U.S. patent application number 14/530569 was filed with the patent office on 2015-10-01 for biomarkers for h-nox delivery of oxygen.
The applicant listed for this patent is OMNIOX, INC.. Invention is credited to STEPHEN P. L. CARY, JENNIFER A. GETZ, ANA KRTOLICA, NATACHA LE MOAN, LAURA SERWER.
Application Number | 20150273024 14/530569 |
Document ID | / |
Family ID | 54188826 |
Filed Date | 2015-10-01 |
United States Patent
Application |
20150273024 |
Kind Code |
A1 |
CARY; STEPHEN P. L. ; et
al. |
October 1, 2015 |
BIOMARKERS FOR H-NOX DELIVERY OF OXYGEN
Abstract
The invention provides methods to monitor tumor oxygenation by
H-NOX proteins. H-NOX proteins extravasate into and preferentially
accumulate in tumor tissue for sustained delivery of oxygen. For
example, the invention provides methods to monitor brain tumor
oxygenation by H-NOX proteins for enhanced treatment of brain
cancers.
Inventors: |
CARY; STEPHEN P. L.; (SAN
MATEO, CA) ; KRTOLICA; ANA; (SAN FRANCISCO, CA)
; LE MOAN; NATACHA; (SAN FRANCISCO, CA) ; SERWER;
LAURA; (BRISBANE, CA) ; GETZ; JENNIFER A.;
(SAN BRUNO, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
OMNIOX, INC. |
SAN CARLOS |
CA |
US |
|
|
Family ID: |
54188826 |
Appl. No.: |
14/530569 |
Filed: |
October 31, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2013/020602 |
Jan 7, 2013 |
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14530569 |
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61898395 |
Oct 31, 2013 |
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61907983 |
Nov 22, 2013 |
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Current U.S.
Class: |
378/65 ;
435/7.23 |
Current CPC
Class: |
G01N 33/57407 20130101;
G01N 2800/52 20130101; Y02A 50/478 20180101; A61N 2005/1098
20130101; A61N 5/10 20130101; A61K 38/41 20130101; G01N 33/53
20130101; Y02A 50/30 20180101 |
International
Class: |
A61K 38/41 20060101
A61K038/41; A61N 5/10 20060101 A61N005/10; G01N 33/574 20060101
G01N033/574 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This work was supported by Grant Nos. 1 R43 CA138006 and 2
R44 CA138006. The U.S. government has rights in any patent issuing
on this application.
Claims
1. A method of treating a hypoxic brain tumor in an individual
comprising a) administering an effective amount of an H-NOX protein
to the individual, b) determining the level of hypoxia in the brain
tumor following administration of the H-NOX protein, and c)
administering an effective amount of radiation to the individual
wherein the tumor hypoxia measured in step b) is reduced compared
to the level of hypoxia in the brain tumor prior to H-NOX
administration.
2. A method of treating a hypoxic brain tumor in an individual
comprising a) determining the level of hypoxia in the brain tumor,
b) administering an effective amount of an H-NOX protein to the
individual, c) determining the level of hypoxia in the brain tumor
following administration of the H-NOX protein, and d) administering
an effective amount of radiation to the individual wherein the
tumor hypoxia measured in step c) is reduced compared to the level
of hypoxia measured in step a).
3. A method of optimizing therapeutic efficacy for treatment of a
hypoxic brain tumor in an individual, the method comprising a)
administering H-NOX to the individual, b) measuring the level of
hypoxia of the tumor one or more times after administration of the
H-NOX protein, c) administering radiation therapy when tumor
hypoxia is reduced compared to the level of hypoxia prior to H-NOX
administration.
4. The method of claim 3, wherein the hypoxia is reduced by at
least about 5%, 10%, 15%, 20%, 25% or 50%.
5. A method of monitoring the efficacy of delivery of O.sub.2 to
hypoxic brain tumor by an H-NOX protein in an individual, the
method comprising a) administering an effective amount of H-NOX
protein to the individual, b) measuring the level of hypoxia in the
tumor at one or more time points after administration of the H-NOX
protein, wherein a reduction of tumor hypoxia compared to the level
of hypoxia in the tumor prior to administration of H-NOX indicates
effective delivery of O.sub.2 to the brain tumor.
6. The method of claim 5, wherein the reduction in tumor hypoxia
indicates that the individual is suitable for administration of
radiation therapy.
7. The method of claim 3, wherein the level of hypoxia in the tumor
is measured one or more of one hour, two hours, three hours, four
hours, eight hours, twelve hours, 24 hours, 48 hours or 72 hours
after administration of H-NOX.
8. A method of monitoring responsiveness or lack of responsiveness
to treatment with a H-NOX in an individual suffering from a brain
tumor comprising measuring the hypoxic state of the tumor following
H-NOX administration, wherein responsiveness is indicated by a
reduction in tumor hypoxia.
9. The method of claim 8, wherein the responsiveness indicates that
the individual is suitable for administration of radiation
therapy.
10. A method of identifying an individual with a brain tumor who is
more likely to exhibit benefit from a therapy comprising an H-NOX
protein, said method comprising a) determining the hypoxia level of
the tumor, a) administering H-NOX to the individual, b) measuring
the level of hypoxia of the tumor, c) wherein about a 5% decrease
in hypoxia indicates the individual is more likely to exhibit
benefit from radiation treatment in combination with H-NOX
treatment.
11. The method of claim 10, wherein the decrease in hypoxia of step
c) is a at least a 10%, a 15%, a 20%, a 25%, a 50%, a 75% or a 100%
decrease in hypoxia.
12. The method of claim 1, wherein tumor hypoxia is measured by one
or more of .sup.18F-fluoromisonidazole (FMISO) tumor uptake,
pimidazole uptake, .sup.18F-fluoroazomycin arabinoside (FAZA)
uptake, a nitroimidazole uptake,
Copper(II)-diacetyl-bis(N.sup.4-methylthiosemicarbazone (Cu-ATSM)
uptake, .sup.19F magnetic resonance imaging of hexafluorobenzene
(C6F6) uptake, .sup.1H MRI of hexamethyldisiloxane uptake, tumor
HIF-1.alpha. expression, tumor Glut-1 expression, tumor LDHA
expression, tumor carbonic anhydrase IX (CA-9) expression, or
lactate and/or pyruvate levels.
13. The method of claim 1, wherein the determination of the level
of hypoxia in the tumor is repeated.
14-15. (canceled)
16. The method of claim 15, further comprising administration of
radiation following administration of H-NOX.
17. (canceled)
18. The method of claim 1, wherein the radiation is
X-radiation.
19. The method of claims 18, wherein the X-radiation is
administered at about 0.5 gray to about 75 gray.
20. The method of claim 1, where the brain cancer is
glioblastoma.
21. (canceled)
22. The method of claim 1, wherein the individual is a human.
23-24. (canceled)
25. The method of claim 1, 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.
26. The method of claim 1, 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.
27. The method of claim 1, wherein the H-NOX comprises one or more
distal pocket mutations.
28. The method of claim 27, wherein the distal pocket mutation is
an amino acid substitution at a site corresponding to L144 of T.
tengcongensis H-NOX.
29. (canceled)
30. The method of claim 26, wherein the amino acid substitution at
position 144 is an L144F substitution.
31-34. (canceled)
35. The method of claim 1, wherein the H-NOX protein is a polymeric
H-NOX protein.
36. The method of claim 35, wherein the polymeric H-NOX protein
comprises monomers, wherein the monomers comprise an H-NOX domain
and a polymerization domain.
37. The method of claim 36, wherein the H-NOX domain is covalently
linked to the polymerization domain.
38. The method of claim 35, wherein the polymeric H-NOX protein is
a trimeric H-NOX protein.
39. The method of claim 38, wherein the trimeric H-NOX protein
comprises one or more trimerization domains.
40. (canceled)
41. The method of claim 39, wherein the trimerization domain is a
foldon domain.
42. The method of claim 41, wherein the foldon domain comprises the
amino acid sequence of SEQ ID NO:4.
43. (canceled)
44. The method of claim 1, wherein the H-NOX protein is covalently
bound to polyethylene glycol.
45-56. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application No. PCT/US2013/020602, filed Jan. 7, 2013. This
application also claims benefit of U.S. provisional patent
application No. 61/898,395, filed on Oct. 31, 2013 and U.S.
provisional patent application No. 61/907,983, filed Nov. 22, 2013.
The contents of each are incorporated herein by reference in its
entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0003] 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:
627042000420SeqList.txt, date recorded: Jun. 12, 2015, size: 49
KB).
TECHNICAL FIELD
[0004] This application pertains to H-NOX proteins and methods of
using them to deliver oxygen to hypoxic tumors and methods to
monitor tumor oxygenation to enhance anti-tumor therapies. H-NOX
proteins provide a new therapeutic tool for delivering O.sub.2 to
humans and, for veterinary purposes, to animals.
BACKGROUND OF THE INVENTION
[0005] 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,
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.
[0006] It was discovered that H-NOX proteins that bind NO but not
O.sub.2 can be converted to H-NOX proteins that bind both NO and
O.sub.2 by the introduction of a single amino acid mutation (see WO
2007/139791 and WO 2007/139767). Thus, the affinity of H-NOX
proteins for O.sub.2 and NO and the ability of H-NOX proteins to
discriminate between O.sub.2 and NO ligands can be altered by the
introduction of one or more amino acid mutations, allowing H-NOX
proteins to be tailored to bind O.sub.2 or NO with desired
affinities. Additional mutations can be introduced to further alter
the affinity for O.sub.2 and/or NO. The H-NOX protein family can
therefore be manipulated to exhibit improved or optimal kinetic and
thermodynamic properties for O.sub.2 delivery. For example, mutant
H-NOX proteins have been generated with altered dissociation
constants and/or off rates for O.sub.2 binding that improve the
usefulness of H-NOX proteins for a variety of clinical and
industrial applications. The ability to tune H-NOX proteins to bind
and deliver O.sub.2 is a therapeutic avenue that addresses and
overcomes the central shortcomings of current O.sub.2 carriers.
[0007] What is needed for certain therapeutic uses is an H-NOX with
a long circulation half-life that can bind and deliver O.sub.2 to
distal tissues for sufficient periods of time. Additionally, H-NOX
proteins extravasate into tumors where they accumulate at different
rates. For example, polymeric H-NOX proteins are tuned to transport
oxygen through normoxic regions of tumors and release oxygen deep
within hypoxic zones within tumors. For brain tumors, the H-NOX
protein may cross the blood-brain bather to deliver O.sub.2 to
hypoxic brain tumors. This combination of features represents a
significant advance in the use of oxygen carriers as modifiers of
the hypoxic niches of tumors to increase the efficacy of
radiotherapy, chemotherapy and other anti-cancer treatments reliant
on oxygenation of tumor cells. Provided herein are methods to
detect tumor oxygenation (e.g. brain tumor oxygenation) to enhance
therapeutic treatments for cancers.
[0008] H-NOX proteins for delivery of O.sub.2 or NO are provided by
U.S. Pat. Nos. 8,404,631 and 8,404,632. Polymeric H-NOX proteins
and H-NOX proteins for treating brain cancer are described in
PCT/US2013/020602. The contents of each are incorporated herein 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 is directed towards methods of delivering,
monitoring, and optimizing the delivery of O.sub.2 to hypoxic
tumors. In some aspects, the invention provides methods of treating
a hypoxic brain tumor in an individual comprising a) administering
an effective amount of an H-NOX protein to the individual, b)
determining the level of hypoxia in the brain tumor following
administration of the H-NOX protein, and c) administering an
effective amount of radiation to the individual wherein the tumor
hypoxia measured in step b) is reduced compared to the level of
hypoxia in the brain tumor prior to H-NOX administration. In some
aspects, the invention provides methods of treating a hypoxic brain
tumor in an individual comprising a) determining the level of
hypoxia in the brain tumor, b) administering an effective amount of
an H-NOX protein to the individual, c) determining the level of
hypoxia in the brain tumor following administration of the H-NOX
protein, and d) administering an effective amount of radiation to
the individual wherein the tumor hypoxia measured in step c) is
reduced compared to the level of hypoxia measured in step a).
[0011] In some aspects, the invention provides methods of
optimizing therapeutic efficacy for treatment of a hypoxic brain
tumor in an individual, the method comprising a) administering
H-NOX to the individual, b) measuring the level of hypoxia of the
tumor one or more times after administration of the H-NOX protein,
c) administering radiation therapy when tumor hypoxia is reduced
compared to the level of hypoxia prior to H-NOX administration. In
some embodiments, the hypoxia is reduced by at least about 5%, 10%,
15%, 20%, 25% or 50%. In some embodiments, the level of tumor
hypoxia is first determined prior to administration of H-NOX.
[0012] In some aspects, the invention provides methods of
monitoring the efficacy of delivery of O.sub.2 to hypoxic brain
tumor by an H-NOX protein in an individual, the method comprising
a) administering an effective amount of H-NOX protein to the
individual, b) measuring the level of hypoxia in the tumor at one
or more time points after administration of the H-NOX protein,
wherein a reduction of tumor hypoxia compared to the level of
hypoxia in the tumor prior to administration of H-NOX indicates
effective delivery of O.sub.2 to the brain tumor. In some
embodiments, the reduction in tumor hypoxia indicates that the
individual is suitable for administration of radiation therapy. In
some embodiments, the hypoxia is reduced by at least about 5%, 10%,
15%, 20%, 25% or 50%. In some embodiments, the level of tumor
hypoxia is first determined prior to administration of H-NOX.
