides may be administered by any
route including but not limited to intravenous, intra-arterial,
intratumoral, intravesicular, inhalation, intraperitoneal,
intrapulmonary, intramuscular, subcutaneous, intra-tracheal,
transmucosal, intraocular, intrathecal, or transdermal
administration.
[0207] In some aspects, sustained delivery of oxygen to a tumor is
desired to inhibit hypoxia-mediated tumor immunity and to enhance
an immune response to the tumor. In some embodiments of the
invention, administration of the O.sub.2 carrier polypeptide (e.g.,
H-NOX protein) is repeated. Administration of the O.sub.2 carrier
polypeptide may be repeated until a robust immune response to the
tumor is established. In some embodiments, administration of the
O.sub.2 carrier polypeptide is repeated at least about any one of
two times, three times, four times, five times, six times, seven
times, eight times, nine times, ten times, twelve times, fourteen
times, twenty times, thirty times, forty times, fifty times or one
hundred times. In some embodiments, administration of the O.sub.2
carrier polypeptide is repeated between about two times and about
twenty times. In some embodiments, administration of the O.sub.2
carrier polypeptide is repeated between any one of about twenty
times and about forty times, any one of about forty times and about
sixty times, any one of about sixty times and about eighty times,
any one of about eighty times and about one hundred times, or any
number of times therebetween. In some embodiments, administration
of the O.sub.2 carrier polypeptide is repeated daily or twice a day
for about 42 to about 84 administrations.
[0208] Exemplary dosing frequencies include, but are not limited
to, at least 1, 2, 3, 4, 5, 6, or 7 times (i.e., daily) a week. In
some embodiments, the O.sub.2 carrier polypeptide (e.g., H-NOX
protein) is administered at least 2, 3, 4, or 6 times a day. In
some embodiments, the O.sub.2 carrier polypeptide is administered
every four, every 8, every 12, every 24 hours, every 48 hours or
two times a week or three times a week. In some embodiments, the
O.sub.2 carrier polypeptide is administered any one of between one
hour and two hours, between two hours and four hours, between four
hours and eight hours, between eight hours and twelve hours, or
between twelve hours and 24 hours. In some embodiments, the O.sub.2
carrier polypeptide is administered every four, every 8, every 12
or every 24 hours for a period of about one to about 10 days. In
some embodiments, the O.sub.2 carrier polypeptide can be
administered, e.g., over a period of a few days or weeks. In some
embodiments, the O.sub.2 carrier polypeptide is administrated for a
longer period, such as a few months or years. The dosing frequency
of the composition may be adjusted over the course of the treatment
based on the judgment of the administering physician.
[0209] In some embodiments, the O.sub.2 carrier polypeptide (e.g.,
H-NOX protein) is administered as a bolus. In some embodiments, the
volume of the bolus is greater than about any of 1 mL, 2 mL, 3 mL,
4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, 20 mL, 25 mL, 50
mL, 75 mL, or 100 mL. In some embodiments, administration of the
bolus dose is repeated as above.
[0210] In some embodiments, the O.sub.2 carrier polypeptide (e.g.,
H-NOX protein) is administered by infusion. In some embodiments,
the infusions is for greater than about any of 15 minutes, 30
minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7
hours, 8 hours, 9 hours, 10 hours, 12 hours, 16 hours, 20 hours or
24 hours. In some embodiments, the infusions is for between about
any of 15 minutes and 30 minutes, 30 minutes and 1 hour, 1 hour and
2 hours, 2 hours and 3 hours, 3 hours and 4 hours, 4 hours and 5
hours, 5 hours and 6 hours, 6 hours and 7 hours, 7 hours and 8
hours, 8 hours and 9 hours, 9 hours and 10 hours, 10 hours and 12
hours, 12 hours and 16 hours, 16 hours and 20 hours or 20 hours and
24 hours. In some embodiments, the infusion rate is greater any of
about 1 mL/hr, 2 mL/hr, 3 mL/hr, 4 mL/hr, 5 mL/hr, 6 mL/hr, 7
mL/hr, 8 mL/hr, 9 mL/hr, 10 mL/hr, 20 mL/hr, 30 mL/hr, 40 mL/hr, 50
mL/hr, 60 mL/hr, 70 mL/hr, 80 mL/hr, 90 mL/hr, 100 mL/hr, 200
mL/hr, 300 mL/hr, 400 mL/hr, 500 mL/hr, 600 mL/hr, 700 mL/hr, 800
mL/hr, 900 mL/hr, 1000 mL/hr, 2000 mL/hr, 3000 mL/hr, 4000 mL/hr,
5000 mL/hr, 6000 mL/hr, 7000 mL/hr, 8000 mL/hr, 9000 mL/hr, 10,000
mL/hr or any rate therebetween. In some embodiments, the infusion
is repeated as above.
[0211] In some embodiments, the O.sub.2 carrier polypeptide (e.g.,
H-NOX protein) is administered at a dose of greater than about any
of 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8
mg/kg, 9 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg,
35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65
mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg,
100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg,
700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, or any dose
therebetween. In some embodiments, the dose is provided as one or
more bolus administrations.
[0212] In some embodiments, the dose is provided as one or more
infusions. In some embodiments the dose is provided in more than
one administration (e.g., a dose of 100 mg/kg may be provided by
two doses of 50 mg/kg).
[0213] In some embodiments of the invention, the O.sub.2 carrier
polypeptide (e.g. an H-NOX protein) is used in combination with
radiation therapy. In some embodiments, the O.sub.2 carrier
polypeptide is administered to the individual any of at least 1, 2,
3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours before
administration of the radiation. In some embodiments, the radiation
is X irradiation. In some embodiments, the dose of X irradiation is
any of about 0.5 Gy to about 75 Gy. In some embodiments, the cycle
of O.sub.2 carrier polypeptide administration and radiation
administration is repeated any one of one, two, three, four, five
or six times. In some embodiments, the cycle of O.sub.2 carrier
polypeptide administration and radiation administration is repeated
after any one of about one week, two weeks, three weeks, four
weeks, five weeks or six weeks. In some embodiments, the
administration of the O.sub.2 carrier polypeptide and radiation
therapy is used in conjunction with another therapy; for example, a
chemotherapy and/or immunotherapy.
[0214] In some embodiments of the invention, the O.sub.2 carrier
polypeptide (e.g. an H-NOX protein) is used in combination with
chemotherapy. In some embodiments, the chemotherapy is a cytotoxin.
Chemotherapeutic agents including cytotoxins are known in the art.
In some embodiments, the cytotoxin is an alkylating agent. In some
embodiments, the cytotoxin is cyclophosphamide or temozolomide. In
some embodiments, the O.sub.2 carrier polypeptide is administered
before administration of the chemotherapy. In some embodiments, the
O.sub.2 carrier polypeptide is administered with administration of
the chemotherapy. In some embodiments, the O.sub.2 carrier
polypeptide is administered after administration of the
chemotherapy. In some embodiments, the O.sub.2 carrier polypeptide
is administered to the individual any of at least 1, 2, 3, 4, 5, 6,
7, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours or is administered
daily or twice a day for 1, 2, 3, 4, 5, 6, or 7 days before
administration of the chemotherapy. In some embodiments, the
O.sub.2 carrier polypeptide is administered to the individual any
of at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, or 24 hours after
administration of the chemotherapy. In some embodiments,
administration of the O.sub.2 carrier polypeptide and/or
administration of the chemotherapy is repeated any one of one, two,
three, four, five, six, seven, eight, nine, ten times or more than
ten times. In some embodiments, administration of the O.sub.2
carrier polypeptide and/or administration of the chemotherapy is
repeated after any one of about one week, two weeks, three weeks,
four weeks, five weeks or six weeks. In some embodiments,
administration of the O.sub.2 carrier polypeptide and the
chemotherapy are on the same dosing cycle. In some embodiments,
administration of the O.sub.2 carrier polypeptide and the
chemotherapy are on different dosing cycles. In some embodiments,
the admiration of H-NOX and radiation therapy is used in
conjunction with another therapy; for example, radiation therapy
and/or immunotherapy.
[0215] In some embodiments, the O.sub.2 carrier polypeptide (e.g.,
an H-NOX protein) is administered to cancer patients prior to
and/or in conjunction with an immunotherapy. In some embodiments,
the immunotherapy is one or more of an anticancer vaccine, an
adoptive immune cell therapy, an agent that targets an immune
checkpoint regulator, an oncolytic virus or a BiTE. In some
embodiments, the immunotherapy targets are one or more of CTLA-4,
PD1, PD-L1, or an immune checkpoint regulator. In some embodiments,
the immunotherapy is a dual PD1/CTLA-4 blockade therapy. In some
embodiments, the immunotherapy is a PDL-1 treatment for patients
with PDL1+ tumors or dual PD1/PD-L1 blockade. Nonlimiting examples
include but are not limited to PD-1 and PDL-1 antagonists such as
antibodies (e.g., Nivolumab). In some embodiments the checkpoint
inhibitor is a CTLA4 antagonist such as an antibody (e.g.,
ipilumumab). In some embodiments, the immunotherapy is an adoptive
T cell therapy including but not limited to chimeric antigen
receptor T cells (e.g., CAR-T cells) or engineered TCR-T cells. In
some embodiments, the immunotherapy is a Bispecific T cell Engagers
(BiTE). In some embodiments, the immunotherapy includes one or more
of anti-lymphocyte activation gene3 (LAG-3) therapy, anti-T cell
immunoglobin mucin-3 (TIM-3) therapy, anti-killer immunoglobin-like
receptors (KIR) therapy, anti-4-1BB (CD137) agonizing/stimulatory
therapy, or glucocorticoid-induced TNFR family related gene (GITR)
agonizing/stimulatory therapy--each alone or in combinations with
each other, and/or in combination with one or more of PD1, PDL-1,
CTLA-4 or other therapies.
[0216] Nonlimiting examples of therapies that target checkpoint
proteins other than PD-1/PDL-1 and CTLA4 negative regulators
include both positive and negative (checkpoint inhibitors)
regulators of immune response and can be antibodies or small
molecules such as IDO (indoleamine-2.3 dioxygenase) pathway
inhibitors such as direct IDO enzymatic activity inhibitors (e.g.
NLG919), IDO effector pathway inhibitors (e.g.
D-1-methyl-tryptophan, Indoximod, NLG8189), TDO (tryptophan
2,3-dioxygenase) inhibitors, or IDO-TDO dual inhibitors;
Lymphocyte-activation gene 3 (LAG-3, CD223) antibody antagonists
(e.g. IMP321, BMS-986016); Killer immunoglobulin-like receptors
(KIRs) antagonists such as antibodies (e.g. lirilumab, IPH2101); T
cell immunoglobulin mucin-3 (TIM-3) antagonists such as antibodies;
B- and T cell attenuator (BTLA, CD272) antagonists such as
antibodies; OX40 (CD134) agonists such as activating/stimulating
antibodies; 4-1BB (CD137) agonists such as stimulatory antibodies
(e.g. BMS-663513); Glucocorticoid-induced TNFR family related gene
(GITR) agonists such as stimulatory antibodies (e.g. TRX518); and
oncolytic viruses.
[0217] In some embodiments, the O.sub.2 carrier polypeptide is
administered before administration of the immunotherapy. In some
embodiments, the O.sub.2 carrier polypeptide is administered with
administration of the immunotherapy. In some embodiments, the
O.sub.2 carrier polypeptide is administered after administration of
the immunotherapy. In some embodiments, the O.sub.2 carrier
polypeptide is administered to the individual any of at least 1, 2,
3, 4, 5, 6, 7, 8, 10, 12, 24, or 48 hours before administration of
the immunotherapy. In some embodiments, the O.sub.2 carrier
polypeptide is administered to the individual any of at least 3, 4,
5, 6, 7 or more days before administration of the immunotherapy. In
some embodiments, the O.sub.2 carrier polypeptide is administered
to the individual any of at least 1, 2, 3, 4, 5, 6, 7, 8, 10, 12,
24 or 48 hours after administration of the immunotherapy. In some
embodiments, the O.sub.2 carrier polypeptide is administered to the
individual any of at least 3, 4, 5, 6, 7 or more days after
administration of the immunotherapy. In some embodiments,
administration of the O.sub.2 carrier polypeptide and/or
administration of the immunotherapy is repeated any one of one,
two, three, four, five, six, seven, eight, nine, ten times or more
than ten times. In some embodiments, administration of the O.sub.2
carrier polypeptide and/or administration of the immunotherapy is
repeated after any one of about one week, two weeks, three weeks,
four weeks, five weeks or six weeks. In some embodiments,
administration of the O.sub.2 carrier polypeptide and the
immunotherapy are on the same dosing cycle. In some embodiments,
administration of the O.sub.2 carrier polypeptide and the
immunotherapy are on different dosing cycles. In some embodiments,
the admiration of H-NOX and radiation therapy is used in
conjunction with another therapy; for example, radiation therapy
and/or chemotherapy.
[0218] In some embodiments, the effectiveness of administration of
the O.sub.2 carrier polypeptide (e.g., H-NOX protein) is monitored;
for example but not limited to tumor hypoxia, expression
hypoxia-associated tumor suppressors and/or activators, presence of
tumor-associated immune cells and/or immune cells directed against
tumor cells and/or local (tumor biopsy, lymph node biopsy) or
systemic (e.g. peripheral blood) cytokine and immune cell profiles.
Methods to determine the level of tumor hypoxia are known in the
art. Examples include but are not limited to measurement of any one
of .sup.18F-fluoromisonidazole (FMISO) tumor uptake, pimidazole
uptake, .sup.18F-fluoroazomycin arabinoside (FAZA) uptake, a
nitroimidazole uptake,
Copper(II)-diacetyl-bis(N4-methylthiosemicarbazone (Cu-ATSM)
uptake, hexafluorobenzene (C6F6) uptake by .sup.19F magnetic
resonance imaging, hexamethyldisiloxane uptake by .sup.1H MRI,
tumor HIF-1.alpha. expression, tumor HIF-2.alpha. expression, tumor
HIF-3.alpha. expression, tumor Glut-1 expression, tumor pH
(pH-weighted MRI) qBOLD, OE-MRI, MOBILE MRI tumor LDHA expression,
tumor carbonic anhydrase IX (CA-9) expression, VEGF expression, or
lactate and/or pyruvate levels. In some embodiments of the methods
of monitoring, treating, and optimization of therapy described
above, tumor hypoxia is measured by .sup.18F-FMISO uptake. In some
embodiments, .sup.18F-FMISO uptake is measured by Positron emission
tomography (PET) scan, computed tomography (CT) scan or computed
axial tomography (CAT) scan. Methods to detect expression of genes
such as HIF-1.alpha., PD-L1 and A2AR are known in the art; for
example, by immunoassay, by immunohistochemistry, by quantitative
PCR, by hybridization (for example, on a gene chip), and the
like.
Kits with H-NOX Proteins
[0219] Also provided are articles of manufacture and kits for the
modulation of tumor immunity in an individual. In some embodiments,
the article of manufacture or kit comprises any of the O.sub.2
carrier polypeptides including any of the H-NOX proteins described
herein including polymeric H-NOX proteins and PEGylated polymeric
H-NOX proteins, and suitable packaging. In some embodiments, the
invention includes a kit with (i) a H-NOX protein (such as a
wild-type or mutant H-NOX protein described herein or formulations
thereof as described herein) and (ii) instructions for using the
kit to deliver O.sub.2 to an individual.
[0220] Suitable packaging for compositions described herein are
known in the art, and include, for example, vials (e.g., sealed
vials), vessels, ampules, bottles, jars, flexible packaging (e.g.,
sealed Mylar or plastic bags), and the like. These articles of
manufacture may further be sterilized and/or sealed. Also provided
are unit dosage forms comprising the compositions described herein.