[0013] In some embodiments of the above aspects, the level of
hypoxia in the tumor is measured one or more of one hour, two
hours, three hours, four hours, eight hours, twelve hours, 24
hours, 48 hours or 72 hours after administration of H-NOX.
[0014] In some aspects, the invention provides methods of
monitoring responsiveness or lack of responsiveness to treatment
with an H-NOX in an individual suffering from a brain tumor
comprising measuring the hypoxic state of the tumor following H-NOX
administration, wherein responsiveness is indicated by a reduction
in tumor hypoxia. In some embodiments, responsiveness indicates
that the individual is suitable for administration of radiation
therapy.
[0015] In some aspects, the invention provides methods of
identifying an individual with a brain tumor who is more likely to
exhibit benefit from a therapy comprising an H-NOX protein, said
method comprising a) determining the hypoxia level of the tumor, b)
administering H-NOX to the individual, c) measuring the level of
hypoxia of the tumor, wherein about a 5% decrease in hypoxia
indicates the individual is more likely to exhibit benefit from
radiation treatment in combination with H-NOX treatment. In some
embodiments, the decrease in hypoxia of step c) is a at least a
10%, a 15%, a 20%, a 25%, a 50%, a 75% or a 100% decrease in
hypoxia.
[0016] In some embodiments of any of the above aspects and
embodiments, tumor hypoxia is measured by one or more 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, .sup.19F magnetic resonance imaging of hexafluorobenzene
(C6F6) uptake, .sup.1H MRI of hexamethyldisiloxane uptake, tumor
HIF-1.alpha. expression, tumor Glut-1 expression, tumor LDHA
expression, tumor carbonic anhydrase IX (CA-9) expression, or
lactate and/or pyruvate levels.
[0017] In some embodiments of any of the above aspects and
embodiments, the determination of the level of hypoxia in the tumor
is repeated. In some embodiments, the determination of tumor
hypoxia is repeated after one or more of one week, two weeks, three
weeks, or four weeks. In further embodiments, the administration of
H-NOX to the individual is repeated if the tumor is hypoxic. In
some embodiments, the methods further comprising administration of
radiation following administration of H-NOX. In yet further
embodiments, the administration or radiation is repeated if the
tumor has reduced hypoxia compared to the tumor prior to
administration of H-NOX.
[0018] In some embodiments of any of the above aspects and
embodiments, the radiation is X-radiation. In some embodiments, the
X-radiation is administered at about 0.5 gray to about 75 gray.
[0019] In some embodiments of any of the above aspects and
embodiments, the brain cancer is glioblastoma. In some embodiments,
the individual is a mammal. In some embodiments, the mammal is a
human. In other embodiments, the mammal is a pet, a laboratory
research animal, or a farm animal. In further 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.
[0020] In some embodiments of any 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 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.
[0021] In some embodiments of any of the above aspects and
embodiments, the H-NOX protein comprises an 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. In some embodiments, the
H-NOX comprises at least two distal pocket mutations. In further
embodiments, the at least two distal pocket mutations are amino
acid substitutions at sites corresponding to W9 and L144 of T.
tengcongensis H-NOX. In other embodiments, the H-NOX is a T.
tengcongensis H-NOX comprising amino acid substitutions at
positions 9 and 144. In some embodiments, the amino acid
substitution at position 9 is a W9F substitution and the amino acid
substitution at position 144 is an L144F substitution.
[0022] In some embodiments of any of the above aspects and
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 other 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 further embodiments, the
trimerization domain is a foldon domain. In further embodiments,
the foldon domain comprises the amino acid sequence of SEQ ID NO:4.
In some embodiments, the trimeric H-NOX protein comprises three
H-NOX monomers wherein each H-NOX monomer is fused to a foldon
domain. In some embodiments, the trimeric H-NOX protein comprises
three Tt L144F H-NOX monomers wherein each Tt L144F H-NOX monomer
is fused to a foldon domain.
[0023] In some embodiments of any of the above aspects and
embodiments, the H-NOX protein is fused to an Fc domain of an
immunoglobulin. In other embodiments, the H-NOX protein is
covalently bound to polyethylene glycol.
[0024] In some embodiments of any of the above aspects and
embodiments, the H-NOX protein does not comprise a guanylyl cyclase
domain.
[0025] In some embodiments of any of the above aspects and
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 other 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] FIGS. 1A and 1B show that H-NOX monomer and H-NOX trimer is
distributed and retained in mice bearing HCT-116 colon-derived
tumors. FIG. 1A shows immunohistochemistry staining of tumors with
H-NOX protein antibody showed persistence of H-NOX trimer in tumors
for 60 minutes as compared to H-NOX monomer which was partially
cleared at 60 minutes. FIG. 1B shows quantification of H-NOX
protein staining intensity in HCT-116 tumor sections. N=6, all
groups. Mean values+/-SEM.
[0027] FIGS. 2A and 2B show that H-NOX monomer and H-NOX trimer
reduced tumor hypoxia in mice bearing HCT-116 colon-derived tumors.
FIG. 2A shows representative tumor section of a 125 mm.sup.3 tumor
isolated from mice treated with vehicle, H-NOX monomer, or H-NOX
trimer. FIG. 2B shows quantification of an anti-pimonidazole
antibody (Hypoxyprobe-1) intensity in tumor sections. N=6, all
groups. Mean values+/-SEM. * indicates hypoxia throughout tumor, **
indicates no hypoxia in tumor.
[0028] FIGS. 3A-3C show tumor penetration and oxygenation by H-NOX
monomer in mice bearing HCT-116 colon-derived tumors. FIG. 3A shows
tumor sections stained with an anti-H-NOX protein antibody. FIG. 3B
shows tumor sections stained with Hypoxyprobe-1. FIG. 3C shows
quantification of the Hypoxyprobe-1 as a function of distance from
the vasculature in the tumors from six mice per group. * indicates
hypoxia throughout tumor, indicates no hypoxia in tumor.
[0029] FIGS. 4A-4G show that H-NOX trimer was distributed and
retained in mice bearing a RIF-1 syngeneic sarcoma tumor
Immunofluorescence images of a representative section from a 400
mm.sup.3 tumor isolated from a mouse 120 minutes after
administration of 750 mg/kg H-NOX trimer (FIG. 4A) or buffer (FIG.
4C), and of a 800 mm.sup.3 tumor isolated from a mouse 120 minutes
after administration of 750 mg/kg H-NOX trimer (FIG. 4B) or buffer
(FIG. 4D). H-NOX protein staining was done with anti-H-NOX
antibody. FIGS. 4E and 4F shows tumor oxygenation by H-NOX trimer
in mice bearing a RIF-1 syngeneic sarcoma tumor. FIG. 4E shows
tumor sections stained with an anti-pimonidazole antibody two hours
after H-NOX or buffer control administration. Whole tumor picture
is shown. FIG. 4F shows tumor sections stained with
anti-pimonidazole antibody (Hypoxyprobe-1) and anti-CD31 antibody
(BD Bioscience) two hours after H-NOX or buffer control
administration. High magnification picture are shown. FIG. 4G shows
biodistribution of H-NOX in RIF1 syngeneic sarcoma tumors. Two
hours after intravenous injection, H-NOX trimer diffuses from the
vasculature into the tumor tissue Immunohistochemistry staining of
tumor sections with H-NOX antibody and CD31 antibody (vasculature
marker, BD Bioscience). No fluorescent staining is detected in mice
injected with buffer.
[0030] FIGS. 5A-5C show H-NOX trimer penetrated tumor in mice
bearing a sarcoma derived tumor and reduced tumor hypoxia. FIG. 5A
shows a western blot membrane was probed with an anti-H-NOX
antibody for detection of H-NOX trimer, with Hypoxyprobe-1 for
detection of hypoxia-associated proteins, or with an anti-actin
antibody for assessment of total protein levels. FIG. 5B shows
quantification of pimonidazole staining intensity in tumor
sections. FIG. 5C shows quantification of anti-HIF-1.alpha.
staining intensity in tumor sections.
[0031] FIGS. 6A and 6B are panels of immunohistochemistry images
showing tumor penetration by H-NOX trimer and reduced brain tumor
hypoxia in mice bearing U251 orthotopic brain tumors. FIG. 6A shows
H-NOX trimer staining with an anti-H-NOX antibody in a U251 tumor
two hours after administration with H-NOX trimer or saline
(control). FIG. 6B shows hypoxyprobe-1 staining in U251 tumors two
hours after administration with H-NOX trimer or saline (control).
Enlarged images from a portion of the tumors are shown.
[0032] FIGS. 7A-7D show tumor penetration by H-NOX trimer and
reduced brain tumor hypoxia in mice bearing U251 orthotopic brain
tumors. FIG. 7A shoes immunofluorescence images of Hypoxyprobe-1
staining in U251 tumors two hours after administration with H-NOX
trimer (right panels) or saline (buffer, left panels). FIG. 7B
shows quantification of Hypoxyprobe-1 staining from the
immunofluorescence images (H-NOX trimer-right panels or saline-left
panels). FIG. 7C shows immunofluorescence images of HIF-1.alpha.
staining in U251 tumor two hours after administration with H-NOX
trimer or saline (buffer). FIG. 7D shows quantification of
HIF-1.alpha. staining from the immunofluorescence images.
[0033] FIGS. 8A-8E show the biodistribution of H-NOX trimer in U251
orthotopic brain tumor and healthy brain. FIG. 8A shows H-NOX
trimer staining with an anti-H-NOX antibody in a U251 tumor two
hours after administration with H-NOX trimer. FIG. 8B shows nuclear
DAPI staining in U251 tumors showing tumor localization in the
brain. FIGS. 8C and 8D show enlarged images from a portion of the
tumors from FIG. 8A show a diffused pattern of H-NOX inside the
tumor and vascular-restricted pattern outside the tumor. FIG. 8E
shows H-NOX trimer staining with an anti-H-NOX antibody and
vasculature staining with anti-CD31 antibody (BD Bioscience) in
healthy mouse brain.
[0034] FIGS. 9A and 9B show real-time fluorescent images of H-NOX
monomer or H-NOX trimer in mouse U251 orthotopic glioblastoma
tumors. FIG. 9A shows H-NOX monomer was cleared by two hours. FIG.
9B shows H-NOX trimer persisted in tumors, peaking at 1-4 hours.
Images acquired by IVIS; arrows indicate areas of fluorescence
above a specific threshold; asterisks indicate peak level of
fluorescence intensity.
[0035] FIGS. 10A-10D show ex vivo fluorescence images of H-NOX
monomer or H-NOX trimer in mouse BT-12 orthotopic glioblastoma
tumors. Brains bearing BT-12 tumors were resected 30 minutes after
750 mg/kg H-NOX monomer administration (FIG. 10A), 60 minutes after
750 mg/kg H-NOX monomer administration (FIG. 10B), 60 minutes after
750 mg/kg H-NOX trimer administration (FIG. 10C), or 60 minutes
after vehicle administration (FIG. 10D).
[0036] FIGS. 11A-11D show real-time fluorescence images of H-NOX
monomer in mouse U251 orthotopic glioblastoma tumors. Imaging was
acquired at 30 minutes (FIG. 11A), 60 minutes (FIG. 11B), 120
minutes (FIG. 11C), and 240 minutes (FIG. 11D) after H-NOX monomer
administration.
[0037] FIGS. 12A-12D show real-time fluorescence images of H-NOX
trimer in mouse U251 orthotopic glioblastoma tumors. Imaging was
acquired at 30 minutes (FIG. 12A), 60 minutes (FIG. 12B), 120
minutes (FIG. 12C), and 240 minutes (FIG. 12D) after H-NOX trimer
administration. Arrows indicate areas of fluorescence; asterisks
indicate peak level of fluorescence intensity.
[0038] FIGS. 13A and 13B show real-time fluorescence images of
H-NOX monomer in mouse U251 orthotopic glioblastoma tumors.
Accumulation of H-NOX monomer in the kidney at 30 minutes (FIG.
13A) and 60 minutes (FIG. 13B) after H-NOX monomer
administration.
[0039] FIGS. 14A-14F show real-time fluorescence images of H-NOX
trimer in mouse GBM-43 orthotopic glioblastoma intracranial and
spinal tumors. Distribution of H-NOX trimer in the spinal column
prior to H-NOX trimer administration (FIG. 14A) and 0.5 hour (FIG.
14B), 1 hour (FIG. 14C), 2 hours (FIG. 14D), 4 hours (FIG. 14E),
and 6 hours (FIG. 14F) after H-NOX trimer administration.
[0040] FIG. 15 shows real-time fluorescence images of H-NOX trimer
in mouse U251 orthotopic glioblastoma intracranial tumors. Top
panel shows the distribution of H-NOX trimer in the brain prior to
H-NOX trimer administration (0 minutes) and at 30 min, 1 hour, 2
hours, 4 hours, 6 hours, and 72 hours after H-NOX trimer
administration. Bottom panel shows the distribution of H-NOX
monomer.
[0041] FIGS. 16A-16F show real-time bioluminescence images of H-NOX
trimer in mouse U251 orthotopic glioblastoma intracranial and
spinal tumors. H-NOX trimer distribution prior to H-NOX trimer
administration (FIG. 16A) and at 30 min (FIG. 16B), 1 hour (FIG.