These unit dosage forms can be stored in a suitable packaging in
single or multiple unit dosages and may also be further sterilized
and sealed. Instructions supplied in the kits of the invention are
typically written instructions on a label or package insert (e.g.,
a paper sheet included in the kit), but machine-readable
instructions (e.g., instructions carried on a magnetic or optical
storage disk) are also acceptable. The instructions relating to the
use of H-NOX proteins generally include information regarding
dosage, dosing schedule, and route of administration for the
intended treatment or industrial use. The kit may further comprise
a description of selecting an individual suitable for
treatment.
[0221] The containers may be unit doses, bulk packages (e.g.,
multi-dose packages) or sub-unit doses. For example, kits may also
be provided that contain sufficient dosages of H-NOX proteins
disclosed herein to provide effective treatment for an individual
for an extended period, such as about any of a week, 2 weeks, 3
weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6
months, 7 months, 8 months, 9 months, or more. Kits may also
include multiple unit doses of H-NOX proteins and instructions for
use and packaged in quantities sufficient for storage and use in
pharmacies, for example, hospital pharmacies and compounding
pharmacies. In some embodiments, the kit includes a dry (e.g.,
lyophilized) composition that can be reconstituted, resuspended, or
rehydrated to form generally a stable aqueous suspension of H-NOX
protein.
Exemplary Methods for Production of H-NOX Proteins
[0222] As noted above, the sequences of several wild-type H-NOX
proteins and nucleic acids are known and can be used to generate
mutant H-NOX domains and nucleic acids of the present invention.
Techniques for the mutation, expression, and purification of
recombinant H-NOX proteins have been described by, e.g., Boon, E.
M. et al. (2005). Nature Chemical Biology 1:53-59 and Karow, D. S.
et al. (Aug. 10, 2004). Biochemistry 43(31):10203-10211, U.S. Pat.
Nos. 8,404,631 and 8,404,632, WO 2007/139791, and WO 2007/139767
which are hereby incorporated by reference in their entireties,
particularly with respect to the mutation, expression, and
purification of recombinant H-NOX proteins. These techniques or
other standard techniques can be used to generate any mutant H-NOX
protein.
[0223] A mutant H-NOX nucleic acid can be incorporated into a
vector, such as an expression vector, using standard techniques.
For example, restriction enzymes can be used to cleave the mutant
H-NOX nucleic acid and the vector. Then, the compatible ends of the
cleaved mutant H-NOX nucleic acid and the cleaved vector can be
ligated. The resulting vector can be inserted into a cell (e.g., an
insect cell, a plant cell, a yeast cell, or a bacterial cell) using
standard techniques (e.g., electroporation) for expression of the
encoded H-NOX protein.
[0224] In particular, heterologous proteins have been expressed in
a number of biological expression systems, such as insect cells,
plant cells, yeast cells, and bacterial cells. Thus, any suitable
biological protein expression system can be utilized to produce
large quantities of recombinant H-NOX protein. In some embodiments,
the H-NOX protein (e.g., a mutant or wild-type H-NOX protein) is an
isolated protein.
[0225] If desired, H-NOX proteins can be purified using standard
techniques. In some embodiments, the protein is at least about 60%,
by weight, free from other components that are present when the
protein is produced. In various embodiments, the protein is at
least about 75%, 90%, or 99%, by weight, pure. A purified protein
can be obtained, for example, by purification (e.g., extraction)
from a natural source, a recombinant expression system, or a
reaction mixture for chemical synthesis. Exemplary methods of
purification include immunoprecipitation, column chromatography
such as immunoaffinity chromatography, magnetic bead immunoaffinity
purification, and panning with a plate-bound antibody, as well as
other techniques known to the skilled artisan. Purity can be
assayed by any appropriate method, e.g., by column chromatography,
polyacrylamide gel electrophoresis, or HPLC analysis. In some
embodiments, the purified protein is incorporated into a
pharmaceutical composition of the invention or used in a method of
the invention. The pharmaceutical composition of the invention may
have additives, carriers, or other components in addition to the
purified protein.
[0226] In some embodiments, the polymeric H-NOX protein comprises
one or more His.sub.6 tags. An H-NOX protein comprising at least
one His.sub.6 tag may be purified using chromatography; for
example, using Ni.sup.2+-affinity chromatography. Following
purification, the His.sub.6 tag may be removed; for example, by
using an exopeptidase. In some embodiments, the invention provides
a purified polymeric H-NOX protein, wherein the polymeric H-NOX
protein was purified through the use of a His.sub.6 tag. In some
embodiments, the purified H-NOX protein is treated with an
exopeptidase to remove the His.sub.6 tags.
[0227] In some embodiments, H-NOX protein comprises one or more
molecules of polyethylene glycol (i.e., PEGylated). Methods to
produce PEGylated proteins are known in the art.
Examples
[0228] The examples, which are intended to be purely exemplary of
the invention and should therefore not be considered to limit the
invention in any way, also describe and detail aspects and
embodiments of the invention discussed above. The examples are not
intended to represent that the experiments below are all or the
only experiments performed. Unless indicated otherwise, temperature
is in degrees Centigrade and pressure is at or near
atmospheric.
Example 1. H-NOX Enables Efficient Oxygenation of Hypoxic Tumor
Microenvironments
[0229] A PEGylated trimeric Thermoanaerobacter tengcongensis (Tt.)
H-NOX bearing a L144F substitution in the distal pocket (FIG. 6B)
was evaluated for the ability to oxygenate tumor microenvironments
and increase radiation sensitity. Administration of the PEGylated
trimeric Tt H-NOX L144F to mice bearing hypoxic tumors induces
rapid and sustained oxygenation of the tumors as directly measured
by the external hypoxia marker, pimonidazole, hypoxia inducible
transcription factor 1 alpha, HIF-1-.alpha., and OxyLite
oxygen-sensing nanofiber (FIGS. 2 and 3, respectively).
[0230] Mice bearing H460 subcutaneous xenograft tumors were
injected i.v. with PEGylated trimer H-NOX (L144F) at 650 mg/kg when
tumor volume reached .about.300-350 mm.sup.3 (.about.10-14 days
after tumor cell subcutaneous implantation). Prior to euthanasia,
mice were injected with the exogenous hypoxia marker pimonidazole
at 60 mg/kg and tumors were harvested. Pimonidazole (FIG. 2A)
(Hypoxyprobe-1) and HIF-1.alpha. (FIG. 2B) levels were measured by
competitive (pimonidazole) and sandwich (HIF-1.alpha., Abcam)
ELISAs, respectively. Graphs show quantification of pimonidazole
and HIF-1.alpha. signals after PEGylated H-NOX (L144F)
administration. Vehicle, 1h and 4h: n=22, 7h: n=18, 12h: n=16, 24h:
n=6. Results from 4 independent experiments. Mean values+/-SEM.
****p<0.0001, ***p<0.001, **p<0.01, *p<0.05 by one-way
ANOVA and Bonferroni's post-hoc tests. (FIG. 2C) Tumors were
assessed for the accumulation of PEGylated H-NOX (L144F) by
sandwich H-NOX ELISA at 1, 4, 7, 16 and 24 hours after injection
and results expressed per gram of tumor tissue. Seven to eight week
old Nu/Nu female mice were subcutaneously implanted with
3.times.10.sup.6 of H460 human lung cancer cells and monitored
until the tumors reached average size of .about.300 mm.sup.3 (10-14
days post-implantation of tumor cells). Mice bearing 200-350
mm.sup.3 xenograft tumors were injected i.v. with bolus vehicle
(formulation buffer: 50 mM succinate, 50 mM NaCl, 3.4 mM EDTA, and
10 mM reduced glutathione at pH 7) or formulation buffer containing
650 mg/kg of PEGylated trimer H-NOX (L144F). To measure tumor
hypoxia, prior to euthanasia, mice were injected with the exogenous
hypoxia marker pimonidazole at 60 mg/kg. Tumors were harvested and
homogenized in an extraction buffer (Abcam kit # ab117996)
supplemented with anti-proteases. Protein concentration was
quantified in each tumor using a Bradford assay. Samples were
assayed for pimonidazole (Hypoxyprobe-1) amount using a competitive
ELISA assay developed by Omniox and for HIF-1.alpha. using the
Abcam ELISA kit (ab117996).
[0231] In H460 lung carcinoma mouse model maximum oxygenation was
achieved between 4h and 8h and it correlated with the peak of H-NOX
(L144F) tumor accumulation as assessed by ELISA (FIG. 2C).
[0232] For assessment of the H-NOX (L144F) tumor accumulation,
tumors were harvested at different timepoints after injection.
Tumors were homogenized in an extraction buffer (Abcam kit #
ab117996) supplemented with anti-proteases and protein
concentration was quantified in each tumor using a Bradford assay.
PEGylated H-NOX (L144F) concentration was quantified by a sandwich
ELISA for H-NOX developed by Omniox and normalized to tumor
weight.
[0233] While supplemental oxygenation of animals successfully
increased oxygenation of mouse tumor tissue at 5-10 mmHg oxygen
concentration, it had no effect on regions with lower oxygen levels
(<5 mmHg). By contrast, PEGylated trimer Tt H-NOX L144F is
capable of increasing oxygenation even in severely hypoxic tumor
tissue (<5 mmHg). This is likely due to PEGylated trimer Tt
H-NOX L144F's superior tissue penetration that enables oxygen
delivery to areas beyond oxygen gradient diffusion limits.
Moreover, while maximum supplemental oxygenation of mouse tumors is
achieved with exposing animals continuously to 95%-100% breathing
oxygen [increasing risk of hyperoxic and inflammatory damage to the
normal tissues (Kallet & Matthay, 2013 Respir Care,
58(1):123-141; Thiel et al., 2005 PLoS Biol, 3(6), e174)], single
bolus i.v. dose of PEGylated trimer TL H-NOX L144F can maintain
tissue oxygenation for more than 7 hours without increasing oxygen
levels in normal tissues. A control Tt H-NOX protein (wild type
variant)--that is not capable of releasing oxygen at oxygen
concentrations present in hypoxic tissues-did not have any effect
on tumor oxygenation (FIG. 3C).
[0234] Seven to eight week old Nu/Nu female mice were
subcutaneously implanted with 3.times.10.sup.6 of H460 human lung
cancer cells and monitored until the tumors reached average size of
.about.500 nm.sup.3(10-18 days post-implantation of tumor cells).
Mice bearing H460 tumors were anesthetized with isoflurane mixed in
20% of oxygen and the OxyLite.TM. probe (Oxford Optronix, UK) was
implanted into H460 subcutaneous xenograft tumors using a
micromanipulator. The OxyLite.TM. consists of the ruthenium
chloride dye held in a polymer matrix of 230 .mu.m in diameter at
the tip. After equilibration for .about.20-30 minutes, pO.sub.2 was
measured using optical fluorescence sensors attached to a
four-channel unit. A low starting pO.sub.2 confirmed entry into
hypoxic tissue away from neighbouring blood vessels (.about.0.2
mmHg; except in FIG. 3D where 5 mmHg). After probe implantation,
probe was left for .about.20-30 min in order for pO.sub.2
measurements to stabilize, and mice were given to respire 100%
O.sub.2 (FIG. 3B, FIG. 3D) or were injected with PEGylated H-NOX
(L144F in FIG. 3A, wt in FIG. 3C) and fluorescent quenching was
recorded.
[0235] The superior ability of PEGylated trimer Tt H-NOX L144F to
deliver oxygen to hypoxic tumor regions relative to the
administration of the hyperoxic gas was further demonstrated by
more efficient radiation tumor cell kill (FIG. 4).
[0236] Mice bearing H460 subcutaneous xenograft tumors (200-350
mm.sup.3) were treated with 10 Gy alone or in combination with 650
mg/kg of PEGylated trimer H-NOX (L144F) injected i.v. 7 hours prior
to irradiation. Tumors were extracted after irradiation and
processed for clonogenic assay. Cell numbers were counted 10-14
days later in triplicate samples from each tumor. Each dot on the
graph represents average surviving fraction for one tumor. Mean
values+/-SEM (n=3 per experiment). Seven to eight week old Nu/Nu
female mice were subcutaneously implanted with 3.times.10.sup.6 of
H460 human lung cancer cells and monitored until the tumors reached
average size of .about.300 mm.sup.3 (10-14 days post-implantation
of tumor cells). Mice bearing 200-350 mm.sup.3 xenograft tumors
were irradiated with 10 Gy alone or in combination with intravenous
delivery of either a bolus vehicle (formulation buffer: 50 mM
succinate, 50 mM NaCl, 3.4 mM EDTA, and 10 mM reduced glutathione
at pH 7) or a formulation buffer containing 650 mg/kg of PEGylated
H-NOX (L144F). Mice were sacrificed after irradiation and tumors
were harvested and processed for an ex-vivo clonogenic assay.
Briefly, tumors cells were minced into fine pieces with a scalpel
and digested for .about.30 minutes with an enzymatic cocktail
containing a mix of collagenase (200 U/ml), hyaluronidase (200
U/ml) and DNAse (10 ml of cocktail/g of tumor). Extracted cells
were then counted and seeded at 500/125/25 cells per well for
untreated and 2000/500/100 cells per well for irradiated tumor
samples in a 6 well plate in duplicate. After 10-12 days, cell
colonies were fixed with PFA and stained with crystal violet. The
number of clones (over 50 cells) was counted and the plating
efficiency was calculated in untreated samples (number of cells
counted in well/number of cells plated x 100). Surviving fraction
was calculated in all samples (number of cells counted/plating
efficiency x 100).
[0237] Following PEGylated trimer Tt H-NOX L144F administration,
there was >15-fold increase in radiation treatment efficacy in
all PEGylated trimer Tt H-NOX L144F-treated tumors that reduced the
surviving fraction of tumor cells from 30% in the 10 Gy treatment
alone group to <2% in the H-NOX (L144F)-pretreated tumor. In the
same experiment, treatment of mice bearing tumors with 100% oxygen
showed variable increase in radiation enhancement (.about.3 fold)
probably resulting from unequal tumor oxygenation between
individual tumors likely due to uneven vascular density between
tumors.
Example 2. H-NOX Acts as an Immunoactivator Enhancing Host
Anti-Tumor Responses
[0238] PEGylated trimer Tt H-NOX L144F-induced oxygenation inhibits
the HIF-1.alpha. pathway (FIG. 2B) and relieves
HIF-1.alpha.-dependent and HIF-1.alpha.-independent tumor
immunosuppression. Mice bearing H460 subcutaneous xenograft tumors
(200-350 mm.sup.3) were either treated with vehicle alone or with
PEGylated trimer H-NOX (L144F) and harvested 7, 16 or 24 hours
after injection for qRT-PCR analysis. Mean values+/-SEM. N=5-6 per
group, *p<0.05 by t-test. Treatment with a single dose of H-NOX
resulted in significant downregulation of HIF-1.alpha. and its
effectors including direct and indirect modulators of the host's
immune response: PD/PDL-1 and VEGF signaling, metabolic and growth
factor regulators (FIG. 5).
[0239] Seven to eight week old Nu/Nu female mice were
subcutaneously implanted with 3.times.10.sup.6 of H460 human lung
cancer cells and monitored until the tumors reached average size of
.about.300 mm.sup.3 (10-14 days post-implantation of tumor cells).