16C), 2 hours (FIG. 16D), 4 hours (FIG. 16E), and 6 hours (FIG.
16F) after H-NOX trimer administration at a dose of 295 mg/kg.
[0042] FIGS. 17A-17F show real-time fluorescence images of H-NOX
trimer in mouse U251 orthotopic glioblastoma tumors. H-NOX trimer
distribution prior to H-NOX trimer administration (FIG. 17A) and at
30 min (FIG. 17B), 1 hour (FIG. 17C), 2 hours (FIG. 17D), 4 hours
(FIG. 17E), and 6 hours (FIG. 17F) after H-NOX trimer
administration at a dose of 30 mg/kg.
[0043] FIGS. 17A-17F show real-time fluorescence images of H-NOX
trimer L144F variant distribution in a U251 orthotopic glioblastoma
mouse model containing small intracranial tumors. H-NOX trimer
L144F variant distribution FIG. 18A) prior to H-NOX trimer
administration and at 30 min (FIG. 18B), 1 hour (FIG. 18C), 2 hours
(FIG. 18D), 4 hours (FIG. 18E), and 6 hours (FIG. 18F) after H-NOX
trimer L144F variant administration at a dose of 30 mg/kg. Small
tumors were 1000.times. fold smaller than large tumors as
determined by bioluminescence (BLI) score.
[0044] FIGS. 19A-19D show fluorescence images of H-NOX trimer
distribution. Ex vivo fluorescence images of a GBM43 orthotopic
glioblastoma mouse model administered 30 mg/kg H-NOX trimer (FIG.
19A) or 750 mg/kg H-NOX trimer (FIG. 19B). Real-time
bioluminescence imaging in a U251 orthotopic glioblastoma mouse
model containing large intracranial tumors (FIG. 19C) or small
intracranial tumors (FIG. 19D) after administration of 295 mg/kg
H-NOX trimer.
[0045] FIG. 20 shows real-time fluorescence images of H-NOX trimer
distribution in two mouse models of orthotopic glioblastoma tumors
(U251 and GBM-43) and one model of an atypical teratoid/rhabdoid
tumor (AT/RT). Images were taken 60 minutes after H-NOX trimer
administration and the color scale for each image was optimized
[0046] FIGS. 21A-21E show ex vivo fluorescence images of H-NOX
protein distribution in the tumor-bearing hemisphere of three mouse
models of orthotopic glioblastoma tumors. FIG. 21A shows H-NOX
trimer distribution 60 minutes after administration in a GBM43
orthotopic glioblastoma mouse model, FIG. 21B shows H-NOX trimer
distribution 6 days after administration in a U251 orthotopic
glioblastoma mouse model, FIG. 21C shows H-NOX monomer distribution
30 minutes after administration in a BT-12 an atypical
teratoid/rhabdoid tumor (AT/RT) mouse model, FIG. 21D shows H-NOX
trimer distribution 60 minutes after administration in a BT-12
orthotopic AT/RT mouse model, and FIG. 21E shows lack of H-NOX
protein signal 30 minutes after vehicle administration in a BT-12
orthotopic AT/RT mouse model.
[0047] FIG. 22 is an immunofluorescence image showing escape of
H-NOX trimer from the vasculature and diffusion throughout a U251
brain tumor in an orthotopic glioblastoma tumor mouse model. Tumor
sections were stained with an anti-H-NOX antibody (top panel) and
an anti-CD31 antibody (vasculature) (bottom panel).
[0048] FIGS. 23A-23D show that a trimeric H-NOX protein accumulates
in and penetrates deep into the tumor tissue in the orthotopic
glioblastoma mouse model. Mice bearing orthotopic glioblastoma
BT-12 (FIG. 23A) or U251 tumors were injected with either 80 kD
H-NOX trimer, OMX-4.80 (FIGS. 23A-D), or 23 kD H-NOX monomer, OMX-4
(FIG. 23A, top panel), via tail vein and monitored by live imaging
of fluorescently labeled protein (FIGS. 23A, 23B) or
immunohistochemical analysis using anti-H-NOX antibody (FIGS. 23C,
23D).
[0049] FIGS. 24A-24D show results of sandwich ELISA assays of H-NOX
trimer in the brain of healthy mice. FIG. 24A shows an ELISA assay
on brain after intravenous injection of H-NOX trimer (750 mg/kg).
FIG. 24B shows an ELISA assay on brain after intravenous injection
of H-NOX trimer (200 mg/kg). FIG. 24C shows the brain/plasma ratio
of H-NOX trimer (750 mg/kg). FIG. 24D shows the brain/plasma ratio
of H-NOX trimer (200 mg/kg). Plasma and brain were collected at 30,
60, 90 and 120 min after H-NOX trimer administration. N=3, all
groups. Mean values+/-SEM.
[0050] FIGS. 25A-25C are a series of graphs showing that H-NOX
trimer sensitized intracranial xenografts to fractionated radiation
therapy in a U251 mouse model of human glioblastoma. FIG. 25A shows
mean bioluminescence imaging (BLI) scores+/-SEM from mice in both
treatment groups, as well as an untreated control group (no H-NOX,
no RT). N=9, all groups. FIG. 25B shows individual BLI scores for
the RT and RT+H-NOX trimer groups on Day 29 (box in A). Line shows
group mean, +\-SEM. The BLI scores of the RT+H-NOX trimer mice were
significantly lower than those from mice treated with RT alone
(p=0.039, Student's t-test). FIG. 25C shows H-NOX trimer group
showed significantly enhanced survival, as compared to mice that
received only radiotherapy (p=0.025, logrank test).
[0051] FIGS. 26A and 26B are a series of graphs showing that H-NOX
trimer sensitized intracranial xenografts to fractionated radiation
therapy in two mouse models of human glioblastoma. FIG. 26A shows
percent survival in a U251 orthotopic glioblastoma mouse model
administered 2 Gy radiation therapy (2 Gy), H-NOX trimer L144F
variant (L144F Trimer), 2 Gy radiation therapy in combination with
H-NOX trimer L144F variant (2 Gy+L144F Trimer), or treatment buffer
(TB). Logrank p-values: 2 Gy versus 2 Gy+L144F Trimer (p=0.158), 2
Gy versus TB (p=0.0612), and L144F Trimer versus TB (p=0.326). FIG.
26B shows percent survival in a GBM43 orthotopic glioblastoma mouse
model administered 2 Gy radiation therapy (2 Gy), 4 Gy radiation
therapy (4 Gy), 8 Gy radiation therapy (8 Gy), 2 cycles of 4 Gy
radiation therapy (4 Gy.times.2), 4 Gy radiation therapy in
combination with H-NOX trimer (4 Gy+H-NOX), or treatment buffer
(untreated). Logrank p-values: 4 Gy versus 4 Gy+H-NOX (p=0.597), 4
Gy versus 4 Gy.times.2 (p=0.038), and 4 Gy.times.2 versus 4
Gy+H-NOX (p=0.111).
[0052] FIGS. 27A-27D show that a single dose of a trimeric H-NOX
protein reduces tumor hypoxia. Mice bearing orthotopic glioblastoma
U251 tumors were treated with either trimeric Tt L144F H-NOX,
OMX-4.80, or buffer alone (top panels of FIGS. 27A and 27D) via
tail vein bolus injection. Tumors were harvested 2 hr-30 hr after
OMX-4.80 administration, and assayed by immunohistochemistry for
pimonidazole (Hypoxyprobe-1 mAb) and total cell nuclear (DAPI)
staining in FIGS. 27A and 27B, or for HIF1.alpha. and tumor cell
marker (HLA) in FIGS. 27C and 27D. FIG. 27A shows representative
tumor sections from mice treated with buffer or trimeric Tt L144F
H-NOX. Hypoxia staining (pimonidazole) is shown in green (left
column of FIG. 27A) and total cell nuclear staining (DAPI) is shown
in dark blue (right column of FIG. 27A). FIG. 27B shows
quantification of pimonidazole staining intensity in tumor
sections. N=5-6 per group. Mean values+/-SEM, p<0.05. FIG. 27C
shows quantification of HIF1.alpha. staining intensity in tumor
sections. N=5-6 per group. Mean values+/-SEM, p<0.05. FIG. 27D
shows representative tumor sections from mice treated with buffer
or OMX-4.80. Hypoxia staining (HIF1.alpha.) is shown in green (left
column of FIG. 27D) and staining of human tumor cells in red (HLA;
right column of FIG. 27D). Only 2 hr and 24 hr time points are
shown.
[0053] FIGS. 28A-28F show treatment with trimeric Tt L144F H-NOX
reduces tumor hypoxia at the invasive edges of the tumor. Mice
bearing orthotopic glioblastoma U251 tumors were treated with
either buffer control (FIG. 28A and FIG. 28C) or trimeric Tt L144F
H-NOX, OMX-4.80, (FIGS. 28B, 28D, 28E, and 28F), via tail vein
injection and received an injection of hypoxia marker, pimonidazole
an hour prior to sacrifice. Brains containing tumors were extracted
and subjected to immunohistochemical analysis using
anti-pimonidazole (FIGS. 28A and 28B), HIF-1.alpha. (FIGS. 28C and
28D), or trimeric Tt L144F H-NOX antibodies (FIGS. 28E and 28F).
Slides were counterstained with DNA marker (DAPI, blue labeled
nuclei) and images merged (FIGS. 28A-28D, and 28F).
[0054] FIGS. 29A-29D show treatment with a trimeric Tt L144F H-NOX
protein enhances efficacy of a single dose of radiation in an
orthothopic glioblastoma model and in a RIF-1 syngeneic tumor
model. FIG. 29A shows mean bioluminescence imaging (BLI)
scores.+-.SEM from mice receiving H-NOX and 10 gray radiation
(triangles) and 10 gray radiation in buffer control (no H-NOX,
circles), as well as an untreated (buffer, no RT, inverted
triangles) control group. N.gtoreq.8, all groups. FIG. 29B shows
that trimeric Tt L144F H-NOX group shows significantly enhanced
survival, as compared to mice that received only radiotherapy
(p<0.05, logrank test). FIG. 29C shows mean bioluminescence
imaging (BLI) scores.+-.SEM from mice receiving H-NOX and 10 gray
radiation (triangles), 10 gray radiation in buffer control
(circles), 15 gray radiation in buffer control (blue inverted
triangles), as well as an untreated (buffer, no RT, black inverted
triangles) control group. N.gtoreq.8, all groups. FIG. 29D shows
tumor volume measurements in RIF1 tumors: trimeric Tt L144F
H-NOX+15 Gy group (red triangles), buffer+15 Gy alone (black
dotted), buffer+25 Gy RT (gray dotted). Untreated control (solid
black line). Inactive H-NOX, OMX-1.80+15 Gray (green line).
Mean.+-.SEM, N=7-9 per group, p<0.01, Student's t-test.
[0055] FIGS. 30A-30G show the nucleic acid and amino acid sequences
of H-NOX proteins. FIG. 30A shows wild-type Thermoanaerobacter
tengcongensis H-NOX (SEQ ID NOs:1 and 2). FIG. 30B shows wildtype
Legionella pneumophilia Orf2 H-NOX (SEQ ID NOs:13 and 14). FIG. 30C
shows wildtype Legionella pneumophilia Orf1 H-NOX (SEQ ID NOs:15
and 16). FIG. 30D shows Homo sapiens .beta.1 (1-385) H-NOX (SEQ ID
NOs:17 and 18). FIG. 30E shows Homo sapiens .beta.2 (1-217) H-NOX
(SEQ ID NOs:19 and 20). FIG. 30F shows Rattus norvegicus .beta.1
H-NOX (SEQ ID NOs:21 and 22). FIG. 30G shows Rattus norvegicus
.beta.2 H-NOX (SEQ ID NOs:23 and 24).
[0056] FIG. 31A shows immunohistochemical staining for pimonidazole
in GL261, U251, GBM43 and GBM6 intracranial tumor models. FIG. 31B
shows immunohistochemical staining for HIF-1.alpha. in GL261, U251,
GBM43 and GBM6 intracranial tumor models.
[0057] FIG. 32A shows a schematic representation of quantitative
oxygen dependencies for OMX-4.80, OMX-1.80, bioreductive activation
of imaging agents (pimonidazole), and biological responses to
hypoxia (HIF-1.alpha.). Three commonly used units for oxygen
concentration are shown on the x axis. This schematic is
theoretical and adapted from the review "Targeting hypoxia in
cancer therapy" by William R. Wilson & Michael P. Hay (Nature
Reviews Cancer 11, 393-410, 2011). FIG. 32B shows a scatterplot
comparing HIF-1.alpha. levels with pimonidazole levels obtained by
immunohistochemical analysis in U251 intracranial tumors from mice
treated with either buffer control or OMX-4.80.
DETAILED DESCRIPTION OF THE INVENTION
[0058] The present invention is based in part on the surprising
discovery that H-NOX proteins preferentially extravasate and
accumulate in tissues thereby providing a long oxygenation window
and circulation half-life to deliver oxygen where needed to treat
disease conditions. For example, H-NOX proteins can cross the
blood-brain bather and accumulate in brain tumors such as gliomas.
As such, H-NOX proteins can be used to deliver oxygen to sensitize
hypoxic tumors to anticancer therapies such as radiation therapy.