Mice bearing 200-350 mm.sup.3 xenograft tumors were injected with
either a bolus vehicle (formulation buffer: 50 mM succinate, 50 mM
NaCl, 3.4 mM EDTA, and 10 mM reduced glutathione at pH 7) or a
formulation buffer containing 650 mg/kg of PEGylated trimer H-NOX
(L144F). To prepare samples for qRT-PCR analysis, mice were
sacrificed and tumors excised. Total RNA was extracted from tumor
samples using the RNeasy kit (QIAGEN) according to the manufacturer
instructions. Reverse transcription and real-time PCR (RT-PCR) on a
StepOnePlus.TM. Real-Time PCR System (Applied Biosystems) were
performed as described. 25 .mu.L reaction was prepared using 2
.mu.L of cDNA template, 12.5 .mu.L of SYBR.RTM. Green PCR Master
Mix (Applied Biosystems) and 1 .mu.L of the following sense and
antisense primers: VEGF: forward, 5'-CAATCGAGACCCTGGTGGA-3' (SEQ ID
NO:23); reverse, 5'-GCACACACTCCAGGCCCT-3' (SEQ ID NO:24); Glut1:
forward, 5'-CAACCAGACATGGGTCCAC-3' (SEQ ID NO:25); reverse,
5'-GTTAACGAAAAGGCCCACAGA-3' (SEQ ID NO:26); PDL1: forward,
5'-GTTGTGGATCCAGTCACCTCT-3' (SEQ ID NO:27); reverse,
5'-GATTCTCAGTGTGCTGGTCAC-3'(SEQ ID NO:28); L7: forward,
5'-CAAGGAGGAAGCTTATCTATGAA-3'(SEQ ID NO:29); reverse,
5'-ATTTGACGAAGGCGAAGAAGCT-3' (SEQ ID NO:30). Thermocycling
conditions were as follows: initial step was 10 min at 95.degree.
C., then 40 cycles of 15 s denaturation at 95.degree. C. followed
by 1 min annealing and extension at 60.degree. C. Results were
analyzed with the StepOne Software v2.0 using the comparative CT
method. Transcripts of gene of interest were normalized against the
transcripts of the mouse ribosomal protein L7 housekeeping gene,
and were presented as fold change relative to the L7 transcript
content.
[0240] For example, PEGylated trimer Tt H-NOX L144F-mediated
downregulation of the HIF-1.alpha. and A2AR adenosinergic signaling
may result in activation and recruitment of effector T cells to the
tumor tissue leading to increased lymphocyte tumor infiltration,
decrease in metastatic tumor growth and tumor regression.
Furthermore, H-NOX-induced oxygenation of tumors may reduce
immunoevasion of tumor cells by inhibiting multiple
hypoxia-dependent mechanisms including downregulation of MHC1 and
upregulation of PDL-1 expression on tumor cell surface, and
activation and recruitment of myeloid-derived suppressor cells
(MDSC) including TAMs that directly suppress immune effector cells
as well as promote angiogenesis and metastasis (see FIG. 1B). H-NOX
treatment may inhibit recruitment of macrophages to TME by
downregulating VEGF, CSF1 and other HIF-1-dependent cytokine
signaling (Chaturvedi et al., 2014 Proc Natl Acad Sci USA,
111(20):E2120-2129; Lewis & Hughes, 2007 Breast Cancer Res,
9(3):209) as well as HIF-1- and HIF-2-mediated macrophage
activation (Fang et al., 2009 Blood, 114(4):844-859; Takeda et al.,
2010 Genes Dev, 24(5):491-501).
[0241] Finally, in stimulating host's anti-tumor immune response,
H-NOX may act as co-activator enhancing other targeted cancer
immunotherapies such as, but not limited to, anti-PD1 (programmed
cell death protein 1), anti-PDL-1 (programmed cell death protein
ligand 1), anti-CTLA-4 or therapies targeting other immune
checkpoints' regulators, anti-cancer vaccines, adoptive immune cell
therapies or combinations thereof. For example, H-NOX may be
administered to cancer patients prior to and in conjunction with
dual PD1/CTLA-4 blockade therapy or in combination with PDL-1
treatment in patients with PDL1+ tumors. It may also act as an
adjuvant to other cancer treatments including, but not limited to,
chemotherapy, radiation therapy or other non-immune targeted or
cell-based therapies that may benefit from active anti-tumor immune
defenses. Indeed, H-NOX may synergize with radiation by
simultaneously stimulating anti-tumor immune response towards
radiation exposed tumor-specific antigens from damaged tumor tissue
(Demaria et al., 2005 Int J Radiat Oncol Biol Phys, 63(3), 655-666)
and oxygen-dependent tumor cell killing (Brown, 2010 Int J Radiat
Biol, 86(11), 907-917). During radiotherapy, H-NOX may also act as
normal tissue radioprotectant by ameliorating hypoxia resulting
from radiation-induced vascular damage.
Example 3. Measurement of Hypoxia and T Cells in B16F10 and CT26
Subcutaneous Tumors and Intracranial GL261-Luciferase Tumors
[0242] Generation of B16F10 and CT26 Subcutaneous Tumors and
Intracranial GL261-Luciferase Tumors.
[0243] Six to eight week old C57BL/6J female mice were
subcutaneously implanted with 1.times.10.sup.6 B16F10 mouse
melanoma cancer cells on the flank (FIG. 7A). Six to eight week old
BALB/c female mice were subcutaneously implanted with
1.times.10.sup.6 CT26 colon tumor cells on the flank (FIG. 7B).
Male C57BL/6J weighting 20 g were injected with 3.times.10.sup.5
GL261-luc cells intracranially into the right caudate nucleus (+0.5
mm A/P, +2.3 mm M/L and -3.2 mm D/V) (FIG. 7C). Intracranial tumors
were allowed to grow for 21 days before sacrifice. Subcutaneous
tumors were measured 3 times a week using calipers and tumor size
was calculated based on the formula: (length x width.sup.2)+0.5.
Once the tumors reached an average size of -300 mm.sup.3 (10-14
days post-implantation of tumor cells), treatment was
initiated.
[0244] Treatment.
[0245] Mice bearing 200-400 mm.sup.3 subcutaneous tumors or day 21
intracranial tumors were injected with the exogenous hypoxia marker
pimonidazole ip. (60 mg/kg, Hypoxyprobe, Burlington Mass.) 1-8
hours prior to sacrifice.
[0246] Immunohistochemistry.
[0247] Rodents were euthanized and tumors resected for
immunohistochemistry (IHC) assay. Tumors were frozen in OCT and
sectioned at 12 .mu.M for IHC processing. Sections were fixed with
4% PFA for 15 minutes at 4.degree. C., then blocked and
permeabilized with 5% BSA, 5% goat serum, and 0.1% Tween 20 for 1-2
hours at room temperature. Sections were then incubated with rabbit
anti-pimonidazole (Hypoxyprobe, 1:100) (FIGS. 7A, 7B, 7C, top
panels) and rat anti-CD3, rat anti-CD4 (Biolegend, 1:50) or rat
anti-CD8 (Biolegend, 1:50) antibodies overnight at 4.degree. C.
(FIGS. 7A, 7B, 7C, middle panels), followed by anti-rabbit or
anti-rat secondary antibodies (1:1000, Jackson Immunoresearch
Laboratories, West Grove, Pa., USA) for 2 hours at room
temperature. The sections were mounted in SlowFade DAPI
(Invitrogen). Sections were imaged with an HD Axiolmager Zeiss
microscope equipped with a CCD digital camera.
[0248] Quantification.
[0249] In each animal, the number of CD3+, CD4+ or CD8+ T cells was
counted in pimonidazole-positive and pimonidazole-negative areas in
2-4 pictures per section in 5 tumor sections spanning 1-1.5 mm of
the tumor thickness. The sum of CD4+ and CD8+ cells in each area
was divided by the sum of pimonidazole-positive and
pimonidazole-negative areas to obtain the total number of T cells
per mm.sup.2 of tumor tissue (FIGS. 7A, 7B, and 7C, bottom panels).
Hypoxic regions of tumors (H) showed 2.5 to over 10-fold less T
cells than normoxic regions (N).
Example 4. H-NOX Treatment of Hypoxic Tumors
[0250] Generation of B16F10 Subcutaneous Tumors.
[0251] Six to eight week old C57BL/6J female mice were
subcutaneously implanted with 1.times.10.sup.6 of B16F10 mouse
melanoma cancer cells on the flank. Tumors were measured 3 times a
week using calipers and tumor size was calculated based on the
formula: (length x width.sup.2)/0.5. Once the tumors reached an
average size of .about.300 mm.sup.3 (10-14 days post-implantation
of tumor cells), treatment was initiated.
[0252] Treatment.
[0253] Mice bearing 200-400 mm.sup.3 tumors were randomized in each
treatment group based on tumor size and injected intratumorally
with vehicle (formulation buffer: 50 mM succinate, 50 mM NaCl, 3.4
mM EDTA, and 10 mM reduced glutathione at pH 7) or 100 .mu.l of
formulation buffer containing 2 mg of PEGylated H-NOX (L144F). One
hour prior to vehicle or H-NOX treatment, mice were injected with
the exogenous hypoxia marker pimonidazole ip. (60 mg/kg,
Hypoxyprobe, Burlington Mass.).
[0254] Immunohistochemistry.
[0255] 6 hours after H-NOX injection, rodents were euthanized and
tumors resected for immunohistochemistry (IHC) assay. Tumors were
frozen in OCT and sectioned at 12 .mu.M for IHC processing.
Sections were fixed with 4% PFA for 15 minutes at 4.degree. C.,
then blocked and permeabilized with 5% BSA, 5% goat serum, and 0.1%
Tween 20 for 1-2 hours at room temperature. Sections were then
incubated with rabbit anti-pimonidazole (Hypoxyprobe, 1:100) or
rabbit anti-carbonic anhydrase IX (CAIX, Novus Biological 1:1000)
and rat anti-CD4 (Biolegend, 1:50) or rat anti-CD8 (Biolegend,
1:50) antibodies overnight at 4.degree. C., followed by anti-rabbit
or anti-rat secondary antibodies (1:1000, Jackson Immunoresearch
Laboratories, West Grove, Pa., USA) for 2 hours at room
temperature. The sections were mounted in SlowFade DAPI
(Invitrogen). Sections were imaged with an HD Axiolmager Zeiss
microscope equipped with a CCD digital camera (FIGS. 8 and 9B).
[0256] Quantification.
[0257] In each animal, the number of CD4+ and CD8+ T cells was
counted in pimonidazole-positive, pimonidazole-negative,
CAIX-positive and CAIX-negative areas in 4 pictures per section in
5 tumor sections spanning 1-1.5 mm of the tumor thickness. The sum
of CD4+ and CD8+ cells in each area was divided by the sum of
pimonidazole-positive, pimonidazole-negative, CAIX-positive and
CAIX-negative areas to obtain the total number of T cell per
mm.sup.2 of tumor tissue. Results shown in FIGS. 8 and 9A
demonstrate that OMX treatment as compared to the vehicle control
(formulation buffer) enhances accumulation of CD4+ and CD8+
lymphocytes in previously pimondazole-negative (FIG. 8) or
CAIX-negative (FIG. 9B) labeled hypoxic regions of the tumors.
Example 5. Measurement of Tumor Hypoxia and Tumor Vessels
[0258] Generation of H460, B16F10 and CT26 Subcutaneous Tumors and
Intracranial GL261-Luciferase Tumors.
[0259] Seven to eight week old Nu/Nu female mice were
subcutaneously implanted with 3.times.10.sup.6 of H460 human lung
cancer cells in the hind limb. Six to eight week old C57BL/6J
female mice were subcutaneously implanted with 1.times.10.sup.6
B16F10 mouse melanoma cancer cells on the flank. Six to eight week
old BALB/c female mice were subcutaneously implanted with
1.times.10.sup.6 CT26 colon tumor cells on the flank. Male C57BL/6J
weighting 20 g were injected with 3.times.10.sup.5 GL261-luc cells
intracranially into the right caudate nucleus (+0.5 mm A/P, +2.3 mm
M/L and -3.2 mm D/V). Intracranial tumors were allowed to grow for
21 days before sacrifice. Subcutaneous tumors were measured 3 times
a week using calipers and tumor size was calculated based on the
formula: (length x width.sup.2)/0.5. Once the tumors reached an
average size of .about.300 mm.sup.3 (10-14 days post-implantation
of tumor cells), treatment was initiated. Treatment. Mice bearing
200-400 mm.sup.3 subcutaneous tumors or day 21 intracranial tumors
were injected with the exogenous hypoxia marker pimonidazole ip.
(60 mg/kg, Hypoxyprobe, Burlington Mass.) 1-8 hours prior to
sacrifice.
[0260] Immunohistochemistry and ELISA.
[0261] Rodents were euthanized and tumors resected for
immunohistochemistry (IHC) and ELISA assays. For ELISA, B16F10,
CT26 and H460 tumors were homogenized in an extraction buffer
(Abcam kit # ab117996) supplemented with anti-proteases. Protein
concentration was quantified in each tumor using a Bradford assay
and samples were assayed for hypoxia levels using a competitive
Pimonidazole (Hypoxyprobe-1, Hypoxyprobe, Burlington Mass.) ELISA
assay. For IHC, GL261 tumors were frozen in OCT and sectioned at 12
.mu.M for IHC processing. Sections were fixed with 100% methanol
for 20 minutes at -20.degree. C., then blocked and permeabilized
with 5% BSA, 5% goat serum, and 0.1% Tween 20 for 1-2 hours at room
temperature. Sections were then incubated with rabbit
anti-pimonidazole (Hypoxyprobe, 1:100) and rat anti-CD31 (BD
Bioscience, 1:50) antibodies overnight at 4.degree. C., followed by
anti-rabbit or anti-rat secondary antibodies (1:1000, Jackson
Immunoresearch Laboratories, West Grove, Pa., USA) for 2 hours at
room temperature. The sections were mounted in SlowFade DAPI
(Invitrogen). Sections were imaged with an HD Axiolmager Zeiss
microscope equipped with a CCD digital camera (FIG. 10).
[0262] Quantification.
[0263] For the pimonidazole ELISA, quantification of the IC.sub.50
values ("Kd") were performed with a 5-parameter fit of the standard
curve and values were normalized according to the protein
concentration in each sample. For GL261 IHC, in each animal, the
percent of pimonidazole+ area within the tumor tissue was
determined using ImageJ (1-2 pictures per section in 5 tumor
sections spanning 1 mm of the tumor thickness) (FIG. 10). These
data show that while there is a range in the levels of hypoxia
between individual animals and between tumor types, its presence is
significant in a majority of the tumors of the examined sizes.
Example 6. Measurement of H-NOX Accumulation in Tumors
[0264] Generation of B16F10 and CT26 Subcutaneous Tumors and
Intracranial GL261-Luciferase Tumors.
[0265] Six to eight week old C57BL/6J female mice were
subcutaneously implanted with 1.times.10.sup.6 B16F10 mouse
melanoma cancer cells on the flank. Six to eight week old BALB/c
female mice were subcutaneously implanted with 1.times.10.sup.6
CT26 colon tumor cells on the flank. Male C57BL/6J weighting 20 g
were injected with 3.times.10.sup.5 GL261-luc cells intracranially
into the right caudate nucleus (+0.5 mm A/P, +2.3 mm M/L and -3.2
mm D/V). Intracranial tumors were allowed to grow for 21 days
before sacrifice. Subcutaneous tumors were measured 3 times a week
using calipers and tumor size was calculated based on the formula:
(length x width.sup.2)+0.5. Once the tumors reached an average size
of -300 mm.sup.3 (10-14 days post-implantation of tumor cells),
treatment was initiated.