The invention includes methods of monitoring the oxygenation of
tumors (e.g. brain tumors) which allows optimization of therapeutic
efficacy for the treatment of hypoxic tumors.
[0059] In some aspects, the invention provides method of treating a
hypoxic brain tumor in an individual where an effective amount of
an H-NOX protein is administered to the individual to deliver
oxygen to the tumor, determining the level of hypoxia in the brain
tumor following administration of the H-NOX protein, and then
administering an effective amount of radiation to the individual
where the tumor hypoxia has decreased as a result of tumor
oxygenation by the H-NOX protein.
[0060] In some aspects, the invention provides methods of
optimizing therapeutic efficacy for treatment of a hypoxic brain
cancer in an individual where H-NOX is administered to the
individual to deliver O.sub.2 to the tumor, the level of hypoxia of
the tumor is measured one or more times after administration of the
H-NOX proteins such that radiation therapy may be administered to
the individual when tumor hypoxia is reduced compared to the level
of hypoxia prior to H-NOX administration.
[0061] In some aspects, the invention provides methods of
monitoring the efficacy of delivery of O.sub.2 to hypoxic brain
tumor by an H-NOX protein in an individual wherein an effective
amount of H-NOX protein is administered to the individual, the
level of hypoxia in the tumor is measured at one or more time
points after administration of the H-NOX protein, wherein a
reduction of tumor hypoxia compared to the level of hypoxia in the
tumor prior to administration of H-NOX indicates effective delivery
of O.sub.2 to the brain tumor.
[0062] In some aspects, the invention provides methods of
monitoring responsiveness or lack of responsiveness to treatment
with an H-NOX in an individual suffering from a brain tumor
comprising measuring the hypoxic state of the tumor following H-NOX
administration, wherein responsiveness is indicated by a reduction
in tumor hypoxia.
[0063] In some aspects, the invention provides methods of
identifying an individual with a brain tumor who is more likely to
exhibit benefit from a therapy. The method includes determining the
hypoxia level of the tumor, administering H-NOX to the individual
to deliver oxygen to the tumor, and measuring the level of hypoxia
of the tumor. A 5% decrease in hypoxia indicates the individual is
more likely to exhibit benefit from radiation treatment.
DEFINITIONS
[0064] 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.
[0065] For use herein, unless clearly indicated otherwise, use of
the terms "a", "an," and the like refers to one or more.
[0066] 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.
[0067] 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."
[0068] It is understood that aspect and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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.
[0077] 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%.
[0078] 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%.
[0079] 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.
[0080] 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.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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.
[0092] 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
[0093] 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.
[0094] 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.
[0095] 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.
[0096] 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".
[0097] 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.
[0098] A "disease" or "disorder" as used herein refers to a
condition where treatment is needed.
[0099] The term "cancer" refers to a malignant proliferative
disorder associated with uncontrolled cell proliferation,
unrestrained cell growth, and decreased cell death via
apoptosis.
[0100] 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.
[0101] 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, any one or more of: alleviation of one or more
symptoms, diminishment of extent of disease, preventing or delaying
spread (e.g., metastasis, for example metastasis to the lung or to
the lymph node) of disease, preventing or delaying recurrence of
disease, delay or slowing of disease progression, amelioration of
the disease state, inhibiting the disease or progression of the
disease, inhibiting or slowing the disease or its progression,
arresting its development, and remission (whether partial or
total). 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.
[0102] 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.
[0103] 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%.
[0104] 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.
[0105] 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.
[0106] "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.
[0107] 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.
[0108] 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. A therapeutically effective amount also
encompasses an amount sufficient to confer benefit, e.g., clinical
benefit.
[0109] 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.
[0110] Responsiveness of a patient can be assessed using any
endpoint indicating a benefit to the patient, including, without
limitation, (1) inhibition, to some extent, of disease progression,
including slowing down and complete arrest; (2) reduction in lesion
size; (3) inhibition (i.e., reduction, slowing down or complete
stopping) of disease cell infiltration into adjacent peripheral
organs and/or tissues; (4) inhibition (i.e. reduction, slowing down
or complete stopping) of disease spread; (5) relief, to some
extent, of one or more symptoms associated with the disorder; (6)
increase in the length of disease-free presentation following
treatment; and/or (8) decreased mortality at a given point of time
following treatment. In the case of H-NOX mediated oxygenation of
hypoxic tissue (e.g. a hypoxic tumor), responsiveness can be
assessed by oxygenation of the tissue or by a reduction in the
hypoxia of the tissue. In some embodiments, any reduction in tissue
hypoxia indicates responsiveness to H-NOX treatment. In some
embodiments, a reduction in tissue hypoxia of about 5% or greater
indicates responsiveness to H-NOX treatment.
[0111] The term "benefit" is used in the broadest sense and refers
to any desirable effect and specifically includes clinical benefit
as defined herein. In some embodiments, tumor oxygenation or a
reduction in tumor hypoxia indicates benefit.
[0112] Clinical benefit can be measured by assessing various
endpoints, e.g., inhibition, to some extent, of disease
progression, including slowing down and complete arrest; reduction
in the number of disease episodes and/or symptoms; reduction in
lesion size; inhibition (i.e., reduction, slowing down or complete
stopping) of disease cell infiltration into adjacent peripheral
organs and/or tissues; inhibition (i.e. reduction, slowing down or
complete stopping) of disease spread; relief, to some extent, of
one or more symptoms associated with the disorder; increase in the
length of disease-free presentation following treatment, e.g.,
progression-free survival; increased overall survival; higher
response rate; and/or decreased mortality at a given point of time
following treatment. In the case of H-NOX mediated oxygenation of
hypoxic tissue (e.g. a hypoxic tumor), clinical benefit can be
assessed by oxygenation of the tissue or by a reduction in the
hypoxia of the tissue. In some embodiments, any reduction in tissue
hypoxia indicates benefit of H-NOX treatment. In some embodiments,
a reduction in tissue hypoxia of about 5% or greater indicates
benefit of H-NOX treatment.
[0113] 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.
[0114] 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.
[0115] A "sterile" formulation is aseptic or essentially free from
living microorganisms and their spores.
[0116] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive or sequential administration in any order.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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.
[0122] As used herein, "HIF-1.alpha." refers to the alpha subunit
of hypoxia inducible factor1. Examples of HIF-1.alpha. include but
are not limited to human HIF-1.alpha. (NCBI RefSeq NP.sub.--001521,
NP.sub.--851397, NM.sub.--181054, and NM.sub.--001530), murine
HIF-1.alpha. (NCBI RefSeq NP.sub.--034561 and NM.sub.--010431), rat
HIF-1.alpha. (NCBI RefSeq NP.sub.--077335 and NM.sub.--024359),
canine HIF-1.alpha. (NCBI RefSeq XP.sub.--865494, XP.sub.--865463,
XM.sub.--860350, and XM.sub.--860370), chimpanzee HIF-1.alpha.
(NCBI RefSeq XP.sub.--001168834, XP.sub.--001168628,
XM.sub.--001168972, and XM.sub.--001168811), monkey HIF-1.alpha.
(NCBI RefSeq XP.sub.--002805105, XP.sub.--002805107,
XM.sub.--002805059, and XM.sub.--002805060), and the like.
[0123] As used herein, "HIF-2.alpha." or "endothelial PAS domain
protein 1" refers to the alpha subunit of hypoxia inducible factor
2. Examples of HIF-2.alpha. include but are not limited to human
HIF-2.alpha. (NCBI RefSeq NP.sub.--001421 and NM.sub.--001430),
murine HIF-2.alpha. (NCBI RefSeq NP.sub.--034267 and
NM.sub.--010137), and rat HIF-2.alpha. (NCBI RefSeq NP.sub.--075578
and NM.sub.--023090), and the like.
[0124] As used herein, "HIF-3.alpha." refers to the alpha subunit
of hypoxia inducible factor 3. Examples of HIF-3.alpha. include but
are not limited to human HIF-3.alpha. (NCBI RefSeq NP.sub.--690009,
NP.sub.--690008, NM.sub.--152796, NM.sub.--152795), murine
HIF-3.alpha. (NCBI RefSeq NP.sub.--001156422, NP.sub.--058564,
NM.sub.--001162950, NM.sub.--016868), rat HIF-3.alpha. (NCBI RefSeq
NP.sub.--071973 and NM.sub.--022528), chimpanzee HIF-3.alpha. (NCBI
RefSeq XP.sub.--512767, XP.sub.--001167388, XM.sub.--001167499, and
XM.sub.--001167448), canine HIF-3.alpha. (NCBI RefSeq
XP.sub.--533636 and XM.sub.--533636), and the like.
[0125] As used herein, "glut1" or "solute carrier family 2
(facilitated glucose transporter), member 1" refers to the glucose
transporter type 1. Examples of glut1 include but are not limited
to human glut1 (NCBI RefSeq NP.sub.--006507 and NM.sub.--006516),
murine glut1 (NCBI RefSeq NP.sub.--035530 and NM.sub.--011400), rat
glut1 (NCBI RefSeq NP.sub.--620182 and NM.sub.--138827), and the
like.
[0126] As used herein, "LDHA" refers to Lactose DeHydrogenase A.
Examples of LDHA include but are not limited to human LDHA (NCBI
RefSeq NP.sub.--001158888, NP.sub.--001158887, NM.sub.--001135239,
NM.sub.--001165416), murine LDHA (NCBI RefSeq NP.sub.--001129541,
NP.sub.--034829 and NM.sub.--001136069, NM.sub.--010699), rat LDHA
(NCBI RefSeq NP.sub.--058721 and NM.sub.--017025), chimpanzee LDHA
(NCBI RefSeq NP.sub.--001029268 and NM.sub.--001034096), canine
LDHA (NCBI RefSeq XP.sub.--534084, XP.sub.--865353,
XM.sub.--534084, and XM.sub.--860260), and the like.
[0127] As used herein, "CA-9", "caix" or "carbonic anhydrase IX"
refers to an isoform of carbonic anhydrase. Examples of carbonic
anhydrase IX include but are not limited to human carbonic
anhydrase IX (NCBI RefSeq NP.sub.--001207 and NM.sub.--001216),
murine carbonic anhydrase IX (NCBI RefSeq NP.sub.--647466 and
NM.sub.--139305), rat carbonic anhydrase IX (NCBI RefSeq
NP.sub.--001101426 and NM.sub.--001107956), chimpanzee carbonic
anhydrase IX (NCBI RefSeq XP.sub.--001167245, XP.sub.--528593,
XM.sub.--528593, and XM.sub.--001167245), canine carbonic anhydrase
IX (NCBI RefSeq XP.sub.--854749, NP.sub.--001138646,
NM.sub.--001145174, and XM.sub.--849656), and the like.
[0128] The term "biomarker" as used herein refers to an indicator,
e.g., predictive, diagnostic, and/or prognostic, which can be
detected in a sample. The biomarker may serve as an indicator of a
particular subtype of a disease or disorder (e.g., cancer)
characterized by certain, molecular, pathological, histological,
and/or clinical features. For example, biomarkers for tumor hypoxia
include but are not limited to .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, .sup.19F magnetic resonance imaging of hexafluorobenzene
(C6F6) uptake, .sup.1H MRI of hexamethyldisiloxane uptake, tumor
HIF-1.alpha. expression, tumor Glut-1 expression, tumor LDHA
expression, tumor carbonic anhydrase IX (CA-9) expression, or
lactate and/or pyruvate levels.
[0129] The "amount" or "level" of a biomarker associated with an
increased clinical benefit to an individual is a detectable level
in a biological sample. These can be measured by methods known to
one skilled in the art and also disclosed herein. The expression
level or amount of biomarker assessed can be used to determine the
response to the treatment.
[0130] The terms "level of expression" or "expression level" in
general are used interchangeably and generally refer to the amount
of a biomarker in a biological sample. "Expression" generally
refers to the process by which information (e.g., gene-encoded
and/or epigenetic) is converted into the structures present and
operating in the cell. Therefore, as used herein, "expression" may
refer to transcription into a polynucleotide, translation into a
polypeptide, or even polynucleotide and/or polypeptide
modifications (e.g., posttranslational modification of a
polypeptide). Fragments of the transcribed polynucleotide, the
translated polypeptide, or polynucleotide and/or polypeptide
modifications (e.g., posttranslational modification of a
polypeptide) shall also be regarded as expressed whether they
originate from a transcript generated by alternative splicing or a
degraded transcript, or from a post-translational processing of the
polypeptide, e.g., by proteolysis. "Expressed genes" include those
that are transcribed into a polynucleotide as mRNA and then
translated into a polypeptide, and also those that are transcribed
into RNA but not translated into a polypeptide (for example,
transfer and ribosomal RNAs).
[0131] "Elevated expression," "elevated expression levels,"
"elevated levels" and "overexpressed" refers to an increased
expression or increased levels of a biomarker in an individual
relative to a control, such as an individual or individuals who are
not suffering from the disease or disorder (e.g., cancer) or an
internal control (e.g., housekeeping biomarker). In some examples,
elevated expression or overexpression is the result of gene
amplification.
[0132] "Reduced expression," "reduced expression levels," or
"reduced levels" refers to a decrease expression or decreased
levels of a biomarker in an individual relative to a control, such
as an individual or individuals who are not suffering from the
disease or disorder (e.g., cancer) or an internal control (e.g.,
housekeeping biomarker).
[0133] The term "housekeeping biomarker" refers to a biomarker or
group of biomarkers (e.g., polynucleotides and/or polypeptides)
which are typically similarly present in all cell types. In some
embodiments, the housekeeping biomarker is a "housekeeping gene." A
"housekeeping gene" refers herein to a gene or group of genes which
encode proteins whose activities are essential for the maintenance
of cell function and which are typically similarly present in all
cell types.
H-NOX Proteins
Overview of H-NOX Protein Family
[0134] 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 O.sub.2, 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 .beta.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
[0135] 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. In some embodiments the trimeric H-NOX domain comprises
three T. tengcongensis W9F/L144F H-NOX domains
[0136] 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, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID
NO:26 or SEQ ID NO:28.
[0137] 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, 125 kDal, to about 150 kDal.
[0138] 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
[0139] 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 .beta.1 subunit). 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 wild-type non-mammalian vertebrate H-NOX proteins,
e.g., X. laevis, O. latipes, O. curivatus, and F. rubripes.
Examples of non-mammalian wild-type NO-binding H-NOX proteins
include wild-type H-NOX proteins of D. melanogaster, A. gambiae,
and M. sexta; examples of non-mammalian wild-type O.sub.2-binding
H-NOX proteins include wild-type H-NOX proteins of C. elegans
gcy-31, gcy-32, gcy-33, gcy-34, gcy-35, gcy-36, and gcy-37; D.
melanogaster CG14885, CG14886, and CG4154; and M. sexta beta-3;
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.
acetobutylicum.
[0140] NCBI Accession numbers for exemplary H-NOX proteins include
the following: Homo sapiens .beta.1 [gi:2746083], Rattus norvegicus
.beta.1 [gi:27127318], Drosophila melangaster .beta.1 [gi:861203],
Drosophila melangaster CG14885-PA [gi:23171476], Caenorhabditis
elegans GCY-35 [gi:52782806], Nostoc punctiforme [gi:23129606],
Caulobacter crescentus [gi:16127222], Shewanella oneidensis
[gi:24373702], Legionella pneumophila (ORF 2) [CUCGC.sub.--272624],
Clostridium acetobutylicum [gi:15896488], and Thermoanaerobacter
tengcongensis [gi:20807169]. Canis lupus H-NOX is provided by
GenBank accession DQ008576. Nucleic acid and amino acid sequences
of exemplary H-NOX proteins and domains are provided in FIG.
30.
[0141] Exemplary H-NOX protein also include the following H-NOX
proteins that are listed by their gene name, followed by their
species abbreviation and Genbank Identifiers (such as the following
protein sequences available as of May 21, 2006; May 22, 2006; May
21, 2007; or May 22, 2007, which are each hereby incorporated by
reference in their entireties): Npun5905_Npu.sub.--23129606,
alr2278_Ana.sub.--17229770, SO2144_Sone.sub.--24373702,
Mdeg1343_Mde.sub.--23027521, VCA0720_Vch.sub.--15601476,
CC2992_Ccr.sub.--16127222, Rsph2043_Rhsp.sub.--22958463
(gi:46192757), Mmc10739_Mcsp.sub.--22999020,
Tar4_Tte.sub.--20807169, Ddes2822_Dde.sub.--23475919,
CAC3243_Cac.sub.--15896488, gcy-31_Ce.sub.--17568389,
CG14885_Dm.sub.--24647455, GUCY1B3_Hs.sub.--4504215,
HpGCS-beta1_Hpul.sub.--14245738, Gycbeta100B_Dm.sub.--24651577,
CG4154_Dm.sub.--24646993 (gi:NP.sub.--650424.2, gi:62484298),
gcy-32_Ce.sub.--13539160, gcy-36_Ce.sub.--17568391 (gi:32566352,
gi:86564713), gcy-35_Ce-17507861 (gi:71990146),
gcy-37_Ce.sub.--17540904 (gi:71985505), GCY1a3_Hs.sub.--20535603,
GCY1a2-Hs.sub.--899477, or GYCa-99B_Dm.sub.--729270 (gi:68067738)
(Lakshminarayan et al. (2003). BMG Genomics 4:5-13). The species
abbreviations used in these names include Ana--Anabaena Sp;
Ccr--Caulobacter crescentus; Cac--Clostridium acetobutylicum;
Dde--Desulfovibrio desulfuricans; Mcsp--Magnetococcus sp.;
Mde--Microbulbifer degradans; Npu--Nostoc punctiforme;
Rhsp--Rhodobacter sphaeroides; Sone--Shewanella oneidensis;
Tte--Thermoanaerobacter tengcongensis; Vch--Vibrio cholerae;
Ce--Caenorhabditis elegans; Dm--Drosophila melanogaster;
Hpul--Hemicentrotus pulcherrimus; Hs--Homo sapiens.
[0142] Other exemplary H-NOX proteins include the following H-NOX
proteins that are listed by their organism name and Pfam database
accession number (such as the following protein sequences available
as of May 21, 2006; May 22, 2006; May 17, 2007; May 21, 2007; or
May 22, 2007, which are each hereby incorporated by reference in
their entireties): Caenorhabditis briggsae Q622M5_CAEBR,
Caenorhabditis briggsae Q61P44_CAEBR, Caenorhabditis briggsae
Q61R54_CAEBR, Caenorhabditis briggsae Q61V90_CAEBR, Caenorhabditis
briggsae Q61A94_CAEBR, Caenorhabditis briggsae Q60TP4_CAEBR,
Caenorhabditis briggsae Q60M10_CAEBR, Caenorhabditis elegans
GCY37_CAEEL, Caenorhabditis elegans GCY31_CAEEL, Caenorhabditis
elegans GCY36_CAEEL, Caenorhabditis elegans GCY32_CAEEL,
Caenorhabditis elegans GCY35_CAEEL, Caenorhabditis elegans
GCY34_CAEEL, Caenorhabditis elegans GCY33_CAEEL, Oryzias curvinotus
Q7T040_ORYCU, Oryzias curvinotus Q75WFO_ORYCU, Oryzias latipes
P79998_ORYLA, Oryzias latipes Q7ZSZ5_ORYLA, Tetraodon nigroviridis
Q4SW38_TETNG, Tetraodon nigroviridis Q4RZ94_TETNG, Tetraodon
nigroviridis Q4S6K5_TETNG, Fugu rubripes Q90VY5_FUGRU, Xenopus
laevis Q6INK9_XENLA, Homo sapiens Q5T8J7_HUMAN, Homo sapiens
GCYA2_HUMAN, Homo sapiens GCYB2_HUMAN, Homo sapiens GCYB1_HUMAN,
Gorilla gorilla Q9N193.sub.--9 PRIM, Pongo pygmaeus Q5RAN8_PONPY,
Pan troglodytes Q9N192_PANTR, Macaca mulatta Q9N194_MACMU,
Hylobates lar Q9N191_HYLLA, Mus musculus Q8BXH3_MOUSE, Mus musculus
GCYB1_MOUSE, Mus musculus Q3UTI4_MOUSE, Mus musculus Q3UH83_MOUSE,
Mus musculus Q6XE41_MOUSE, Mus musculus Q80YP4_MOUSE, Rattus
norvegicus Q80WX7_RAT, Rattus norvegicus Q80WX8_RAT, Rattus
norvegicus Q920Q1_RAT, Rattus norvegicus Q54A43_RAT, Rattus
norvegicus Q80WY0_RAT, Rattus norvegicus Q80WY4_RAT, Rattus
norvegicus Q8CH85_RAT, Rattus norvegicus Q80WY5_RAT, Rattus
norvegicus GCYB1_RAT, Rattus norvegicus Q8CH90_RAT, Rattus
norvegicus Q91XJ7_RAT, Rattus norvegicus Q80WX9_RAT, Rattus
norvegicus GCYB2_RAT, Rattus norvegicus GCYA2_RAT, Canis familiaris
Q4ZHR9_CANFA, Bos taurus GCYB1_BOVIN, Sus scrofa Q4ZHR7_PIG,
Gryllus bimaculatus Q59HN5_GRYBI, Manduca sexta 077106_MANSE,
Manduca sexta 076340_MANSE, Apis mellifera Q5UAFO_APIME, Apis
mellifera Q5FANO_APIME, Apis mellifera Q6L5L6_APIME, Anopheles
gambiae str PEST Q7PYK9_ANOGA, Anopheles gambiae str PEST
Q7Q9W6_ANOGA, Anopheles gambiae str PEST Q7QF31_ANOGA, Anopheles
gambiae str PEST Q7PS01_ANOGA, Anopheles gambiae str PEST
Q7PFY2_ANOGA, Anopheles gambiae Q7KQ93_ANOGA, Drosophila
melanogaster Q24086_DROME, Drosophila melanogaster GCYH_DROME,
Drosophila melanogaster GCYH_DROME, Drosophila melanogaster
GCYDA_DROME, Drosophila melanogaster GCYDB_DROME, Drosophila
melanogaster Q9VA09_DROME, Drosophila pseudoobscura Q29CE1_DROPS,
Drosophila pseudoobscura Q296C7_DROPS, Drosophila pseudoobscura
Q296C8_DROPS, Drosophila pseudoobscura Q29BU7_DROPS, Aplysia
californica Q7YWK7_APLCA, Hemicentrotus pulcherrimus Q95NK5_HEMPU,
Chlamydomonas reinhardtii, Q5YLC2_CHLRE, Anabaena sp Q8YUQ7_ANASP,
Flavobacteria bacterium BBFL7 Q26GR8.sub.--9 BACT, Psychroflexus
torquis ATCC 700755 Q1VQE5.sub.--9 FLAO, marine gamma
proteobacterium HTCC2207 Q1YPJ5.sub.--9 GAMM, marine gamma
proteobacterium HTCC2207 Q1YTK4.sub.--9 GAMM, Caulobacter
crescentus Q9A451_CAUCR, Acidiphilium cryptum JF-5 Q2DG60_ACICY,
Rhodobacter sphaeroides Q3JOU9_RHOS4, Silicibacter pomeroyi
Q5LPV1_SILPO, Paracoccus denitrificans PD1222, Q3PC67_PARDE,
Silicibacter sp TM1040 Q3QNY2.sub.--9 RHOB, Jannaschia sp
Q28ML8_JANSC, Magnetococcus sp MC-1 Q3XT27.sub.--9 PROT, Legionella
pneumophila Q5WXPO_LEGPL, Legionella pneumophila Q5WTZ5_LEGPL,
Legionella pneumophila Q5X268_LEGPA, Legionella pneumophila
Q5X2R2_LEGPA, Legionella pneumophila subsp pneumophila
Q5ZWM9_LEGPH, Legionella pneumophila subsp pneumophila
Q5ZSQ8_LEGPH, Colwellia psychrerythraea Q47Y43_COLP3,
Pseudoalteromonas atlantica T6c Q3CSZ5_ALTAT, Shewanella oneidensis
Q8EF49_SHEON, Saccharophagus degradans Q21E20_SACD2, Saccharophagus
degradans Q21ER7_SACD2, Vibrio angustum S14 Q1ZWE5.sub.--9 VIBR,
Vibrio vulnificus Q8DAE2_VIBVU, Vibrio alginolyticus 12G01
Q1VCP6_VIBAL, Vibrio sp DAT722 Q2FA22.sub.--9 VIBR, Vibrio
parahaemolyticus Q87NJ1_VIBPA, Vibrio fischeri Q5E1F5_VIBF1, Vibrio
vulnificus Q7MJS8_VIBVY, Photobacterium sp SKA34 Q2C6Z5.sub.--9
GAMM, Hahella chejuensis Q2SFY7_HAHCH, Oceanospirillum sp MED92
Q2BKV0.sub.--9 GAMM, Oceanobacter sp RED65 Q1NO35.sub.--9 GAMM,
Desulfovibrio desulfuricans Q310U7_DESDG, Halothermothrix orenii H
168 Q2AIW5.sub.--9 FIRM, Thermoanaerobacter tengcongensis
Q8RBX6_THETN, Caldicellulosiruptor saccharolyticus DSM 8903
Q2ZH17_CALSA, Clostridium acetobutylicum Q97E73_CLOAB, Alkaliphilus
metalliredigenes QYMF Q3C763.sub.--9 CLOT, Clostridium tetani
Q899J9_CLOTE, and Clostridium beijerincki NCIMB 8052 Q2WVN0_CLOBE.
These sequences are predicted to encode H-NOX proteins based on the
identification of these proteins as belonging to the H-NOX protein
family using the Pfam database as described herein.
[0143] 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.
[0144] 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.
[0145] 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
[0146] 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).
[0147] 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.
[0148] 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.
[0149] 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 F4L H7G A8E L9W Q30G E33P N61G
C78H A109F I145Y I145H K151E I157F E183F
[0150] 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 L9X F70X M73X F77X C78X I145X
I149X
[0151] 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 an 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] The residue number for a mutation indicates the position in
the sequence of the particular H-NOX protein being described. For
example, T. tengcongensis ISA 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.
[0157] 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
[0158] 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).
[0159] 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.
Polymerization Domains
[0160] 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.
[0161] 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
[0162] 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 provided by SEQ ID NO:4.
[0163] The isolated foldon domain folds into a single
.beta.-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,
6.0M 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.
[0164] 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.
[0165] 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
[0166] 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.
[0167] 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).
[0168] 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.
[0169] 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.
[0170] 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.