[0266] Treatment. Mice bearing 200-400 mm.sup.3 subcutaneous tumors
were randomized in each treatment group based on tumor size and
injected intravenously (650 mg/kg), subcutaneously (650 mg/kg), or
intratumorally (2 mg, 100 .mu.l) with vehicle (formulation buffer:
50 mM succinate, 50 mM NaCl, 3.4 mM EDTA, and 10 mM reduced
glutathione at pH 7) or formulation buffer containing PEGylated
H-NOX (L144F). Mice bearing day 21 intracranial tumors were
randomized in each treatment group based on bioluminescent signal
measured with the Xenogen IVIS spectrum and injected intravenously
with formulation buffer alone or containing 750 mg/kg of H-NOX
(L144F).
[0267] Measurement of PEGylated H-NOX (L144F) Accumulation in
Subcutaneous Tumor Tissue.
[0268] Tumors were harvested 6h (B16F10) or 8h (CT26) after H-NOX
or vehicle injection. Tumors were homogenized in an extraction
buffer (Abcam kit # ab117996) supplemented with anti-proteases and
protein concentration was quantified in each tumor using a Bradford
assay. PEGylated H-NOX (L144F) concentration was quantified by a
sandwich ELISA ELISA (detection sensitivity at 1 ng/ml) for H-NOX
and normalized to tumor weight to express H-NOX amount in .mu.g/g
tumor tissue. Quantification of H-NOX levels in tumor lysates was
determined by 5-parameter fit of the standard curve.
[0269] Biodistribution of H-NOX (L144F) in GL261 by IHC.
[0270] 2h after H-NOX injection, rodents were euthanized and tumors
resected for immunohistochemistry (IHC) assay. Tumors were frozen
in OCT and sectioned at 12 .mu.M for IHC processing. Sections were
fixed with 100% methanol for 20 minutes at -20.degree. C., then
blocked and permeabilized with 5% BSA, 5% goat serum, and 0.1%
Tween 20 for 1-2 hours at room temperature. Sections were then
incubated with rabbit anti-H-NOX (1:500, custom-made rabbit
polyclonal produced by AnaSpec Inc, Fremont, Calif.) and rat
anti-CD31 (BD Bioscience, 1:50) antibodies overnight at 4.degree.
C., followed by anti-rabbit or anti-rat secondary antibodies
(1:1000, Jackson Immunoresearch Laboratories, West Grove, Pa., USA)
for 2 hours at room temperature. The sections were mounted in
SlowFade DAPI (Invitrogen). Sections were imaged with an HD
Axiolmager Zeiss microscope equipped with a CCD digital camera. In
each animal, the percent of H-NOX-positive area within the tumor
tissue was determined using ImageJ (1-2 pictures per section in 5
tumor sections spanning 1 mm of the tumor thickness).
Example 7. Measurement of Hypoxia and T Cells in Canine Oral
Melanoma Tumors
[0271] Canine Oral Melanoma.
[0272] Pet dogs with oral melanoma tumors were recruited for the
study with owners' consent and injected intravenously (slow
infusion) with PEGylated H-NOX (L144F) 4h prior to surgery. Tissue
extracted from surgery was analyzed by IHC.
[0273] Immunohistochemistry.
[0274] 4 hours after H-NOX injection, tumors were resected, frozen
in OCT and sectioned at 12 .mu.M for IHC processing. Sections were
fixed with 4% PFA for 15 minutes at 4.degree. C., then blocked and
permeabilized with 5% BSA, 5% goat serum, and 0.1% Tween 20 for 1-2
hours at room temperature. Sections were then incubated with rabbit
anti-carbonic anhydrase IX (CAIX, Novus Biological 1:1000) or
rabbit anti-H-NOX (1:500, custom-made rabbit polyclonal produced by
AnaSpec Inc, Fremont, Calif.) and rat anti-CD4 (Abd Serotech, 1:50)
or rat anti-CD8 (Abd Serotech, 1:50) antibodies overnight at
4.degree. C., followed by anti-rabbit or anti-rat secondary
antibodies (1:1000, Jackson Immunoresearch Laboratories, West
Grove, Pa., USA) for 2 hours at room temperature. The sections were
mounted in SlowFade DAPI (Invitrogen). Sections were imaged with an
HD Axiolmager Zeiss microscope equipped with a CCD digital camera
(FIG. 11). Images revealed presence of high lymphocyte numbers in
tumor regions that expressed hypoxia marker CAIX indicative of
hypoxic state prior to OMX administration suggesting that OMX
treatment relieved immunosuppressive microenvironment and allowed
lymphocyte infiltration.
Example 8. Correlation of Tumor Volume, Tumor Hypoxia and Reduced T
Cell Infiltration
[0275] 4T1-Luc Tumor Model.
[0276] 8 week-old female BALB/c mice were purchased from Charles
River Labs. Luciferase-expressing 4T1 mouse breast tumor cells
(4T1-Fluc-Neo; Imanis Life Sciences) were grown in RPMI medium
supplemented with 10% fetal bovine serum, 1.times.
penicilin/streptomycin, and 0.7 mg/ml G-418 (InvivoGen). Cells were
trypsinized and resuspended in a 50:50 mixture of medium:Matrigel
(Corning), and 2.times.10.sup.5 cells in 100 .mu.l volume was
injected subcutaneously into mice. At day 10 and day 14
post-implantation, tumors were measured and volumes calculated
(length x width x height x 0.523), mice were injected
simultaneously with 120 mg/kg pimonidazole i.p. (PIMO, Hypoxyprobe)
and 30 mg/kg EF5 i.v. (Hypoxia Imaging Center), sacrificed 90 min
post-PIMO/EF5 injection, and tumors were harvested. Harvested
tumors were frozen in OCT for immunostaining, as well as
dissociated into single cells using a gentleMACS dissociator
followed by incubation with 0.75 mg/ml collagenase/dispase (Roche)
at 37.degree. C. with shaking for 45 min. Dissociated cells were
passed through 70 .mu.m filters.
[0277] Flow Cytometry.
[0278] Unfixed dissociated cells were stained with antibodies for T
cells (hamster anti-mouse CD3-AlexaFluor 488, clone 145-2C11,
eBioscience; rat anti-mouse CD4-APC, clone RM4-5, BD Biosciences;
rat anti-mouse CD8-PE, clone 53-6.7, BD Biosciences), and flow
cytometry was performed using a FACSCalibur. Spleens were used as T
cell positive controls for gating purposes. Also after filtration
of dissociated cells through 70 .mu.m filters, cells were stained
with viability dye 570 (BD Biosciences), fixed with formalin and
methanol, stained with antibodies for hypoxia markers (rabbit
anti-pimonidazole, Hypoxyprobe, followed by donkey anti-rabbit
AlexaFluor 647; mouse anti-EF5 conjugated to AlexaFluor 488,
Hypoxia Imaging Center), and analyzed on a FACSCalibur. Flow
cytometry data were analyzed using FlowJo.
[0279] Immunofluorescence Staining.
[0280] Frozen sections were cut at 10 .mu.m, fixed with 4% PFA,
stained with primary antibodies (rat anti-mouse CD4, rat anti-mouse
CD8, rabbit anti-PIMO), followed by secondary antibodies (donkey
anti-rat AlexaFluor 594, donkey anti-PIMO AlexaFluor 488), and
counterstained with DAPI.
[0281] As shown in FIGS. 13A-13K, larger tumor size correlates with
enhanced hypoxia and reduced lymphocyte infiltration in
subcutaneous 4T1-Luc syngeneic tumors. FIG. 12A shows tumor volumes
on day 10 and day 14 post-implantation. FIG. 12B shows the fraction
of lymphocytes within the viable cell population and FIG. 12C shows
the absolute lymphocyte cell numbers within the viable population.
Negative correlations between tumor volume and percentage
lymphocytes (FIG. 12D) and between percentage hypoxia and
percentage lymphocytes (FIG. 12F) were demonstrated whereas the
relationship between tumor volume and percentage hypoxia showed a
positive correlation (FIG. 12E). Negative correlations were also
seen between tumor volume and percentage CD3-positive T cells (FIG.
12G), between tumor volume and percentage CD4-positive T cells
(FIG. 12H), between tumor volume and percentage CD8-positive T
cells (FIG. 12I), between tumor volume and percentage
CD3-CD4-double-positive T cells (FIG. 12J), and between tumor
volume and percentage CD3-CD8-double-positive T cells (FIG.
12K).
[0282] FIGS. 13A-13F show that hypoxic tumor regions are
immunosuppressive and exhibit reduced T cell infiltration in
subcutaneous 4T1-Luc syngeneic mouse tumors.
TABLE-US-00003 SEQUENCES Tt WT
ATGAAGGGGACAATCGTCGGGACATGGATAAAGACCCTGAGGGACCTTTACGGGAATGATGTGGTTGATGAATC-
TTT
AAAAAGTGTGGGTTGGGAACCAGATAGGGTAATTACACCTCTGGAGGATATTGATGACGATGAGGTTAGGAGAA-
TTT
TTGCTAAGGTGAGTGAAAAAACTGGTAAAAATGTCAACGAAATATGGAGAGAGGTAGGAAGGCAGAACATAAAA-
ACT
TTCAGCGAATGGTTTCCCTCCTATTTTGCAGGGAGAAGGCTAGTGAATTTTTTAATGATGATGGATGAGGTACA-
CCT
ACAGCTTACCAAGATGATAAAAGGAGCCACTCCTCCAAGGCTTATTGCAAAGCCTGTTGCAAAAGATGCCATTG-
AAA
TGGAGTACGTTTCTAAAAGAAAGATGTACGATTACTTTTTAGGGCTTATAGAGGGTAGTTCTAAATTTTTCAAG-
GAA
GAAATTTCAGTGGAAGAGGTCGAAAGAGGCGAAAAAGATGGCTTTTCAAGGCTAAAAGTCAGGATAAAATTTAA-
AAA CCCCGTITTTGAGTGA (SEQ ID NO: 1)
MKGTIVGTWIKTLRDLYGNDVVDESLKSVGWEPDRVITPLEDIDDDEVRRIFAKVSEKTGKNVNEIWREVGRQN-
IKT
FSEWFPSYFAGRRLVNFLMMMDEVHLQLTKMIKGATPPRLIAKPVAKDAIEMEYVSKRKMYDYFLGLIEGSSKF-
FKE EISVEEVERGEKDGFSRLKVRIKFKNPVFE (SEQ ID NO: 2) foldon domain
GGTTATATTCCTGAAGCTCCAAGAGATGGGCAAGCTTACGTTCGTAAAGATGGCGAATGGGTATTACTTTCTAC
CTTTTTA (SEQ ID NO: 3) GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 4)
L2 WT
ATGATGTCTATGAAAGGAATCATATTCAACGAATTTCTCAATTTTGTAGAAAAAAGTGAATCCTACACCCTGGT
AGATCAAATTATTATGGATAGTCATTTGAAGTCCCATGGTGCCTACACGTCTATCGGTACATACTCTCCCAAAG
AATTATTTCAATTGGTTAAAGCGCTTGCTATGAAAAATGGCAAACCAACATCAGTGATTTTACAAGAATATGGT
GAGTATTTGTTTGAGGTTTTTGCAAAAAAATATCCTCAATTTTTCAGGGAAAAAAAGTCGGTGTTTCAATTTTT
GGAAGCGCTTGAAACACATATTCATTTCGAAGTGAAAAAATTGTATGACTATACTGAACTACCCCATTTTGAAT
GCCAATATCACAGTCAAAATCAAATGGAAATGATTTACACTTCTTCGCGTCCTTTGGCCGATTTTGCGGAAGGT
TTAATAAAAGGTTGTATTAAATATCATAAAGAAAACATGACTATTGTTCGTGAAAATCTGCCTGCAAAAACAGG
CTTTAAGGTAAGATTTGTATTAACAAAAGGCGATCCTGATGAGTGA (SEQ ID NO: 9)
MMSMKGIIFNEFLNFVEKSESYTLVDQIIMDSHLKSHGAYTSIGTYSPKELFQLVKALAMKNGKPTSVILQEYG