[0171] In some embodiments the recombinant monomeric H-NOX protein
comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ
ID NO:10 or SEQ ID NO:12.
Characteristics of Wild-Type and Mutant H-NOX Proteins
[0172] 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.
[0173] 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.-1, about 1.0 to
about 16.0 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 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.).
[0174] 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.
[0175] 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 2 .mu.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.
[0176] 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.).
[0177] 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.
[0178] 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.
[0179] 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.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, 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.
[0180] 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
O.sub.2 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.
[0181] 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.
[0182] 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).
[0183] 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.).
[0184] 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
[0185] 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
[0186] 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)
[0187] 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 Fe.sup.11--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)
[0188] 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
[0189] 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.
Calculated K.sub.D
[0190] 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
[0191] 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
[0192] 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
[0193] 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.
[0194] 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
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] In some embodiments, the nucleic acid comprises the nucleic
acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or
SEQ ID NO:11.
[0204] 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.
[0205] 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, SEQ ID NO:7, SEQ ID
NO:9, or SEQ ID NO:11.
Formulations of H-NOX Proteins
[0206] 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.
[0207] 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.
[0208] 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, 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] An exemplary dose of hemoglobin as a blood substitute is
from about 10 mg to about 5 grams or more of extracellular
hemoglobin per kilogram of patient body weight. Thus, 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.
[0213] 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.
[0214] 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.
Therapeutic Applications of H-NOX Proteins
[0215] Any of the wild-type or mutant H-NOX proteins, including
polymeric H-NOX proteins, or pharmaceutical compositions described
herein may be used in therapeutic applications.
[0216] Particular H-NOX proteins, including polymeric H-NOX
proteins, can be selected for such applications based on the
desired 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 for the particular
indication being treated. H-NOX proteins can be used to treat
cardiovascular disease, neurological disease, tumor hypoxia, loss
of blood, or wounds. For example, an O.sub.2-binding H-NOX protein
can be used in most situations where red blood cells or plasma
expanders are currently utilized.
[0217] H-NOX proteins, including polymeric H-NOX proteins, can be
used as an adjunct with radiation or chemotherapy for the treatment
of cancer. In some embodiments, an H-NOX protein is used as a
radiation therapy adjuvant in solid tumors (e.g., individuals with
poor pre-metastatic prognoses) or as a PDT therapy adjuvant in
surface tumors (e.g., colon, lung, or skin cancer, or cancer in
another accessible surface or location). H-NOX proteins can be used
to treat anemia by providing additional oxygen-carrying capacity in
a patient who is suffering from anemia. Exemplary neurological
indications include ischemic stroke, traumatic brain injury, and
spinal cord injury. The methods and compositions are applicable to
both acute (providing rapid oxygen to tissues or a specific site,
e.g. acute myocardial infarction, acute local or systemic tissue
oxygenation, or blood transfusion), and chronic situations (e.g.
post-acute recovery from cardiac infarction).
[0218] In a particular aspect, the invention provides methods of
using H-NOX proteins to deliver O.sub.2 to brain tumors (e.g. a
glioblastoma). In some embodiments, the administration of H-NOX is
used as an adjunct to radiation therapy or chemotherapy. In some
embodiments, the invention provides methods to treat a brain cancer
(e.g. a glioblastoma) in an individual by administering an
effective amount of an H-NOX protein and administering an effective
amount of radiation to the individual. In some embodiments, the
invention provides methods to reduce brain tumor growth (e.g.
glioblastoma growth) in an individual by administering an effective
amount of an H-NOX protein and administering an effective amount of
radiation to the individual. In some embodiments, the H-NOX protein
is a polymeric H-NOX protein (e.g. a trimeric H-NOX protein). In
some embodiments, the polymeric H-NOX protein comprises one or more
H-NOX domains comprising a mutation at a position corresponding to
L144 of T. tengcongensis H-NOX. In some embodiments, the polymeric
H-NOX protein comprises one or more H-NOX domains comprising a
mutation corresponding to a L144F mutation of T. tengcongensis
H-NOX. In some embodiments, the polymeric H-NOX protein comprises
one or more H-NOX domains comprising a mutation at positions
corresponding to W9 and L144 of T. tengcongensis H-NOX. In some
embodiments, the polymeric H-NOX protein comprises one or more
H-NOX domains comprising mutations corresponding to a W9F/L144F
mutation of T. tengcongensis H-NOX. In some embodiments, the H-NOX
domain is a human H-NOX domain. In some embodiments, the H-NOX
domain is a canine H-NOX domain. In some embodiments, the polymeric
H-NOX protein comprises a L144F T. tengcongensis H-NOX domain. In
some embodiments, the polymeric H-NOX protein comprises a W9F/L144F
T. tengcongensis H-NOX domain and a foldon domain.
[0219] In various embodiments, the invention features a method of
delivering O.sub.2 to an individual (e.g., a mammal, such as a
primate (e.g., a human, a monkey, a gorilla, an ape, a lemur,
etc.), a bovine, an equine, a porcine, a canine, or a feline) by
administering to an individual in need thereof a wild-type or
mutant H-NOX protein, including a polymeric H-NOX protein in an
amount sufficient to deliver O.sub.2 to the individual. In some
embodiments, the invention provides methods of carrying or
delivering blood gas to an individual such as a mammal, comprising
the step of delivering (e.g., transfusing, etc.) to the blood of
the individual (e.g., a mammal) one or more of H-NOX compositions.
Methods for delivering O.sub.2 carriers to blood or tissues (e.g.,
mammalian blood or tissues) are known in the art. In various
embodiments, the H-NOX protein is an apoprotein that is capable of
binding heme or is a holoprotein with heme bound. The H-NOX protein
may or may not have heme bound prior to the administration of the
H-NOX protein to the individual. In some embodiments, O.sub.2 is
bound to the H-NOX protein before it is delivered to the
individual. In other embodiments, O.sub.2 is not bound to the H-NOX
protein prior to the administration of the protein to the
individual, and the H-NOX protein transports O.sub.2 from one
location in the individual to another location in the
individual.
[0220] Wild-type and mutant H-NOX proteins, including polymeric
H-NOX proteins, with a relatively low K.sub.D for O.sub.2 (such as
less than about 80 nM or less than about 50 nM) are expected to be
particularly useful to treat tissues with low oxygen tension (such
as tumors, some wounds, or other areas where the oxygen tension is
very low, such as a p50 below 1 mm Hg). The high affinity of such
H-NOX proteins for O.sub.2 may increase the length of time the
O.sub.2 remains bound to the H-NOX protein, thereby reducing the
amount of O.sub.2 that is released before the H-NOX protein reaches
the tissue to be treated.
[0221] In some embodiments for the direct delivery of an H-NOX
protein with bound O.sub.2 to a particular site in the body (such
as a glioblastoma), the k.sub.off for O.sub.2 is more important
than the K.sub.D value because O.sub.2 is already bound to the
protein (making the k.sub.on less important) and oxygen needs to be
released at or near a particular site in the body (at a rate
influenced by the k.sub.off). In some embodiments, the k.sub.off
may also be important when H-NOX proteins are in the presence of
red cells in the circulation, where they facilitate diffusion of
O.sub.2 from red cells, and perhaps prolonging the ability of
diluted red cells to transport O.sub.2 to further points in the
vasculature.
[0222] In some embodiments for the delivery of an H-NOX protein
that circulates in the bloodstream of an individual, the H-NOX
protein binds O.sub.2 in the lungs and releases O.sub.2 at one or
more other sites in the body. For some of these applications, the
K.sub.D value is more important than the k.sub.off since O.sub.2
binding is at or near equilibrium. In some embodiments for extreme
hemodilution, the K.sub.D more important than the k.sub.off when
the H-NOX protein is the primary O.sub.2 carrier because the H-NOX
protein will bind and release O.sub.2 continually as it travels
through the circulation. Since hemoglobin has a p50 of 14 mm Hg,
red cells (which act like capacitors) have a p50 of .about.30 mm
Hg, and HBOCs have been developed with ranges between 5 mm Hg and
90 mm Hg, the optimal K.sub.D range for H-NOX proteins may
therefore be between .about.2 mm Hg to .about.100 mm Hg for some
applications.
[0223] Polymeric H-NOX proteins can also be used for imaging. In
particular, light imaging (e.g., optical coherence tomography; see,
for example, Villard, J. W. (2002). Circulation 105:1843-1849,
which is incorporated by reference in its entirety particularly
with respect to optical coherence tomography) is obfuscated by
erythrocytes. Perfusion with an H-NOX solution allows for clearer
images of the circulation and vessel walls because the H-NOX
protein is much smaller than erythrocytes.
[0224] H-NOX proteins, including polymeric H-NOX proteins, and
pharmaceutical compositions of the invention can be administered to
an individual by any conventional means such as by oral, topical,
intraocular, intrathecal, intrapulmonary, intra-tracheal, or
aerosol administration; by transdermal or mucus membrane
adsorption; or by injection (e.g., subcutaneous, intravenous,
intra-arterial, intravesicular, or intramuscular injection). H-NOX
proteins may also be included in large volume parenteral solutions
for use as blood substitutes. In exemplary embodiments, the H-NOX
protein is administered to the blood (e.g., administration to a
blood vessel such as a vein, artery, or capillary), a wound, a
tumor, a hypoxic tissue, or a hypoxic organ of the individual.
[0225] In some embodiments, a sustained continuous release
formulation of the composition is used. Administration of an H-NOX
protein can occur, e.g., for a period of seconds to hours depending
on the purpose of the administration. For example, as a blood
delivery vehicle, an exemplary time course of administration is as
rapid as possible. Other exemplary time courses include about any
of 10, 20, 30, 40, 60, 90, or 120 minutes. Exemplary infusion rates
for H-NOX solutions as blood replacements are from about 30 mL/hour
to about 13,260 mL/hour, such as about 100 mL/hour to about 3,000
mL/hour. An exemplary total dose of H-NOX protein is about 900
mg/kg administered over 20 minutes at 13,260 mL/hour. An exemplary
total dose of H-NOX protein for a swine is about 18.9 grams.
[0226] 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, an H-NOX protein is administered at least 2, 3,
4, or 6 times a day. The H-NOX protein can be administered, e.g.,
over a period of a few days or weeks. In some embodiments, the
H-NOX protein 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.
[0227] In some embodiments of the invention, the H-NOX protein
(e.g. a polymeric H-NOX protein) is used as an adjunct to radiation
therapy or chemotherapy. For example, for the treatment of
glioblastoma. In some embodiments, the H-NOX is administered to the
individual any of at least 1, 2, 3, 4, 5 or 6 hours before
administration of the radiation or chemotherapy. 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 H-NOX administration and radiation
administration is repeated any one of one, two, three, four, five
or six times. In some embodiments, the cycle of H-NOX
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 admiration of H-NOX
and radiation therapy is used in conjunction with another therapy;
for example, a chemotherapy.
[0228] As noted above, the selection of dosage amounts for H-NOX
proteins depends upon the dosage form utilized, the frequency and
number of administrations, the condition being treated, and the
particular purpose to be achieved according to the determination of
the ordinarily skilled artisan in the field. In some embodiments,
an effective amount of an H-NOX protein for administration to human
is between a few grams to over 350 grams.
[0229] In some embodiments, two or more different H-NOX proteins
are administered simultaneously, sequentially, or concurrently. In
some embodiments, another compound or therapy useful for the
delivery of O.sub.2 is administered simultaneously, sequentially,
or concurrently with the administration of one or more H-NOX
proteins.
[0230] Other exemplary therapeutic applications for which H-NOX
proteins can be used 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
therapeutic applications for O.sub.2 carriers.
Biomarkers to Monitor H-NOX Mediated Oxygenation
[0231] In some aspects, the invention provides methods for
monitoring oxygenation of hypoxic tumors by H-NOX proteins. In some
embodiments, the invention provides methods of treating a hypoxic
brain tumor (e.g., a glioblastoma) in an individual comprising
administering an effective amount of an H-NOX protein, determining
the level of hypoxia in the brain tumor, and administering an
effective amount of radiation to the individual where the level of
tumor hypoxia is reduced following administration of the H-NOX
protein compared to the level of tumor hypoxia measured in the
tumor prior to H-NOX administration. In some embodiments, the level
of hypoxia in the brain tumor is determined prior to administration
of H-NOX. In some embodiments, radiation is administered to the
individual when the hypoxia of the tumor is reduced by at least
about any of 5%, 10%, 15%, 20%, 25%, 50%, 75% or 100%.
[0232] In some aspects, the invention provides methods for
optimizing therapeutic efficacy for treatment of hypoxic brain
tumor (e.g., a glioblastoma). An effective amount of an H-NOX
protein is administered to the individual and the level of hypoxia
in the brain tumor is determined one or more times following
administration of the H-NOX protein. An effective amount of
radiation is administered to the individual when the level of tumor
hypoxia is reduced following administration of the H-NOX protein
compared to the level of tumor hypoxia measured in the tumor prior
to H-NOX administration. In some embodiments, the level of hypoxia
in the brain tumor is determined prior to administration of H-NOX.
In some embodiments, radiation is administered to the individual
when the hypoxia of the tumor is reduced by at least about any of
5%, 10%, 15%, 20%, 25%, 50%, 75% or 100%. In some embodiments,
radiation is administered to the individual when the level of tumor
hypoxia is at or near a minimum following administration of H-NOX.