EYLFEVFAKKYPQFFREKKSVFQFLEALETHIHFEVKKLYDYTELPHFECQYHSQNQMEMIYTSSRPLADFAEG
LIKGCIKYHKENMTIVRENLPAKTGFKVRFVLTKGDPDE (SEQ ID NO: 10) L1 WT
ATGAAAGGTATCGTTTTTACCTCCTTAAATGACATGATTATAGAACAATTTGGCATAGAAACCTGGGACCAACT
CGTATCCTCACTAGACCTTCCAAGTGGTGGAAGTTATACAGCAGGCGGCACTTACTOGGATACAGAATTTCAGC
AATTGATTAAGGCCATTGCGAAGAGGACCAATCAGCACGCTTCTGTTTTTTTAGAGGCCTTTGGTGAATACATG
TTTCCTATCTTATCGAGTAAGTGCGCAATTTTTTTAAAAAAGGACATGACATTAAAAGAATTTTTAAAAAGCAT
TGATGGAACAATTCATGTGGAAGTAGAAAAGTTATACCCAGATGAAACATTACCTACCATTAGCTATGAAGAGC
CTGCTGCAAACCAATTGGTTATGGTGTATCGATCGCATAGAAGACTCTGTCATTTTGCAATGGGGCTCATCCAG
GGAGCAGCGCAACATTTTAAAAAGAAAATTACCATTAAGCAGACTCACTGCATGTTAAAAAAAGATGATCATTG
TCGTTTGGAGATTACCTTTGAGTGA (SEQ ID NO: 11)
MKGIVFTSLNDMIIEQFGIETWDQLVSSLDLPSGGSYTAGGTYSDTEFQQLIKAIAKRTNQHASVFLEAFGEYM
FPILSSKCAIFLKKDMTLKEFLKSIDGTIHVEVEKLYPDETLPTISYEEPAANQLVMVYRSHRRLCHFAMGLIQ
GAAQHFKKKITIKQTHCMLKKDDHCRLEITFE (SEQ ID NO: 12) Homo sapiens WT
(1-385)
ATGTACGGATTTGTGAATCACGCCCTGGAGTTGCTGGTGATCCGCAATTACGGCCCCGAGGTGTGGGAAGACAT
CAAAAAAGAGGCACAGTTAGATGAAGAAGGACAGTTTCTTGTCAGAATAATATATGATGACTCCAAAACTTATG
ATTTGGTTGCTGCTGCAAGCAAAGTCCTCAATCTCAATGCTGGAGAAATCCTCCAAATGTTTGGGAAGATGTTT
TTCGTCTTTTGCCAAGAATCTGGTTATGATACAATCTTGCGTGTCCTGGGCTCTAATGTCAGAGAATTTCTACA
GAACCTTGATGCTCTGCACGACCACCTTGCTACCATCTACCCAGGAATGCGTGCACCTTCCTTTAGGTGCACTG
ATGCAGAAAAGGGCAAAGGACTCATTTTGCACTACTACTCAGAGAGAGAAGGACTTCAGGATATTGTCATTGGA
ATCATCAAAACAGTGGCACAACAAATCCATGGCACTGAAATAGACATGAAGGTTATTCAGCAAAGAAATGAAGA
ATGTGATCATACTCAATTTTTAATTGAAGAAAAAGAGTCAAAAGAAGAGGATTTTTATGAAGATCTTGACAGAT
TTGAAGAAAATGGTACCCAGGAATCACGCATCAGCCCATATACATTCTGCAAAGCTTTTCCTTTTCATATAATA
TTTGACCGGGACCTAGTGGTCACTCAGTGTGGCAATGCTATATACAGAGTTCTCCCCCAGCTCCAGCCTGGGAA
TTGCAGCCTTCTGTCTGTCTTCTCGCTGGTTCGTCCTCATATTGATATTAGTTTCCATGGGATCCTTTCTCACA
TCAATACTGTTTTTGTATTGAGAAGCAAGGAAGGATTGTTGGATGTGGAGAAATTAGAATGTGAGGATGAACTG
ACTGGGACTGAGATCAGCTGCTTACGTCTCAAGGGTCAAATGATCTACTTACCTGAAGCAGATAGCATACTTTT
TCTATGTTCACCAAGTGTCATGAACCTGGACGATTTGACAAGGAGAGGGCTGTATCTAAGTGACATCCCTCTGC
ATGATGCCACGCGCGATCTTGTTCTTTTGGGAGAACAATTTAGAGAGGAATACAAACTCACCCAAGAACTGGAA
ATCCTCACTGACAGGCTACAGCTCACGTTAAGAGCCCTGGAAGATTGA (SEQ ID NO: 13)
MYGFVNHALELLVIRNYGPEVWEDIKKEAQLDEEGQFLVRIIYDDSKTYDLVAAASKVLNLNAGEILQMFGKMF
FVFCQESGYDTILRVLGSNVREFLQNLDALHDHLATIYPGMRA-SFRCIDAEKGKGLILHYYSEREGLQDIVIG
IIKTVAQQIHGTEIDMKVIQQRNEECDHTQFLIEEKESKEEDFYEDLDRFEENGTOESRISPYTFCKAFPFHII
FDRDLVVTQCGNAIYRVLPQLQPGNCSLLSVFSLVRPHIDISFHGILSHINTVFVLRSKEGLLDVEKLECEDEL
TGTEISCLRLKGQMIYLPEADSILFLCSPSVMNLDDLTRAGLYLSDIPLHDATADLVLLGEQFREEYKLIQELE
ILTDRLOLTLRALED (SEQ ID NO: 14) Homo sapiens .beta.2 (1-217)
ATGTATGGATTCATCAACACCTGCCTGCAGTCTCTTGTGACAGAGAAATTTGGTGAGGAGACATGGGAGAASCT
GAAGGCTCOTGCAGAAGTGCAAGATGTCTTCATGACCTACACCGTGTATGATGACATCATCACCATTAAGCTCA
TCCAAGAAGCCTGCAAGGTTCTGGATGTGTCCATGGAAGCCATTCTGAAGCTCTTTGGCGAATACTTCTTTAAG
TTCTGTAAGATGTCTGGCTATGACAGGATGCTGCGGACACTTGGAGGAAATCTCACCGAGTTTATTGAAAACCT
AGATGCACTCCACAGTTACCTGGCACTGTCCTATCAGGAAATGAACGCACCATCCTTTCGAGTGGAGGAAGGAG
CTGACGGGGCGATGCTTCTCCACTACTACTCAGACAGACATGGTCTGTGTCACATTGTACCAGGTATCATTGAA
GCTGTGGCCAAGGACTTCTTTGACACTGATGTGGCCATGAGTATCCTGGATATGAACGAAGAGGTGGAAAGGAC
AGGGAAGAAAGAACATGTTGTGTTTCTGGTCGTGCAGAAGGCTCACAGACAGATAAGAGGAGCAAAGGCAAGCC
GGCCACAAGGCAGTGAGGACAGCCAGGCAGACCAGGAGGCTCTCCAGGGAACACTCCTT (SEQ ID
NO: 15)
MYGFINTCLQSLVTEKFGEETWEKLKAPAEVQDVFMTYTVYDDIITIKLIQEACKVLDVSMEAILKLFGEYFFK
FCKMSGYDRMLATLGGNLIEFIENLDALHSYLALSYQEMNAPSYAVEEGADGAMLLHYYSDRHGLCHIVPGIIE
AVAKDFFDTDVAMSILDMNEEVERTGKKEHVVFLVVQKAHRQIRGAKASRPQGSEDSQADQEALQGILL
(SEQ ID NO: 16) Rattus norvezieus .beta.1 (1-385)
ATGTACGGTTTTGTGAACCATGCCCTGGAGCTGCTGGTGATCCGCAATTACGGTCCCGAGGTGTGGGAAGACAT
CAAAAAAGAGGCGCAGCTGGATGAAGAAGGCCAGTTTCTTGTGAGAATAATCTACGATGATTCCAAAACCTATG
ACTTGGTGGCTGCTGCGAGCAAAGTCCTCAACCTCAATGCTGGTGAAATCCTGCAGATGTTTGGGAAGATGTTT
TTCGTCTTCTGTCAAGAGTCTGGCTATGATACCATCTTGCGTGTCCTGGGATCTAATGTCAGGGAGTTTTTGCA
GAACCTCGACGCCCTGCACGACCACCTCGCCACCATCTACCCAGGGATGCGCGCACCTTCCTTCCGGTGCACCG
ATGCAGAAAAAGGCAAAGGGCTCATTCTGCACTACTACTCGGAAAGAGAGGGGCTTCAGGACATTGTGATCGGG
ATTATCAAGACTGTAGCTCAACAGATCCATGGCACTGAGATAGACATGAAGGTTATTCAGCAAAGAAGTGAAGA
ATGTGATCATACCCAATTTTTAATTGAAGAAAAAGAATCAAAAGAAGAGGATTTTTATGAAGATCTGGACAGGT
TTGAAGAGAACGGTACCCAGGACTCCCGTATCAGCCCGTACACCTTCTGCAAAGCGTTTCCTTTTCACATCATA
TTTGACCGGGACCTAGTAGTCACGCAGTGTGGAAATGCTATCTACAGAGTGCTCCCCCAGCTCCAGCCTGGGAA
GTGCAGCCTTCTGTCTGTCTTCTCTCTGGTCCGCCCTCATATTGACATCAGTTTCCACGGGATTCTTTCACACA
TCAATACCGTCTTTGTACTGAGAAGCAAGGAAGGGTTGCTGGATGTTGAGAAACTTGAATGTGAGGATGAACTG
ACTGGGGCAGAGATTAGCTGCCTCCGTCTCAAAGGCCAAATGATCTATTTACCGGAAGGAGATAGCATCCTCTT
CCTCTGTTCACCAAGTGTGATGAACTTGGATGACCTAACAAGAAGAGGCCTGTACCTGAGTGACATCCCTCTCC
ATGATGCTACACGAGACCTGGTCCTTTTGGGAGAACAGTTCCGGGAGGAGTACAAACTGACACAAGAGCTGGAA
ATCCICACAGACAGGCTGCAGCTCACACTGAGGGCTTTGGAGGATTGA (SEQ ID NO: 17)
MYGFVNHALELLVIRNYGPEVWEDIKKEAQLDEEGQFLVRIIYDDSKTYDLVAAASKVLNLNAGEILQMFGKMF
FVFCQESGYDTILRVLGSNVAEFLQNLDALHDHLATIYPGMRAPSFRCTDAEKGKGLILHYYSEREGLQDIVIG
IIKTVAQQIHGTEIDMKVIQQRSEECDHTQFLIEEKESKEEDFYEDLDRFEENGTQCSRISPYTFCKAFPFHII
FDRDLVVTQCGNAIYRVLPQLQPGKCSLLSVFSLVRPHIDISFHGILSHINTVFVLRSKEGLLDVEKLECEDEL
TGAEISCLRLKGQMIYLPEADSILFLCSPSVMNLDDLTRAGLYLSDIPLHDATRDLVLLGEOFREEYKLTQELE
ILTDRLQLTLRALED (SEQ ID NO: 18) Rattus norvegicus .beta.1 (1-385)
ATGTACGGTTTTGTGAACCATGCCCTGGAGCTGCTGGTGATCCGCAATTACGGTCCCGAGGTGTGGGAAGACAT
CAAAAAAGAGGCGCAGCTGGATGAAGAAGGCCAGTTTCTTGTGAGAATAATCTACGATGATTCCAAAACCTATG
ACTTGGTGGCTGCTGCGAGCAAAGTCCTCAACCTCAATGCTGGTGAAATCCTGCAGATGTTTGGGAAGATGTTT
TTCGTCTTCTGTCAAGAGTCTGGCTATGATACCATCTTGCGTGTCCTGGGATCTAATGTCAGGGAGTTTTTGCA
GAACCTCGACGCCCTGCACGACCACCTCGCCACCATCTACCCAGGGATGCGCGCACCTTCCTTCCGGTGCACCG
ATGCAGAAAAAGGCAAAGGGCTCATTCTGCACTACTACTCGGAAAGAGAGGGGCTTCAGGACATTGTGATCGGG
ATTATCAAGACTGTAGCTCAACAGATCCATGGCACTGAGATAGACATGAAGGTTATTCAGCAAAGAAGTGAAGA
ATGTGATCATACCCAATTTTTAATTGAAGAAAAAGAATCAAAAGAAGAGGATTTTTATGAAGATCTGGACAGGT
TTGAAGAGAACGGTACCCAGGACTCCCGTATCAGCCCGTACACCTTCTGCAAAGCGTTTCCTTTTCACATCATA
TTTGACCGGGACCTAGTAGTCACGCAGTGTGGAAATGCTATCTACAGAGTGCTCCCCCAGCTCCAGCCTGGGAA
GTGCAGCCTTCTGTCTGTCTTCTCTCTGGTCCGCCCTCATATTGACATCAGTTTCCACGGGATTCTTTCACACA
TCAATACCGTCTTTGTACTGAGAAGCAAGGAAGGGTTGCTGGATGTTGAGAAACTTGAATGTGAGGATGAACTG
ACTGGGGCAGAGATTAGCTGCCTCCGTCTCAAAGGCCAAATGATCTATTTACCGGAAGCAGATAGCATCCTCTT
CCTCTGTTCACCAAGTGTGATGAACTTGGATGACCTAACAAGAAGAGGCCTGTACCTGAGTGACATCCCTCTCC
ATGATGCTACACGAGACCTGGTCCTTTTGGGAGAACAGTTCCGGGAGGAGTACAAACTGACACAAGAGCTGGAA
ATCCTCACAGACAGGCTGCAGCTCACACTGAGGGCTTTGGAGGATTGA (SEQ ID NO: 19)
MYGEVNHALELLVIRNYGPEVWEDIKKEAQLDEEGQFLVRIIYDDSKTYDLVAAASKVLNLNAGEILQMFGKMF
FVFCQESGYDTILRVLGSNVREFLONLDALHDHLATIYPGMRAPSFRCTDAEKGKGLILHYYSEREGLQDIVIG
IIKTVAQQIHGTEIDMKVIQQRSEECDHTQFLIEEKESKEEDFYEDLDRFEENGTQDSRISPYTFCKAFPFHII
FDRDLVVTQCGNAIYRVLPQLQPGKCSLLSVFSLVRPHIDISFHGILSHINTVFVLRSKEGLLDVEKLECEDEL
TGAEISCLRLKGQMIYLPEADSILFLCSPSVMNLDDLTRRGLYLSDIPLHDATRDLVLLGEQFREEYKLTQELE
ILTDRLQLTLRALED (SEQ ID NO: 20) Rattus norvegicus .beta.2
ATGTATGGATTCATCAACACCTGCCTGCAGTCTCTTGTGACAGAGAAATTTGGTGAGGAGACATGGGAGAAGCT
GAAGGCTCCTGCAGAAGTGCAAGATGTCTTCATGACCTACACCGTGTATGATGACATCATCACCATTAAGCTCA
TCCAAGAAGCCTGCAAGGTTCTGGATGTGTCCATGGAAGCCATTCTGAAGCTCTTTGGCGAATACTTCTTTAAG
TTCTGTAAGATGTCTGGCTATGACAGGATGCTGCGGACACTTGGAGGAAATCTCACCGAGTTTATTGAAAACCT
AGATGCACTCCACAGTTACCTGGCACTGTCCTATCAGGAAATGAACGCACCATCCTTTCGAGTGGAGGAAGGAG
CTGACGGGGCGATCCTTCTCCACTACTACTCAGACAGACATGGTCTGTGTCACATTCTACCAGGTATCATTGAA
GCTGTGGCCAAGGACTTCTTTGACACTGATGTGGCCATGAGTATCCTGGATATGAACGAAGAGGTGGAAAGGAC
AGGGAAGAAAGAACATGTTGTGTTTCTGGTCGTGCAGAAGGCTCACAGACAGATAAGAGGAGCAAAGGCAAGCC
GGCCACAAGGCAGTGAGGACAGCCAGGCAGACCAGGAGGCTCTCCAGGGAACACTCCTTCGGATGAAGGAGAGA
TATTTAAACATCCCTGTTTGCCCTGGGGAGAAATCTCACTCAACTGCTGTGAGGGCATCGGTCCTTTTTGGAAA
AGGGCCCCTCAGGGACACCTTCCAGCCCGTCTATCCTGAGAGACTATGGGTCGAAGAGGAGGTGTTCTGTGATG
CTTTTCCTTTCCACATTGTCTTTGATGAAGCACTAAGGGTCAAGCAAGCTGGAGTGAATATTCAGAAGTATGTC
CCTGGAATCTTAACCCAGAAGTTTGCACTAGATGAGTATTTTTCCATCATCCACCCTCAAGTTACTTTCAACAT
CTCCAGCATCTGCAAGTTCATTAACAGTCAGTTTGTCTTGAAGACAAGAAAAGAAATGATGCCCAAAGCAAGGA
AGAGCCAGCCGATGCTCAAACTCCGGGGTCAGATGATCTGGATGGAGTCTCTGAGGTGCATGATCTTCATGTGT
TCCCCAAACGTCCGCAGCCTGCAAGAGCTGGAAGAGAGCAAGATGCATCTTTCTGATATCGCTCCGCACGACAC
GACCAGGGATCTCATCCTCCTCAACCAGCAGAGGCTGGCAGAGATGGAGCTGTCCTGCCAACTGGAAAAGAAGA
AGGAGGAGTTGCGTGTCCTTTCCAATCACCTGGCCATCGAGAAGAAGAAGACAGAGACCTTGCTGTATGCCATG
CTGCCTGAACATGTGGCCAACCAACTCAAGGAGGGCAGAAAGGTGGCTGCAGGAGAATTTGAAACATGTACAAT
CCTTTTCAGCGATGTTGTGACATTTACCAACATCTGTGCAGCCTGTGAACCTATCCAAATCGTGAACATGCTGA
ATTCAATGTACTCCAAGTTTGACAGGTTAACCAGTGTCCATGATGTCTACAAAGTAGAAACAATAGGGGATGCT
TACATGGTGGTGGGTGGAGTACCAGTACCCGTTGAAAGCCATGCTCAAAGAGTCGCCAATTTTGCTCTGGGGAT
GAGAATTTCTGCAAAAGAAGTGATGAATCCTGTCACTGGGGAACCTATCCAGATCAGAGTGGGAATCCACACTG
GACCAGTCTTAGCAGGTGTTGTGGGAGACAAGATGCCTCGGTACTGCTTGTTTGGTGACACTGTAAACACAGCC
TCTAGGATGGAAAGTCACGGGCTTCCCAGCAAAGTGCATCTGAGCCCCACAGCCCACAGAGCCCTGAAAAACAA
AGGGTTTGAAATTGTCAGGAGAGGCGAGATCGAAGTGAAGGGGAAAGGAAAGATGACCACATACTTTCTGATCC
AGAACCTGAATGCCACCGAGGATGAGATAATGGGGCGACCTTCAGCCCCCGCTGATGGGAAGGAAGTATGTACT
CCCGGAAACCAAGTCAGGAAGTCCCCTGCTGTCCCGAGGAACACAGACCATCAGCAACAAGTCTACAAAGGAGA
CCCAGCAGACGCTTCTAATGAAGTCACACTTGCTGGGAGCCCAGTGGCAGGGCGAAACTCCACAGATGCAGTCA
ATAACCAGCCATCACCAGATGAGACCAAGACAAGTGTCGTTGCTAGTGGCCCTGTGCTGTCTGCTTTCTGTGTT
GTGCTGTGA (SEQ ID NO: 21)
MYGFINTCLQSLVTEKFGEETWEKLKAPAEVQDVFMTYTVYDDIITIKLIQEACKVLDVSMEAILKLFGEYFFK
FCKMSGYDRMLRTLGGNLTEFIENLDALHSYLALSYQEMNAPSFRVEEGADGAMLLHYYSDRHGLCHIVPGIIE
AVAKDFFDTDVAMSILDMNEEVERTGKKEHVVFLVVQKAHRQIRGAKASRPQGSEDSQADQEALQGTLLRMKER
YLNIPVCPGEKSHSTAVRASVLFGKGPLRDTFQPVYPERLWVEEEVFCDAFPFHIVFDEALRVKQAGVNIQKYV
PGILTQKFALDEYFSIIHPQVTFNISSICKFINSQFVLKTRKEMMPKARKSQPMLKLRGQMIWMESLRCMIFMC
LFEHVANOLKEGRKVAAGEFETCTILFSDVVIFTNICAACEPDOIVNMLNSMYSKFCRLTSVHDVYKVETIGDA
YMVVGGVPVPVESHAQRVANFALGMRISAKEVMNPVTGEPIQRDVGIHTGPVLAGVVGDKMPRYCLFGDTVNTA
SRMESHGLPSKVHLSPTAHRALKNKGFEIVARGEIEVKGKGKMTTYFLIQNLNATECEIMGRPSAPADGKEVCT
PGNOVRKSPAVPRNTDEQQQVYKGDPADASNEVTLAGSPVAGRNSTDAVNNQPSPDETKTSVVASGPVLSAFCV
VL (SEQ ID NO: 22)
Sequence CWU 1
1
301555DNAThermoanaerobacter tengcongensis 1atgaagggga caatcgtcgg
gacatggata aagaccctga gggaccttta cgggaatgat 60gtggttgatg aatctttaaa
aagtgtgggt tgggaaccag atagggtaat tacacctctg 120gaggatattg
atgacgatga ggttaggaga atttttgcta aggtgagtga aaaaactggt
180aaaaatgtca acgaaatatg gagagaggta ggaaggcaga acataaaaac
tttcagcgaa 240tggtttccct cctattttgc agggagaagg ctagtgaatt
ttttaatgat gatggatgag 300gtacacctac agcttaccaa gatgataaaa
ggagccactc ctccaaggct tattgcaaag 360cctgttgcaa aagatgccat
tgaaatggag tacgtttcta aaagaaagat gtacgattac 420tttttagggc
ttatagaggg tagttctaaa tttttcaagg aagaaatttc agtggaagag
480gtcgaaagag gcgaaaaaga tggcttttca aggctaaaag tcaggataaa
atttaaaaac 540cccgtttttg agtga 5552184PRTThermoanaerobacter
tengcongensis 2Met Lys Gly Thr Ile Val Gly Thr Trp Ile Lys Thr Leu
Arg Asp Leu1 5 10 15 Tyr Gly Asn Asp Val Val Asp Glu Ser Leu Lys
Ser Val Gly Trp Glu 20 25 30 Pro Asp Arg Val Ile Thr Pro Leu Glu
Asp Ile Asp Asp Asp Glu Val 35 40 45 Arg Arg Ile Phe Ala Lys Val
Ser Glu Lys Thr Gly Lys Asn Val Asn 50 55 60 Glu Ile Trp Arg Glu
Val Gly Arg Gln Asn Ile Lys Thr Phe Ser Glu65 70 75 80 Trp Phe Pro
Ser Tyr Phe Ala Gly Arg Arg Leu Val Asn Phe Leu Met 85 90 95 Met
Met Asp Glu Val His Leu Gln Leu Thr Lys Met Ile Lys Gly Ala 100 105
110 Thr Pro Pro Arg Leu Ile Ala Lys Pro Val Ala Lys Asp Ala Ile Glu
115 120 125 Met Glu Tyr Val Ser Lys Arg Lys Met Tyr Asp Tyr Phe Leu
Gly Leu 130 135 140 Ile Glu Gly Ser Ser Lys Phe Phe Lys Glu Glu Ile
Ser Val Glu Glu145 150 155 160 Val Glu Arg Gly Glu Lys Asp Gly Phe
Ser Arg Leu Lys Val Arg Ile 165 170 175 Lys Phe Lys Asn Pro Val Phe
Glu 180 381DNAThermoanaerobacter tengcongensis 3ggttatattc
ctgaagctcc aagagatggg caagcttacg ttcgtaaaga tggcgaatgg 60gtattacttt
ctaccttttt a 81427PRTThermoanaerobacter tengcongensis 4Gly Tyr Ile
Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys1 5 10 15 Asp
Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 20 25
5690DNAThermoanaerobacter tengcongensis 5atgaagggga caatcgtcgg
gacatggata aagaccctga gggaccttta cgggaatgat 60gtggttgatg aatctttaaa
aagtgtgggt tgggaaccag atagggtaat tacacctctg 120gaggatattg
atgacgatga ggttaggaga atttttgcta aggtgagtga aaaaactggt
180aaaaatgtca acgaaatatg gagagaggta ggaaggcaga acataaaaac
tttcagcgaa 240tggtttccct cctattttgc agggagaagg ctagtgaatt
ttttaatgat gatggatgag 300gtacacctac agcttaccaa gatgataaaa
ggagccactc ctccaaggct tattgcaaag 360cctgttgcaa aagatgccat
tgaaatggag tacgtttcta aaagaaagat gtacgattac 420tttttagggt
ttatagaggg tagttctaaa tttttcaagg aagaaatttc agtggaagag
480gtcgaaagag gcgaaaaaga tggcttttca aggctaaaag tcaggataaa
atttaaaaac 540cccgtttttg agtataagaa aaatctcgag ggcagcggcg
gttatattcc tgaagctcca 600agagatgggc aggcttacgt tcgtaaagat
ggcgaatggg tattactttc taccttttta 660aggggtagtc accatcacca
tcaccattga 6906225PRTThermoanaerobacter tengcongensis 6Met Lys Gly
Thr Ile Val Gly Thr Trp Ile Lys Thr Leu Arg Asp Leu1 5 10 15 Tyr
Gly Asn Asp Val Val Asp Glu Ser Leu Lys Ser Val Gly Trp Glu 20 25
30 Pro Asp Arg Val Ile Thr Pro Leu Glu Asp Ile Asp Asp Asp Glu Val
35 40 45 Arg Arg Ile Phe Ala Lys Val Ser Glu Lys Thr Gly Lys Asn
Val Asn 50 55 60 Glu Ile Trp Arg Glu Val Gly Arg Gln Asn Ile Lys
Thr Phe Ser Glu65 70 75 80 Trp Phe Pro Ser Tyr Phe Ala Gly Arg Arg
Leu Val Asn Phe Leu Met 85 90 95 Met Met Asp Glu Val His Leu Gln
Leu Thr Lys Met Ile Lys Gly Ala 100 105 110 Thr Pro Pro Arg Leu Ile
Ala Lys Pro Val Ala Lys Asp Ala Ile Glu 115 120 125 Met Glu Tyr Val
Ser Lys Arg Lys Met Tyr Asp Tyr Phe Leu Gly Phe 130 135 140 Ile Glu
Gly Lys Phe Phe Lys Glu Glu Ile Glu Glu Val Glu Arg Gly145 150 155
160 Glu Lys Asp Gly Phe Ser Arg Leu Lys Val Arg Ile Lys Phe Lys Asn
165 170 175 Pro Val Phe Glu Tyr Lys Lys Asn Leu Glu Gly Ser Gly Gly
Tyr Ile 180 185 190 Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg
Lys Asp Gly Glu 195 200 205 Trp Val Leu Leu Ser Thr Phe Leu Arg Gly
Ser His His His His His 210 215 220 His225
7663DNAThermoanaerobacter tengcongensis 7atgaagggga caatcgtcgg
gacatggata aagaccctga gggaccttta cgggaatgat 60gtggttgatg aatctttaaa
aagtgtgggt tgggaaccag atagggtaat tacacctctg 120gaggatattg
atgacgatga ggttaggaga atttttgcta aggtgagtga aaaaactggt
180aaaaatgtca acgaaatatg gagagaggta ggaaggcaga acataaaaac
tttcagcgaa 240tggtttccct cctattttgc agggagaagg ctagtgaatt
ttttaatgat gatggatgag 300gtacacctac agcttaccaa gatgataaaa
ggagccactc ctccaaggct tattgcaaag 360cctgttgcaa aagatgccat
tgaaatggag tacgtttcta aaagaaagat gtacgattac 420tttttagggt
ttatagaggg tagttctaaa tttttcaagg aagaaatttc agtggaagag
480gtcgaaagag gcgaaaaaga tggcttttca aggctaaaag tcaggataaa
atttaaaaac 540cccgtttttg agtataagaa aaatctcgag ggcagcggcg
gttatattcc tgaagctcca 600agagatgggc aggcttacgt tcgtaaagat
ggcgaatggg tattactttc taccttttta 660tga
6638167PRTThermoanaerobacter tengcongensis 8Met Lys Gly Thr Ile Gly
Thr Ile Lys Thr Leu Arg Asp Leu Gly Asn1 5 10 15 Asp Asp Glu Leu
Lys Gly Glu Asp Arg Ile Thr Leu Glu Asp Ile Asp 20 25 30 Asp Asp
Glu Arg Arg Ile Phe Ala Lys Glu Lys Thr Gly Lys Asn Asn 35 40 45
Glu Ile Arg Glu Gly Arg Gln Asn Ile Lys Thr Phe Glu Phe Phe Ala 50
55 60 Gly Arg Arg Leu Asn Phe Leu Met Met Met Asp Glu His Leu Gln
Leu65 70 75 80 Thr Lys Met Ile Lys Gly Ala Thr Arg Leu Ile Ala Lys
Ala Lys Asp 85 90 95 Ala Ile Glu Met Glu Lys Arg Lys Met Asp Phe
Leu Gly Phe Ile Glu 100 105 110 Gly Lys Phe Phe Lys Glu Glu Ile Glu
Glu Glu Arg Gly Glu Lys Asp 115 120 125 Gly Phe Arg Leu Lys Arg Ile
Lys Phe Lys Asn Phe Glu Lys Lys Asn 130 135 140 Leu Glu Gly Gly Gly
Ile Glu Ala Arg Asp Gly Gln Ala Arg Lys Asp145 150 155 160 Gly Glu
Leu Leu Thr Phe Leu 165 9564DNALegionella pneumophila 9atgatgtcta
tgaaaggaat catattcaac gaatttctca attttgtaga aaaaagtgaa 60tcctacaccc
tggtagatca aattattatg gatagtcatt tgaagtccca tggtgcctac
120acgtctatcg gtacatactc tcccaaagaa ttatttcaat tggttaaagc
gcttgctatg 180aaaaatggca aaccaacatc agtgatttta caagaatatg
gtgagtattt gtttgaggtt 240tttgcaaaaa aatatcctca atttttcagg
gaaaaaaagt cggtgtttca atttttggaa 300gcgcttgaaa cacatattca
tttcgaagtg aaaaaattgt atgactatac tgaactaccc 360cattttgaat
gccaatatca cagtcaaaat caaatggaaa tgatttacac ttcttcgcgt
420cctttggccg attttgcgga aggtttaata aaaggttgta ttaaatatca
taaagaaaac 480atgactattg ttcgtgaaaa tctgcctgca aaaacaggct
ttaaggtaag atttgtatta 540acaaaaggcg atcctgatga gtga
56410187PRTLegionella pneumophila 10Met Met Ser Met Lys Gly Ile Ile
Phe Asn Glu Phe Leu Asn Phe Val1 5 10 15 Glu Lys Ser Glu Ser Tyr
Thr Leu Val Asp Gln Ile Ile Met Asp Ser 20 25 30 His Leu Lys Ser
His Gly Ala Tyr Thr Ser Ile Gly Thr Tyr Ser Pro 35 40 45 Lys Glu
Leu Phe Gln Leu Val Lys Ala Leu Ala Met Lys Asn Gly Lys 50 55 60
Pro Thr Ser Val Ile Leu Gln Glu Tyr Gly Glu Tyr Leu Phe Glu Val65
70 75 80 Phe Ala Lys Lys Tyr Pro Gln Phe Phe Arg Glu Lys Lys Ser
Val Phe 85 90 95 Gln Phe Leu Glu Ala Leu Glu Thr His Ile His Phe
Glu Val Lys Lys 100 105 110 Leu Tyr Asp Tyr Thr Glu Leu Pro His Phe
Glu Cys Gln Tyr His Ser 115 120 125 Gln Asn Gln Met Glu Met Ile Tyr
Thr Ser Ser Arg Pro Leu Ala Asp 130 135 140 Phe Ala Glu Gly Leu Ile
Lys Gly Cys Ile Lys Tyr His Lys Glu Asn145 150 155 160 Met Thr Ile
Val Arg Glu Asn Leu Pro Ala Lys Thr Gly Phe Lys Val 165 170 175 Arg
Phe Val Leu Thr Lys Gly Asp Pro Asp Glu 180 185 11543DNALegionella
pneumophila 11atgaaaggta tcgtttttac ctccttaaat gacatgatta
tagaacaatt tggcatagaa 60acctgggacc aactcgtatc ctcactagac cttccaagtg
gtggaagtta tacagcaggc 120ggcacttact cggatacaga atttcagcaa
ttgattaagg ccattgcgaa gaggaccaat 180cagcacgctt ctgttttttt
agaggccttt ggtgaataca tgtttcctat cttatcgagt 240aagtgcgcaa
tttttttaaa aaaggacatg acattaaaag aatttttaaa aagcattgat
300ggaacaattc atgtggaagt agaaaagtta tacccagatg aaacattacc
taccattagc 360tatgaagagc ctgctgcaaa ccaattggtt atggtgtatc
gatcgcatag aagactctgt 420cattttgcaa tggggctcat ccagggagca
gcgcaacatt ttaaaaagaa aattaccatt 480aagcagactc actgcatgtt
aaaaaaagat gatcattgtc gtttggagat tacctttgag 540tga
54312180PRTLegionella pneumophila 12Met Lys Gly Ile Val Phe Thr Ser
Leu Asn Asp Met Ile Ile Glu Gln1 5 10 15 Phe Gly Ile Glu Thr Trp
Asp Gln Leu Val Ser Ser Leu Asp Leu Pro 20 25 30 Ser Gly Gly Ser
Tyr Thr Ala Gly Gly Thr Tyr Ser Asp Thr Glu Phe 35 40 45 Gln Gln
Leu Ile Lys Ala Ile Ala Lys Arg Thr Asn Gln His Ala Ser 50 55 60
Val Phe Leu Glu Ala Phe Gly Glu Tyr Met Phe Pro Ile Leu Ser Ser65
70 75 80 Lys Cys Ala Ile Phe Leu Lys Lys Asp Met Thr Leu Lys Glu
Phe Leu 85 90 95 Lys Ser Ile Asp Gly Thr Ile His Val Glu Val Glu
Lys Leu Tyr Pro 100 105 110 Asp Glu Thr Leu Pro Thr Ile Ser Tyr Glu
Glu Pro Ala Ala Asn Gln 115 120 125 Leu Val Met Val Tyr Arg Ser His
Arg Arg Leu Cys His Phe Ala Met 130 135 140 Gly Leu Ile Gln Gly Ala
Ala Gln His Phe Lys Lys Lys Ile Thr Ile145 150 155 160 Lys Gln Thr
His Cys Met Leu Lys Lys Asp Asp His Cys Arg Leu Glu 165 170 175 Ile
Thr Phe Glu 180 131158DNAHomo sapiens 13atgtacggat ttgtgaatca
cgccctggag ttgctggtga tccgcaatta cggccccgag 60gtgtgggaag acatcaaaaa
agaggcacag ttagatgaag aaggacagtt tcttgtcaga 120ataatatatg
atgactccaa aacttatgat ttggttgctg ctgcaagcaa agtcctcaat