In some embodiments, the level of hypoxia of the brain tumor is
measured at one or more of about one hour, two hours, three hours,
four hours, five hours, six hours, seven hours, eight hours, nine
hours, ten hours, eleven hours, twelve hours, fourteen hours,
sixteen hours, eighteen hours, 24 hours, 36 hours, 48 hours, 60
hours or 72 hours after administration of H-NOX.
[0233] In some aspects, the invention provides methods to monitor
the efficacy of delivery of O.sub.2 to a hypoxic brain tumor (e.g.,
a glioblastoma) by an H-NOX protein. An H-NOX protein is
administered to the individual and the level of tumor hypoxia is
measured at one or more time points after administration of the
H-NOX protein. A reduction in tumor hypoxia following
administration of the H-NOX protein compared to the level of tumor
hypoxia measured in the tumor prior to H-NOX administration
indicates effective delivery of O.sub.2 to the brain tumor. In some
embodiments, the level of hypoxia in the brain tumor is determined
prior to administration of H-NOX. In some embodiments, the level of
hypoxia of the brain tumor is measured at one or more of about one
hour, two hours, three hours, four hours, five hours, six hours,
seven hours, eight hours, nine hours, ten hours, eleven hours,
twelve hours, fourteen hours, sixteen hours, eighteen hours, 24
hours, 36 hours, 48 hours, 60 hours or 72 hours after
administration of H-NOX. In some embodiments, a reduction in tumor
hypoxia by at least about any of 5%, 10%, 15%, 20%, 25%, 50% 75% or
100% indicates that the individual is suitable for administration
of anticancer therapy. In some embodiments, a reduction in tumor
hypoxia by at least about any of 5%, 10%, 15%, 20%, 25%, 50% 75% or
100% indicates that the individual is suitable for administration
of radiation therapy.
[0234] In some aspects, the invention provides methods to monitor
responsiveness or lack of responsiveness to treatment with an H-NOX
in an individual suffering from a brain tumor (e.g., a
glioblastoma). The hypoxic state of the tumor is measured following
H-NOX administration. Responsiveness is indicated by a reduction in
tumor hypoxia or oxygenation of the tumor. In some embodiments, the
level of tumor hypoxia is measured at one or more time points after
administration of the H-NOX protein. In some embodiments, the level
of hypoxia in the brain tumor is determined prior to administration
of H-NOX. In some embodiments, the level of hypoxia of the brain
tumor is measured at one or more of about one hour, two hours,
three hours, four hours, five hours, six hours, seven hours, eight
hours, nine hours, ten hours, eleven hours, twelve hours, fourteen
hours, sixteen hours, eighteen hours, 24 hours, 36 hours, 48 hours,
60 hours or 72 hours after administration of H-NOX. In some
embodiments, a reduction in tumor hypoxia by at least about any of
5%, 10%, 15%, 20%, 25%, 50% 75% or 100% indicates responsiveness to
treatment with an H-NOX.
[0235] In some aspects, the invention provides methods of
identifying an individual with a brain tumor (e.g., a glioblastoma)
who is more likely to exhibit benefit from a therapy comprising an
H-NOX protein. The hypoxia level of the tumor is determined An
effective amount of an H-NOX protein is administered to the
individual and the level of hypoxia in the brain tumor is
determined one or more times following administration of the H-NOX
protein. A decrease in tumor hypoxia by about 5% indicates that the
individual is more likely to exhibit benefit from H-NOX treatment
combined with radiation treatment. In some embodiments, the
individual is more likely to exhibit benefit from H-NOX treatment
combined with anticancer treatment when the hypoxia of the tumor is
reduced by at least about any of 5%, 10%, 15%, 20%, 25%, 50%, 75%
or 100%. In some embodiments, the individual is more likely to
exhibit benefit from H-NOX treatment combined with radiation
treatment when the hypoxia of the tumor is reduced by at least
about any of 5%, 10%, 15%, 20%, 25%, 50%, 75% or 100%. In some
embodiments, the level of hypoxia of the brain tumor is measured at
one or more of about one hour, two hours, three hours, four hours,
five hours, six hours, seven hours, eight hours, nine hours, ten
hours, eleven hours, twelve hours, fourteen hours, sixteen hours,
eighteen hours, 24 hours, 36 hours, 48 hours, 60 hours or 72 hours
after administration of H-NOX.
[0236] In some embodiments of the invention, the determination of
tumor hypoxia is repeated after about one or more of one week, two
weeks, three weeks, four weeks, five weeks, six weeks, two months,
three months, four months, five months, six months, of one year. In
some embodiments, administration of H-NOX is repeated if the tumor
is hypoxic. In some embodiments, the administration of H-NOX is
repeated if the tumor has increased hypoxia compared to the tumor
after an initial administration of H-NOX. In some embodiments, the
increased hypoxia is an increase by any of about 5%, 10%, 15%, 20%,
25%, 50%, 75%, 100% compared to the tumor after an initial
administration of H-NOX. In some embodiments, radiation is
administered to the individual one or more times after a repeat
administration of H-NOX. In some embodiments, radiation is
administered to the individual one or more times after a repeat
administration of H-NOX where a decrease in tumor hypoxia is seen
with one or more repeat administrations of H-NOX.
[0237] 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,
.sup.18F-fluorodeoxyglucose (FDG) 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 LDHA
expression, tumor carbonic anhydrase IX (CA-9) 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. 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.
[0238] In some embodiments of the methods of monitoring, treating
and/or optimizing therapeutic efficacy described above, the H-NOX
protein is any of the H-NOX protein described herein. In some
embodiments, the H-NOX protein comprises one or more distal pocket
mutations (e.g. one distal pocket mutation, two distal pocket
mutations, three distal pocket mutations, four distal pocket
mutations, five distal pocket mutations). In some embodiments, the
H-NOX protein is a polymeric H-NOX protein (e.g. a trimeric H-NOX
protein). In some embodiments, the polymeric H-NOX protein
comprises one or more H-NOX domains comprising a mutation at a
position corresponding to L144 of T. tengcongensis H-NOX. In some
embodiments, the polymeric H-NOX protein comprises one or more
H-NOX domains comprising a mutation corresponding to a L144F
mutation of T. tengcongensis H-NOX. In some embodiments, the
polymeric H-NOX protein comprises one or more H-NOX domains
comprising a mutation at positions corresponding to W9 and L144 of
T. tengcongensis H-NOX. In some embodiments, the polymeric H-NOX
protein comprises one or more H-NOX domains comprising mutations
corresponding to a W9F/L144F mutation of T. tengcongensis H-NOX. In
some embodiments, the H-NOX domain is a human H-NOX domain. In some
embodiments, the H-NOX domain is a canine H-NOX domain. In some
embodiments, the polymeric H-NOX protein comprises a L144F T.
tengcongensis H-NOX domain. In some embodiments, the polymeric
H-NOX protein comprises a W9F/L144F T. tengcongensis H-NOX domain
and a foldon domain. In some embodiments, the H-NOX protein is a
trimeric H-NOX protein. In some embodiments, the trimeric H-NOX
protein comprises at least one T. tengcongensis H-NOX domain. In
some embodiments, the trimeric H-NOX protein comprises at least one
L144F T. tengcongensis H-NOX domain. In some embodiments, the
trimeric H-NOX protein comprises at least one L144F T.
tengcongensis H-NOX domain and at least one foldon domain. In some
embodiments, the trimeric H-NOX protein comprises three L144F T.
tengcongensis H-NOX domains, each fused to a foldon domain. In some
embodiments, the H-NOX protein does not comprise a guanylyl cyclase
domain.
[0239] In some embodiments of the methods of monitoring, treating
and/or optimizing therapeutic efficacy described above, the H-NOX
protein is modified with polyethylene glycol (e.g., pegylated). In
some embodiments, the H-NOX protein is fused to another
polypeptide; for example but not limited to albumin or an Fc region
of an immunoglobulin.
[0240] In some embodiments of the methods of monitoring, treating
and/or optimizing therapeutic efficacy described above, radiation
is administered to the individual following H-NOX administration.
In some embodiments, the radiation is X-radiation or gamma
radiation. In some embodiments, the dose of X irradiation is any of
about 0.5 gy to about 75 gy. In some embodiments, the dose of X
irradiation is at least about any of 0.5 gy, 1 gy, 2 gy, 3 gy, 4
gy, 5 gy, 6 gy, 7 gy, 8 gy, 9 gy, 10 gy, 15 gy, 20 gy, 25 gy, 50
gy, or 75 gy. In some embodiments, the cycle of H-NOX
administration and radiation administration is repeated any one of
one, two, three, four, five or six times. In some embodiments, the
level of tumor hypoxia is determined after H-NOX administration and
before administration of radiation (e.g., X-radiation). In some
embodiments, the cycle of H-NOX 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 radiation therapy is external beam radiation
therapy. In some embodiments, the radiation therapy is
3-dimensional conformal radiation therapy (3D-CRT),
intensity-modulated radiation therapy (IMRT), image-guided
radiation therapy (IGRT), tomotherapy or stereotactic
radiosurgery.
[0241] As noted above, the selection of dosage amounts for H-NOX
proteins depends upon the dosage form utilized, the frequency and
number of administrations, the condition being treated, and the
particular purpose to be achieved according to the determination of
the ordinarily skilled artisan in the field. In some embodiments,
an effective amount of an H-NOX protein for administration to human
is between a few grams to over 350 grams. In some embodiments, the
dosage of H-NOX is determined by the methods of monitoring tumor
oxygenation described above; for example, the amount of H-NOX
administered to the individual may be increased if the level of
tumor hypoxia does not decrease significantly. In some embodiments,
the amount of H-NOX administered to the individual may be decreased
if the level of tumor hypoxia decreases significantly in response
to H-NOX administration.
[0242] In some embodiments of the methods of monitoring, treating
and/or optimizing therapeutic efficacy described above, the
individual is mammal, such as a primate (e.g., a human, a monkey, a
gorilla, an ape, a lemur, etc.), a bovine, an equine, a porcine, a
canine, or a feline. In some embodiments, the individual is pet, a
laboratory research animal or a farm animal.
[0243] In some embodiments of the methods of monitoring, treating
and/or optimizing therapeutic efficacy described above, the methods
comprise the step of delivering (e.g., transfusing, etc.) to the
blood of the individual (e.g., a mammal) one or more of H-NOX
compositions. Methods for delivering O.sub.2 carriers to blood or
tissues (e.g., mammalian blood or tissues) are known in the art. In
various embodiments, the H-NOX protein is an apoprotein that is
capable of binding heme or is a holoprotein with heme bound. The
H-NOX protein may or may not have heme bound prior to the
administration of the H-NOX protein to the individual. In some
embodiments, O.sub.2 is bound to the H-NOX protein before it is
delivered to the individual. In other embodiments, O.sub.2 is not
bound to the H-NOX protein prior to the administration of the
protein to the individual, and the H-NOX protein transports O.sub.2
from one location in the individual to another location in the
individual.
[0244] In some embodiments of the methods of monitoring, treating
and/or optimizing therapeutic efficacy described above, the brain
tumor is a glioblastoma, an astrocytoma, a meningioma, an
ependymoma, a medulloblatomoa, a pineocytoma, a pineoblastoma, or a
craniopharyngioma.
Kits with H-NOX Proteins
[0245] Also provided are articles of manufacture and kits that
include any of the H-NOX proteins described herein including
polymeric H-NOX proteins, and suitable packaging. In some
embodiments, the invention includes a kit with (i) an 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. In various
embodiments, the invention features a kit with (i) an 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 for any of the industrial uses described herein
(e.g., use of an H-NOX protein as a reference standard for
analytical instrumentation needing such a reference standard,
enhancement of cell growth in cell culture by maintaining or
increasing O.sub.2 levels in vitro, addition of O.sub.2 to a
solution, or removal of O.sub.2 from a solution).
[0246] In some embodiments, kits are provided for use in the
treatment of brain cancer (e.g. glioblastoma). In some embodiments,
the kit comprises a polymeric H-NOX protein. In some embodiments,
the kit comprises an effective amount of a polymeric H-NOX protein
comprising two or more wild-type or mutant H-NOX domains. In some
embodiments, the kit comprises 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 kit comprises a trimeric H-NOX protein comprising
three monomers, each monomer comprising a mutation corresponding to
a T. tengcongensis L144F H-NOX mutation and a trimerization domain.
In some embodiments, the kit comprises a trimeric H-NOX protein
comprising three monomers, each monomer comprising a mutation
corresponding to a T. tengcongensis W9F/L144F H-NOX mutation and a
trimerization domain. In some embodiments, the trimeric H-NOX
protein comprises human H-NOX domains. In some embodiments, the
trimeric H-NOX protein comprises canine H-NOX domains. In some
embodiments, the kit 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 kit
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 kit comprises a trimeric
H-NOX protein comprising three monomers, each monomer comprising a
T. tengcongensis L144F H-NOX domain and a foldon domain.
[0247] 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 as to 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 or treatment.
[0248] 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
[0249] The present invention also provides methods for the
production of any of the polymeric H-NOX proteins described herein.