180ctcaatgctg gagaaatcct ccaaatgttt gggaagatgt ttttcgtctt
ttgccaagaa 240tctggttatg atacaatctt gcgtgtcctg ggctctaatg
tcagagaatt tctacagaac 300cttgatgctc tgcacgacca ccttgctacc
atctacccag gaatgcgtgc accttccttt 360aggtgcactg atgcagaaaa
gggcaaagga ctcattttgc actactactc agagagagaa 420ggacttcagg
atattgtcat tggaatcatc aaaacagtgg cacaacaaat ccatggcact
480gaaatagaca tgaaggttat tcagcaaaga aatgaagaat gtgatcatac
tcaattttta 540attgaagaaa aagagtcaaa agaagaggat ttttatgaag
atcttgacag atttgaagaa 600aatggtaccc aggaatcacg catcagccca
tatacattct gcaaagcttt tccttttcat 660ataatatttg accgggacct
agtggtcact cagtgtggca atgctatata cagagttctc 720ccccagctcc
agcctgggaa ttgcagcctt ctgtctgtct tctcgctggt tcgtcctcat
780attgatatta gtttccatgg gatcctttct cacatcaata ctgtttttgt
attgagaagc 840aaggaaggat tgttggatgt ggagaaatta gaatgtgagg
atgaactgac tgggactgag 900atcagctgct tacgtctcaa gggtcaaatg
atctacttac ctgaagcaga tagcatactt 960tttctatgtt caccaagtgt
catgaacctg gacgatttga caaggagagg gctgtatcta 1020agtgacatcc
ctctgcatga tgccacgcgc gatcttgttc ttttgggaga acaatttaga
1080gaggaataca aactcaccca agaactggaa atcctcactg acaggctaca
gctcacgtta 1140agagccctgg aagattga 115814385PRTHomo sapiens 14Met
Tyr Gly Phe Val Asn His Ala Leu Glu Leu Leu Val Ile Arg Asn1 5 10
15 Tyr Gly Pro Glu Val Trp Glu Asp Ile Lys Lys Glu Ala Gln Leu Asp
20 25 30 Glu Glu Gly Gln Phe Leu Val Arg Ile Ile Tyr Asp Asp Ser
Lys Thr 35 40 45 Tyr Asp Leu Val Ala Ala Ala Ser Lys Val Leu Asn
Leu Asn Ala Gly 50 55 60 Glu Ile Leu Gln Met Phe Gly Lys Met Phe
Phe Val Phe Cys Gln Glu65 70 75 80 Ser Gly Tyr Asp Thr Ile Leu Arg
Val Leu Gly Ser Asn Val Arg Glu 85 90 95 Phe Leu Gln Asn Leu Asp
Ala Leu His Asp His Leu Ala Thr Ile Tyr 100 105 110 Pro Gly Met Arg
Ala Pro Ser Phe Arg Cys Thr Asp Ala Glu Lys Gly 115 120 125 Lys Gly
Leu Ile Leu His Tyr Tyr Ser Glu Arg Glu Gly Leu Gln Asp 130 135 140
Ile Val Ile Gly Ile Ile Lys Thr Val Ala Gln Gln Ile His Gly Thr145
150 155 160 Glu Ile Asp Met Lys Val Ile Gln Gln Arg Asn Glu Glu Cys
Asp His 165 170 175 Thr Gln Phe Leu Ile Glu Glu Lys Glu Ser Lys Glu
Glu Asp Phe Tyr 180 185 190 Glu Asp Leu Asp Arg Phe Glu Glu Asn Gly
Thr Gln Glu Ser Arg Ile 195 200 205 Ser Pro Tyr Thr Phe Cys Lys Ala
Phe Pro Phe His Ile Ile Phe Asp 210 215 220 Arg Asp Leu Val Val Thr
Gln Cys Gly Asn Ala Ile Tyr Arg Val Leu225 230 235 240 Pro Gln Leu
Gln Pro Gly Asn Cys Ser Leu Leu Ser Val Phe Ser Leu 245 250 255 Val
Arg Pro His Ile Asp Ile Ser Phe His Gly Ile Leu Ser His Ile 260 265
270 Asn Thr Val Phe Val Leu Arg Ser Lys Glu Gly Leu Leu Asp Val Glu
275 280 285 Lys Leu Glu Cys Glu Asp Glu Leu Thr Gly Thr Glu Ile Ser
Cys Leu 290 295 300 Arg Leu Lys Gly Gln Met Ile Tyr Leu Pro Glu Ala
Asp Ser Ile Leu305 310 315 320 Phe Leu Cys Ser Pro Ser Val Met Asn
Leu Asp Asp Leu Thr Arg Arg 325 330 335 Gly Leu Tyr Leu Ser Asp Ile
Pro Leu His Asp Ala Thr Arg Asp Leu 340 345 350 Val Leu Leu Gly Glu
Gln Phe Arg Glu Glu Tyr Lys Leu Thr Gln Glu 355 360 365 Leu Glu Ile
Leu Thr Asp Arg Leu Gln Leu Thr Leu Arg Ala Leu Glu 370 375 380
Asp385 15651DNAHomo sapiens 15atgtatggat tcatcaacac ctgcctgcag
tctcttgtga cagagaaatt tggtgaggag 60acatgggaga agctgaaggc tcctgcagaa
gtgcaagatg tcttcatgac ctacaccgtg 120tatgatgaca tcatcaccat
taagctcatc caagaagcct gcaaggttct ggatgtgtcc 180atggaagcca
ttctgaagct ctttggcgaa tacttcttta agttctgtaa gatgtctggc
240tatgacagga tgctgcggac acttggagga aatctcaccg agtttattga
aaacctagat 300gcactccaca gttacctggc actgtcctat caggaaatga
acgcaccatc ctttcgagtg 360gaggaaggag ctgacggggc gatgcttctc
cactactact cagacagaca tggtctgtgt 420cacattgtac caggtatcat
tgaagctgtg gccaaggact tctttgacac tgatgtggcc 480atgagtatcc
tggatatgaa cgaagaggtg gaaaggacag ggaagaaaga acatgttgtg
540tttctggtcg tgcagaaggc tcacagacag ataagaggag caaaggcaag
ccggccacaa 600ggcagtgagg acagccaggc agaccaggag gctctccagg
gaacactcct t 65116217PRTHomo sapiens 16Met Tyr Gly Phe Ile Asn Thr
Cys Leu Gln Ser Leu Val Thr Glu Lys1 5 10 15 Phe Gly Glu Glu Thr
Trp Glu Lys Leu Lys Ala Pro Ala Glu Val Gln 20 25 30 Asp Val Phe
Met Thr Tyr Thr Val Tyr Asp Asp Ile Ile Thr Ile Lys 35 40 45 Leu
Ile Gln Glu Ala Cys Lys Val Leu Asp Val Ser Met Glu Ala Ile 50 55
60 Leu Lys Leu Phe Gly Glu Tyr Phe Phe Lys Phe Cys Lys Met Ser
Gly65 70
75 80 Tyr Asp Arg Met Leu Arg Thr Leu Gly Gly Asn Leu Thr Glu Phe
Ile 85 90 95 Glu Asn Leu Asp Ala Leu His Ser Tyr Leu Ala Leu Ser
Tyr Gln Glu 100 105 110 Met Asn Ala Pro Ser Phe Arg Val Glu Glu Gly
Ala Asp Gly Ala Met 115 120 125 Leu Leu His Tyr Tyr Ser Asp Arg His
Gly Leu Cys His Ile Val Pro 130 135 140 Gly Ile Ile Glu Ala Val Ala
Lys Asp Phe Phe Asp Thr Asp Val Ala145 150 155 160 Met Ser Ile Leu
Asp Met Asn Glu Glu Val Glu Arg Thr Gly Lys Lys 165 170 175 Glu His
Val Val Phe Leu Val Val Gln Lys Ala His Arg Gln Ile Arg 180 185 190
Gly Ala Lys Ala Ser Arg Pro Gln Gly Ser Glu Asp Ser Gln Ala Asp 195
200 205 Gln Glu Ala Leu Gln Gly Thr Leu Leu 210 215 171158DNARattus
norvegicus 17atgtacggtt ttgtgaacca tgccctggag ctgctggtga tccgcaatta
cggtcccgag 60gtgtgggaag acatcaaaaa agaggcgcag ctggatgaag aaggccagtt
tcttgtgaga 120ataatctacg atgattccaa aacctatgac ttggtggctg
ctgcgagcaa agtcctcaac 180ctcaatgctg gtgaaatcct gcagatgttt
gggaagatgt ttttcgtctt ctgtcaagag 240tctggctatg ataccatctt
gcgtgtcctg ggatctaatg tcagggagtt tttgcagaac 300ctcgacgccc
tgcacgacca cctcgccacc atctacccag ggatgcgcgc accttccttc
360cggtgcaccg atgcagaaaa aggcaaaggg ctcattctgc actactactc
ggaaagagag 420gggcttcagg acattgtgat cgggattatc aagactgtag
ctcaacagat ccatggcact 480gagatagaca tgaaggttat tcagcaaaga
agtgaagaat gtgatcatac ccaattttta 540attgaagaaa aagaatcaaa
agaagaggat ttttatgaag atctggacag gtttgaagag 600aacggtaccc
aggactcccg tatcagcccg tacaccttct gcaaagcgtt tccttttcac
660atcatatttg accgggacct agtagtcacg cagtgtggaa atgctatcta
cagagtgctc 720ccccagctcc agcctgggaa gtgcagcctt ctgtctgtct
tctctctggt ccgccctcat 780attgacatca gtttccacgg gattctttca
cacatcaata ccgtctttgt actgagaagc 840aaggaagggt tgctggatgt
tgagaaactt gaatgtgagg atgaactgac tggggcagag 900attagctgcc
tccgtctcaa aggccaaatg atctatttac cggaagcaga tagcatcctc
960ttcctctgtt caccaagtgt gatgaacttg gatgacctaa caagaagagg
cctgtacctg 1020agtgacatcc ctctccatga tgctacacga gacctggtcc
ttttgggaga acagttccgg 1080gaggagtaca aactgacaca agagctggaa
atcctcacag acaggctgca gctcacactg 1140agggctttgg aggattga
115818385PRTRattus norvegicus 18Met Tyr Gly Phe Val Asn His Ala Leu
Glu Leu Leu Val Ile Arg Asn1 5 10 15 Tyr Gly Pro Glu Val Trp Glu
Asp Ile Lys Lys Glu Ala Gln Leu Asp 20 25 30 Glu Glu Gly Gln Phe
Leu Val Arg Ile Ile Tyr Asp Asp Ser Lys Thr 35 40 45 Tyr Asp Leu
Val Ala Ala Ala Ser Lys Val Leu Asn Leu Asn Ala Gly 50 55 60 Glu
Ile Leu Gln Met Phe Gly Lys Met Phe Phe Val Phe Cys Gln Glu65 70 75
80 Ser Gly Tyr Asp Thr Ile Leu Arg Val Leu Gly Ser Asn Val Arg Glu
85 90 95 Phe Leu Gln Asn Leu Asp Ala Leu His Asp His Leu Ala Thr
Ile Tyr 100 105 110 Pro Gly Met Arg Ala Pro Ser Phe Arg Cys Thr Asp
Ala Glu Lys Gly 115 120 125 Lys Gly Leu Ile Leu His Tyr Tyr Ser Glu
Arg Glu Gly Leu Gln Asp 130 135 140 Ile Val Ile Gly Ile Ile Lys Thr
Val Ala Gln Gln Ile His Gly Thr145 150 155 160 Glu Ile Asp Met Lys
Val Ile Gln Gln Arg Ser Glu Glu Cys Asp His 165 170 175 Thr Gln Phe
Leu Ile Glu Glu Lys Glu Ser Lys Glu Glu Asp Phe Tyr 180 185 190 Glu
Asp Leu Asp Arg Phe Glu Glu Asn Gly Thr Gln Asp Ser Arg Ile 195 200
205 Ser Pro Tyr Thr Phe Cys Lys Ala Phe Pro Phe His Ile Ile Phe Asp
210 215 220 Arg Asp Leu Val Val Thr Gln Cys Gly Asn Ala Ile Tyr Arg
Val Leu225 230 235 240 Pro Gln Leu Gln Pro Gly Lys Cys Ser Leu Leu
Ser Val Phe Ser Leu 245 250 255 Val Arg Pro His Ile Asp Ile Ser Phe
His Gly Ile Leu Ser His Ile 260 265 270 Asn Thr Val Phe Val Leu Arg
Ser Lys Glu Gly Leu Leu Asp Val Glu 275 280 285 Lys Leu Glu Cys Glu
Asp Glu Leu Thr Gly Ala Glu Ile Ser Cys Leu 290 295 300 Arg Leu Lys
Gly Gln Met Ile Tyr Leu Pro Glu Ala Asp Ser Ile Leu305 310 315 320
Phe Leu Cys Ser Pro Ser Val Met Asn Leu Asp Asp Leu Thr Arg Arg 325
330 335 Gly Leu Tyr Leu Ser Asp Ile Pro Leu His Asp Ala Thr Arg Asp
Leu 340 345 350 Val Leu Leu Gly Glu Gln Phe Arg Glu Glu Tyr Lys Leu
Thr Gln Glu 355 360 365 Leu Glu Ile Leu Thr Asp Arg Leu Gln Leu Thr
Leu Arg Ala Leu Glu 370 375 380 Asp385 191158DNARattus norvegicus
19atgtacggtt ttgtgaacca tgccctggag ctgctggtga tccgcaatta cggtcccgag
60gtgtgggaag acatcaaaaa agaggcgcag ctggatgaag aaggccagtt tcttgtgaga
120ataatctacg atgattccaa aacctatgac ttggtggctg ctgcgagcaa
agtcctcaac 180ctcaatgctg gtgaaatcct gcagatgttt gggaagatgt
ttttcgtctt ctgtcaagag 240tctggctatg ataccatctt gcgtgtcctg
ggatctaatg tcagggagtt tttgcagaac 300ctcgacgccc tgcacgacca
cctcgccacc atctacccag ggatgcgcgc accttccttc 360cggtgcaccg
atgcagaaaa aggcaaaggg ctcattctgc actactactc ggaaagagag
420gggcttcagg acattgtgat cgggattatc aagactgtag ctcaacagat
ccatggcact 480gagatagaca tgaaggttat tcagcaaaga agtgaagaat
gtgatcatac ccaattttta 540attgaagaaa aagaatcaaa agaagaggat
ttttatgaag atctggacag gtttgaagag 600aacggtaccc aggactcccg
tatcagcccg tacaccttct gcaaagcgtt tccttttcac 660atcatatttg
accgggacct agtagtcacg cagtgtggaa atgctatcta cagagtgctc
720ccccagctcc agcctgggaa gtgcagcctt ctgtctgtct tctctctggt
ccgccctcat 780attgacatca gtttccacgg gattctttca cacatcaata
ccgtctttgt actgagaagc 840aaggaagggt tgctggatgt tgagaaactt
gaatgtgagg atgaactgac tggggcagag 900attagctgcc tccgtctcaa
aggccaaatg atctatttac cggaagcaga tagcatcctc 960ttcctctgtt
caccaagtgt gatgaacttg gatgacctaa caagaagagg cctgtacctg
1020agtgacatcc ctctccatga tgctacacga gacctggtcc ttttgggaga
acagttccgg 1080gaggagtaca aactgacaca agagctggaa atcctcacag
acaggctgca gctcacactg 1140agggctttgg aggattga 115820385PRTRattus
norvegicus 20Met Tyr Gly Phe Val Asn His Ala Leu Glu Leu Leu Val
Ile Arg Asn1 5 10 15 Tyr Gly Pro Glu Val Trp Glu Asp Ile Lys Lys
Glu Ala Gln Leu Asp 20 25 30 Glu Glu Gly Gln Phe Leu Val Arg Ile
Ile Tyr Asp Asp Ser Lys Thr 35 40 45 Tyr Asp Leu Val Ala Ala Ala
Ser Lys Val Leu Asn Leu Asn Ala Gly 50 55 60 Glu Ile Leu Gln Met
Phe Gly Lys Met Phe Phe Val Phe Cys Gln Glu65 70 75 80 Ser Gly Tyr
Asp Thr Ile Leu Arg Val Leu Gly Ser Asn Val Arg Glu 85 90 95 Phe
Leu Gln Asn Leu Asp Ala Leu His Asp His Leu Ala Thr Ile Tyr 100 105
110 Pro Gly Met Arg Ala Pro Ser Phe Arg Cys Thr Asp Ala Glu Lys Gly
115 120 125 Lys Gly Leu Ile Leu His Tyr Tyr Ser Glu Arg Glu Gly Leu
Gln Asp 130 135 140 Ile Val Ile Gly Ile Ile Lys Thr Val Ala Gln Gln
Ile His Gly Thr145 150 155 160 Glu Ile Asp Met Lys Val Ile Gln Gln
Arg Ser Glu Glu Cys Asp His 165 170 175 Thr Gln Phe Leu Ile Glu Glu
Lys Glu Ser Lys Glu Glu Asp Phe Tyr 180 185 190 Glu Asp Leu Asp Arg
Phe Glu Glu Asn Gly Thr Gln Asp Ser Arg Ile 195 200 205 Ser Pro Tyr
Thr Phe Cys Lys Ala Phe Pro Phe His Ile Ile Phe Asp 210 215 220 Arg
Asp Leu Val Val Thr Gln Cys Gly Asn Ala Ile Tyr Arg Val Leu225 230
235 240 Pro Gln Leu Gln Pro Gly Lys Cys Ser Leu Leu Ser Val Phe Ser
Leu 245 250 255 Val Arg Pro His Ile Asp Ile Ser Phe His Gly Ile Leu
Ser His Ile 260 265 270 Asn Thr Val Phe Val Leu Arg Ser Lys Glu Gly
Leu Leu Asp Val Glu 275 280 285 Lys Leu Glu Cys Glu Asp Glu Leu Thr
Gly Ala Glu Ile Ser Cys Leu 290 295 300 Arg Leu Lys Gly Gln Met Ile
Tyr Leu Pro Glu Ala Asp Ser Ile Leu305 310 315 320 Phe Leu Cys Ser
Pro Ser Val Met Asn Leu Asp Asp Leu Thr Arg Arg 325 330 335 Gly Leu
Tyr Leu Ser Asp Ile Pro Leu His Asp Ala Thr Arg Asp Leu 340 345 350
Val Leu Leu Gly Glu Gln Phe Arg Glu Glu Tyr Lys Leu Thr Gln Glu 355
360 365 Leu Glu Ile Leu Thr Asp Arg Leu Gln Leu Thr Leu Arg Ala Leu
Glu 370 375 380 Asp385 212229DNARattus norvegicus 21atgtatggat
tcatcaacac ctgcctgcag tctcttgtga cagagaaatt tggtgaggag 60acatgggaga
agctgaaggc tcctgcagaa gtgcaagatg tcttcatgac ctacaccgtg
120tatgatgaca tcatcaccat taagctcatc caagaagcct gcaaggttct
ggatgtgtcc 180atggaagcca ttctgaagct ctttggcgaa tacttcttta
agttctgtaa gatgtctggc 240tatgacagga tgctgcggac acttggagga
aatctcaccg agtttattga aaacctagat 300gcactccaca gttacctggc
actgtcctat caggaaatga acgcaccatc ctttcgagtg 360gaggaaggag
ctgacggggc gatgcttctc cactactact cagacagaca tggtctgtgt
420cacattgtac caggtatcat tgaagctgtg gccaaggact tctttgacac
tgatgtggcc 480atgagtatcc tggatatgaa cgaagaggtg gaaaggacag
ggaagaaaga acatgttgtg 540tttctggtcg tgcagaaggc tcacagacag
ataagaggag caaaggcaag ccggccacaa 600ggcagtgagg acagccaggc
agaccaggag gctctccagg gaacactcct tcggatgaag 660gagagatatt
taaacatccc tgtttgccct ggggagaaat ctcactcaac tgctgtgagg
720gcatcggtcc tttttggaaa agggcccctc agggacacct tccagcccgt
ctatcctgag 780agactatggg tcgaagagga ggtgttctgt gatgcttttc
ctttccacat tgtctttgat 840gaagcactaa gggtcaagca agctggagtg
aatattcaga agtatgtccc tggaatctta 900acccagaagt ttgcactaga
tgagtatttt tccatcatcc accctcaagt tactttcaac 960atctccagca
tctgcaagtt cattaacagt cagtttgtct tgaagacaag aaaagaaatg
1020atgcccaaag caaggaagag ccagccgatg ctcaaactcc ggggtcagat
gatctggatg 1080gagtctctga ggtgcatgat cttcatgtgt tccccaaacg
tccgcagcct gcaagagctg 1140gaagagagca agatgcatct ttctgatatc
gctccgcacg acacgaccag ggatctcatc 1200ctcctcaacc agcagaggct
ggcagagatg gagctgtcct gccaactgga aaagaagaag 1260gaggagttgc
gtgtcctttc caatcacctg gccatcgaga agaagaagac agagaccttg
1320ctgtatgcca tgctgcctga acatgtggcc aaccaactca aggagggcag
aaaggtggct 1380gcaggagaat ttgaaacatg tacaatcctt ttcagcgatg
ttgtgacatt taccaacatc 1440tgtgcagcct gtgaacctat ccaaatcgtg
aacatgctga attcaatgta ctccaagttt 1500gacaggttaa ccagtgtcca
tgatgtctac aaagtagaaa caatagggga tgcttacatg 1560gtggtgggtg
gagtaccagt acccgttgaa agccatgctc aaagagtcgc caattttgct
1620ctggggatga gaatttctgc aaaagaagtg atgaatcctg tcactgggga
acctatccag 1680atcagagtgg gaatccacac tggaccagtc ttagcaggtg
ttgtgggaga caagatgcct 1740cggtactgct tgtttggtga cactgtaaac
acagcctcta ggatggaaag tcacgggctt 1800cccagcaaag tgcatctgag
ccccacagcc cacagagccc tgaaaaacaa agggtttgaa 1860attgtcagga
gaggcgagat cgaagtgaag gggaaaggaa agatgaccac atactttctg
1920atccagaacc tgaatgccac cgaggatgag ataatggggc gaccttcagc
ccccgctgat 1980gggaaggaag tatgtactcc cggaaaccaa gtcaggaagt
cccctgctgt cccgaggaac 2040acagaccatc agcaacaagt ctacaaagga
gacccagcag acgcttctaa tgaagtcaca 2100cttgctggga gcccagtggc
agggcgaaac tccacagatg cagtcaataa ccagccatca 2160ccagatgaga
ccaagacaag tgtcgttgct agtggccctg tgctgtctgc tttctgtgtt
2220gtgctgtga 222922742PRTRattus norvegicus 22Met Tyr Gly Phe Ile
Asn Thr Cys Leu Gln Ser Leu Val Thr Glu Lys1 5 10 15 Phe Gly Glu
Glu Thr Trp Glu Lys Leu Lys Ala Pro Ala Glu Val Gln 20 25 30 Asp
Val Phe Met Thr Tyr Thr Val Tyr Asp Asp Ile Ile Thr Ile Lys 35 40
45 Leu Ile Gln Glu Ala Cys Lys Val Leu Asp Val Ser Met Glu Ala Ile
50 55 60 Leu Lys Leu Phe Gly Glu Tyr Phe Phe Lys Phe Cys Lys Met
Ser Gly65 70 75 80 Tyr Asp Arg Met Leu Arg Thr Leu Gly Gly Asn Leu
Thr Glu Phe Ile 85 90 95 Glu Asn Leu Asp Ala Leu His Ser Tyr Leu
Ala Leu Ser Tyr Gln Glu 100 105 110 Met Asn Ala Pro Ser Phe Arg Val
Glu Glu Gly Ala Asp Gly Ala Met 115 120 125 Leu Leu His Tyr Tyr Ser
Asp Arg His Gly Leu Cys His Ile Val Pro 130 135 140 Gly Ile Ile Glu
Ala Val Ala Lys Asp Phe Phe Asp Thr Asp Val Ala145 150 155 160 Met
Ser Ile Leu Asp Met Asn Glu Glu Val Glu Arg Thr Gly Lys Lys 165 170
175 Glu His Val Val Phe Leu Val Val Gln Lys Ala His Arg Gln Ile Arg
180 185 190 Gly Ala Lys Ala Ser Arg Pro Gln Gly Ser Glu Asp Ser Gln
Ala Asp 195 200 205 Gln Glu Ala Leu Gln Gly Thr Leu Leu Arg Met Lys
Glu Arg Tyr Leu 210 215 220 Asn Ile Pro Val Cys Pro Gly Glu Lys Ser
His Ser Thr Ala Val Arg225 230 235 240 Ala Ser Val Leu Phe Gly Lys
Gly Pro Leu Arg Asp Thr Phe Gln Pro 245 250 255 Val Tyr Pro Glu Arg
Leu Trp Val Glu Glu Glu Val Phe Cys Asp Ala 260 265 270 Phe Pro Phe
His Ile Val Phe Asp Glu Ala Leu Arg Val Lys Gln Ala 275 280 285 Gly
Val Asn Ile Gln Lys Tyr Val Pro Gly Ile Leu Thr Gln Lys Phe 290 295
300 Ala Leu Asp Glu Tyr Phe Ser Ile Ile His Pro Gln Val Thr Phe
Asn305 310 315 320 Ile Ser Ser Ile Cys Lys Phe Ile Asn Ser Gln Phe
Val Leu Lys Thr 325 330 335 Arg Lys Glu Met Met Pro Lys Ala Arg Lys
Ser Gln Pro Met Leu Lys 340 345 350 Leu Arg Gly Gln Met Ile Trp Met
Glu Ser Leu Arg Cys Met Ile Phe 355 360 365 Met Cys Ser Pro Asn Val
Arg Ser Leu Gln Glu Leu Glu Glu Ser Lys 370 375 380 Met His Leu Ser
Asp Ile Ala Pro His Asp Thr Thr Arg Asp Leu Ile385 390 395 400 Leu
Leu Asn Gln Gln Arg Leu Ala Glu Met Glu Leu Ser Cys Gln Leu 405 410
415 Glu Lys Lys Lys Glu Glu Leu Arg Val Leu Ser Asn His Leu Ala Ile
420 425 430 Glu Lys Lys Lys Thr Glu Thr Leu Leu Tyr Ala Met Leu Pro
Glu His 435 440 445 Val Ala Asn Gln Leu Lys Glu Gly Arg Lys Val Ala
Ala Gly Glu Phe 450 455 460 Glu Thr Cys Thr Ile Leu Phe Ser Asp Val
Val Thr Phe Thr Asn Ile465 470 475 480 Cys Ala Ala Cys Glu Pro Ile
Gln Ile Val Asn Met Leu Asn Ser Met 485 490 495 Tyr Ser Lys Phe Asp
Arg Leu Thr Ser Val His Asp Val Tyr Lys Val 500 505 510 Glu Thr Ile
Gly Asp Ala Tyr Met Val Val Gly Gly Val Pro Val Pro 515 520 525 Val
Glu Ser His Ala Gln Arg Val Ala Asn Phe Ala Leu Gly Met Arg 530 535
540 Ile Ser Ala Lys Glu Val Met Asn Pro Val Thr Gly Glu Pro Ile
Gln545 550 555 560 Ile Arg Val Gly Ile His Thr Gly Pro Val Leu Ala
Gly Val Val Gly 565 570 575 Asp Lys Met Pro Arg Tyr Cys Leu Phe Gly
Asp Thr Val Asn Thr Ala 580 585 590 Ser Arg Met Glu Ser His Gly Leu
Pro Ser Lys Val His Leu Ser Pro 595 600 605 Thr Ala His Arg Ala Leu
Lys Asn Lys Gly Phe Glu Ile Val Arg Arg 610 615 620 Gly Glu Ile Glu
Val Lys Gly Lys Gly Lys Met Thr Thr Tyr Phe Leu625 630 635 640 Ile
Gln Asn Leu Asn Ala Thr Glu Asp Glu Ile Met Gly Arg Pro Ser 645 650
655 Ala Pro Ala Asp Gly Lys Glu Val Cys Thr
Pro Gly Asn Gln Val Arg 660 665 670 Lys Ser Pro Ala Val Pro Arg Asn
Thr Asp His Gln Gln Gln Val Tyr 675 680 685 Lys Gly Asp Pro Ala Asp
Ala Ser Asn Glu Val Thr Leu Ala Gly Ser 690 695 700 Pro Val Ala Gly
Arg Asn Ser Thr Asp Ala Val Asn Asn Gln Pro Ser705 710 715 720 Pro
Asp Glu Thr Lys Thr Ser Val Val Ala Ser Gly Pro Val Leu Ser 725 730
735 Ala Phe Cys Val Val Leu 740 2319PRTArtificial SequenceSynthetic
Construct 23Cys Ala Ala Thr Cys Gly Ala Gly Ala Cys Cys Cys Thr Gly
Gly Thr1 5 10 15 Gly Gly Ala2418PRTArtificial SequenceSynthetic
Construct 24Gly Cys Ala Cys Ala Cys Ala Cys Thr Cys Cys Ala Gly Gly
Cys Cys1 5 10 15 Cys Thr2519PRTArtificial SequenceSynthetic
Construct 25Cys Ala Ala Cys Cys Ala Gly Ala Cys Ala Thr Gly Gly Gly
Thr Cys1 5 10 15 Cys Ala Cys2621PRTArtificial SequenceSynthetic
Construct 26Gly Thr Thr Ala Ala Cys Gly Ala Ala Ala Ala Gly Gly Cys
Cys Cys1 5 10 15 Ala Cys Ala Gly Ala 20 2721PRTArtificial
SequenceSynthetic Construct 27Gly Thr Thr Gly Thr Gly Gly Ala Thr
Cys Cys Ala Gly Thr Cys Ala1 5 10 15 Cys Cys Thr Cys Thr 20
2821PRTArtificial SequenceSynthetic Construct 28Gly Ala Thr Thr Cys
Thr Cys Ala Gly Thr Gly Thr Gly Cys Thr Gly1 5 10 15 Gly Thr Cys
Ala Cys 20 2923PRTArtificial SequenceSynthetic Construct 29Cys Ala
Ala Gly Gly Ala Gly Gly Ala Ala Gly Cys Thr Thr Ala Thr1 5 10 15
Cys Thr Ala Thr Gly Ala Ala 20 3022PRTArtificial SequenceSynthetic
Construct 30Ala Thr Thr Thr Gly Ala Cys Gly Ala Ala Gly Gly Cys Gly
Ala Ala1 5 10 15 Gly Ala Ala Gly Cys Thr 20
* * * * *