In some embodiments, the method involves culturing a cell that has
a nucleic acid encoding a polymeric H-NOX protein under conditions
suitable for production of the polymeric H-NOX protein. In various
embodiments, the polymeric H-NOX is also purified (such as
purification of the H-NOX protein from the cells or the culture
medium). In some embodiments, the method involves culturing a cell
that has a nucleic acid encoding a monomer H-NOX protein comprising
an H-NOX domain and a polymerization domain. The monomers then
associate in vivo or in vitro to form a polymeric H-NOX protein. A
polymeric H-NOX protein comprising heterologous H-NOX domains may
be generated by co-introducing two or more nucleic acids encoding
monomeric H-NOX proteins with the desired H-NOX domains and where
in the two or more monomeric H-NOX proteins comprise the same
polymerization domain.
[0250] In some embodiments, a polymeric H-NOX protein comprising
heterologous H-NOX domains is prepared by separately preparing
polymeric H-NOX proteins comprising homologous monomeric H-NOX
subunits comprising the desired H-NOX domains and a common
polymerization domain. The different homologous H-NOX proteins are
mixed at a desired ratio of heterologous H-NOX subunits, the
homologous polymeric H-NOX proteins are dissociated (e.g. by heat,
denaturant, high salt, etc.), then allowed to associate to form
heterologous polymeric H-NOX proteins. The mixture of heterologous
polymeric H-NOX proteins may be further purified by selecting for
the presence of the desired subunits at the desired ratio. For
example, each different H-NOX monomer may have a distinct tag to
assist in purifying heterologous polymeric H-NOX proteins and
identifying and quantifying the heterologous subunits.
[0251] 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, which is
hereby incorporated by reference in its entirety, 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.
[0252] In particular, mutant H-NOX proteins described herein can be
generated a number of methods that are known in the art. Mutation
can occur at either the amino acid level by chemical modification
of an amino acid or at the codon level by alteration of the
nucleotide sequence that codes for a given amino acid. Substitution
of an amino acid at any given position in a protein can be achieved
by altering the codon that codes for that amino acid. This can be
accomplished by site-directed mutagenesis using, for example: (i)
the Amersham technique (Amersham mutagenesis kit, Amersham, Inc.,
Cleveland, Ohio) based on the methods of Taylor, J. W. et al. (Dec.
20, 1985). Nucleic Acids Res. 13(24):8749-8764; Taylor, J. W. et
al. (Dec. 20, 1985). Nucleic Acids Res. 13(24):8765-8785; Nakamaye,
K. L. et al. (Dec. 22, 1986). Nucleic Acids Res. 14(24):9679-9698;
and Dente et al. (1985). in DNA Cloning, Glover, Ed., IRL Press,
pages 791-802, (ii) the Promega kit (Promega Inc., Madison, Wis.),
or (iii) the Biorad kit (Biorad Inc., Richmond, Calif.), based on
the methods of Kunkel, T. A. (January 1985). Proc. Natl. Acad. Sci.
USA 82(2):488-492; Kunkel, T. A. (1987). Methods Enzymol.
154:367-382; Kunkel, U.S. Pat. No. 4,873,192, which are each hereby
incorporated by reference in their entireties, particularly with
respect to the mutagenesis of proteins. Mutagenesis can also be
accomplished by other commercially available or non-commercial
means, such as those that utilize site-directed mutagenesis with
mutant oligonucleotides.
[0253] Site-directed mutagenesis can also be accomplished using
PCR-based mutagenesis such as that described in Zhengbin et al.
(1992). pages 205-207 in PCR Methods and Applications, Cold Spring
Harbor Laboratory Press, New York; Jones, D. H. et al. (February
1990). Biotechniques 8(2):178-183; Jones, D. H. et al. (January
1991). Biotechniques 10(1):62-66, which are each hereby
incorporated by reference in their entireties, particularly with
respect to the mutagenesis of proteins. Site-directed mutagenesis
can also be accomplished using cassette mutagenesis with techniques
that are known to those of skill in the art.
[0254] A mutant H-NOX nucleic acid and/or polymerization domain 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.
[0255] 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.
[0256] 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.
[0257] 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.
EXAMPLES
[0258] 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 Proteins Demonstrated Tumor Penetration and Oxygenation in an
In Vivo Mouse Model of Cancer
[0259] Oxygen is a critical factor that enhances radiation-induced
DNA damage and tumor killing. Low oxygen levels or hypoxia within
solid tumors can blunt the therapeutic effects of tumor therapy.
For example in hypoxic regions of the tumor, radiation therapy has
been found to be three times less effective as compared to tumor
regions with normal oxygen levels. As a result, many patients with
tumors containing regions of hypoxia often show incomplete
responses to conventional tumor therapy and have poor prognosis for
survival. The correlation of hypoxia with poor patient outcomes has
been observed in a wide range of tumors arising from, among others,
prostate, sarcoma, pancreatic, head-and-neck, cervical, and brain
cancers. See Moeller, B J et al. (2007) Cancer Metastasis Rev
26:241-248; Vaupel, P, (2004) Semin Radiat Oncol, 14:198-206;
Varlotto, J, et al. (2005) Int J Radiat Oncol Biol Phys, 63:25-36;
Rockwell, S, et al. (2009) Curr Mol Med. 9:442-458; which are all
incorporated herein in their entirety by reference.
[0260] To determine the ability of H-NOX proteins to penetrate
tumors, groups of 6 mice bearing subcutaneous HCT116 colon-derived
tumors were injected via the tail vein with 750 mg/kg of a H-NOX
monomer, 750 mg/kg of a T. tengcongensis L144F H-NOX trimer or
saline control. The mice were subsequently sacrificed at 30 minutes
or 60 minutes post-injection. The tumors were resected, sectioned,
stained with an anti-H-NOX antibody, and imaged for H-NOX staining
intensity (FIG. 1A). Quantification of the stained HCT-116 tumor
sections demonstrated that the 23 kDa T. tengcongensis L144F H-NOX
monomer accumulated in tumors by 30 minutes and exhibited partial
clearance by 60 minutes (FIG. 1B). In comparison, the 80 kDa L144F
H-NOX trimer accumulated in tumors by 30 minutes and continued to
persist in the tumors at 60 minutes post-injection with
accumulation peaking at 4 hours post-injection (FIG. 1B).
[0261] To determine if the H-NOX proteins reduced hypoxia in the
tumors, groups of 6 mice bearing subcutaneous HCT116 colon-derived
tumors were injected via the tail vein with 750 mg/kg of a L144F
H-NOX monomer, 750 mg/kg of a L144F H-NOX trimer or saline control.
Prior to euthanasia, mice were given hypoxia marker pimonidazole
via intraperitoneal injection and active vasculature marker
DiOC7.sub.3 via intravenous injection. Tumors were harvested at
either 30 minutes or 60 minutes after H-NOX protein injection, and
assayed by immunohistochemistry for pimonidazole with
Hydroxyprobe-1 monoclonal antibody and total vasculature with
anti-CD31 antibody (FIG. 2A). Quantification of the stained HCT-116
tumor sections demonstrated that in contrast to control-treated
mice the 23 kDa H-NOX monomer decreased hypoxia 30 minutes
post-injection but that there was no recovery in hypoxia 60 minutes
post-injection (FIG. 2B). In comparison, the 80 kDa L144F H-NOX
trimer did not appear to reduce hypoxia at 30 minutes
post-injection, but substantially reduced hypoxia at 60 minutes
post-injection (FIG. 2B). Further experiments confirmed that in
mice bearing subcutaneous HCT116 colon-derived tumors the H-NOX
monomer distributed throughout the tumor tissue (FIG. 3A, bottom
panel) and relieved tumor hypoxia at distances far from the
vasculature as detected by anti-pimonidazole antibody (FIG. 3B,
bottom panel). The Hypoxyprobe-1 (anti-pimonidazole antibody) stain
was quantified in tumor tissue isolated from six mice by amount of
staining as a function of distance from the vasculature. It was
found that the average Hypoxyprobe-1 staining was reduced from
about 13 .mu.M in saline treated mice to 5 .mu.M in H-NOX monomer
treated mice at a distance of about 150 .mu.m from the nearest
blood vessel (FIG. 3C). These results were further confirmed in
mice bearing murine RIF-1 sarcoma xenografts.
[0262] Mice bearing RIF-1 sarcoma tumors were injected via the tail
vein with 750 mg/kg of T. tengcongensis L144F H-NOX trimer or
saline control. Prior to euthanasia, mice were given hypoxia marker
pimonidazole via intraperitoneal injection. Tumors were harvested
at 120 minutes after L144F H-NOX trimer injection, and assayed by
immunofluorescence imaging for H-NOX trimer distribution (FIG. 4),
or by western blot for pimonidazole with Hydroxyprobe-1 monoclonal
antibody, hypoxia-inducible factor 1 (HIF-1.alpha.) with
anti-HIF-1.alpha. antibody, H-NOX protein with an anti-H-NOX
antibody, and total protein with anti-actin antibody (FIG. 5).
Immunofluorescence staining demonstrated distribution of L144F
H-NOX trimer in tumor sections prepared from large isolated tumors
approximately 400 mm.sup.3 and 800 mm.sup.3 in size (FIG. 4).
Western blot analysis of cell lysates from harvested tumors of
treated mice demonstrated that the L144F H-NOX trimer localized to
tumor tissue and that these tumors had decreased pimonidazole
protein adducts as compared to untreated mice (FIG. 5A).
Quantification of the western blots further confirmed low levels of
pimonidazole protein adducts as well as low levels of HIF-1.alpha.
protein in the tumors of treated mice as compared to saline treated
mice (FIGS. 5B and 5C).
Example 2
11-NOX Proteins Demonstrated Tumor Penetration and Oxygenation in
an In Vivo Mouse Model of Glioblastoma
[0263] To further characterize the ability of H-NOX proteins to
penetrate into tumor tissue, three mouse models of glioblastoma
were used to assess the distribution of T. tengcongensis L144F
H-NOX monomer and T. tengcongensis L144F H-NOX trimer in brain
tumors. BT-12 cells, a childhood atypical teratoid/rhabdoid infant
brain tumor line that is highly invasive into the spinal column,
were used to generate a mouse model of child glioblastoma, GBM-43
cells were used for generating a radioresistant model of adult
glioblastoma, and U251 cells were used to generate a hypoxic model
of adult glioblastoma. The glioblastoma mouse models were generated
as previous described. See Ozawa, T, et al., (2010) J Vis Exp, July
13; (41) which is incorporated in its entirety herein by reference.
Briefly, BT-12 cells, U251 cells, or GBM-42 cells were harvested
for intracranial injection and resuspended in Dulbecco's Modified
Eagle Medium (DMEM) at a concentration of about 1.times.10.sup.8
cells per mL. Mice were anesthetized by intraperitoneal (IP)
injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). The
anesthetic depth was monitored prior to the first incision as well
as at regular intervals through the procedure, using the pedal
withdrawal reflex by pinching the foot pad on both feet. A 1 cm
sagittal incision was made along the scalp, and the skull suture
lines were exposed. A small hole was created by puncture with a 25
g needle, at 3 mm lateral and 0.5 mm anterior of the bregma. Using
a sterile Hamilton syringe (Stoelting), 3.times.10.sup.5 cells in 3
.mu.l was injected at a depth of 3 mm over a 60 second period.
After injection, the syringe was held in place for 1 minute and
then slowly removed. The skull was cleaned with 3% hydrogen
peroxide and then sealed with bone wax before closing the scalp
using 7 mm surgical staples (Stoelting). Mice received a
subcutaneous injection of 0.1 mg/kg buprenorphine, were placed on a
heating pad and monitored until they regained mobility for use in
these studies.
[0264] To determine if L144F H-NOX trimers could penetrate brain
tissue, mice bearing U251 orthotopic brain tumors were injected via
tail vein with either 750 mg/kg T. tengcongensis L144F H-NOX
monomer or 750 mg/kg T. tengcongensis L144F H-NOX trimer. Prior to
euthanasia, mice were given the hypoxia marker pimonidazole by
intraperitoneal injection. For immunohistochemistry analysis,
brains were isolated, sectioned and stained for pimonidazole with
Hydroxyprobe-1 monoclonal antibody, hypoxia-inducible factor 1
(HIF-1.alpha.) with anti-HIF-1.alpha. antibody, H-NOX protein with
an anti-H-NOX antibody, and HLA-ABC protein with an anti-HLA-ABC
antibody (NvusBiological rat monoclonal antibody clone #YTH862.2)
about two hours after H-NOX protein administration. A set of brain
tissue samples was further stained with secondary antibodies
conjugated to anti-rabbit antibody conjugated with FITC (green
channel) manufactured by Jackson ImmunoResearch and DAPI for
immunofluorescence imaging. Mice treated with H-NOX trimer
demonstrated increased staining for H-NOX as compared to control
treated mice indicating that the H-NOX trimer penetrated brain
tissue (FIG. 6A). In addition, decreased staining for pimonidazole
with Hydroxyprobe-1 monoclonal antibody showed that H-NOX trimer
administration substantially reduced hypoxia at 60 minutes
post-injection (FIG. 6B). Decreased staining for pimonidazole and
HIF-1.alpha. protein was further observed in immunofluorescence
images (FIGS. 7A and 7C). Quantification of the immunofluorescence
images demonstrated low levels of pimonidazole staining as well as
low levels of HIF-1.alpha. protein staining in the tumors of L144F
H-NOX trimer treated mice as compared to saline treated mice (FIGS.
7B and 7D).
[0265] FIG. 8 shows the biodistribution of H-NOX trimer in U251
orthotopic brain tumor and healthy brain. Fluorescent imaging of
H-NOX trimer at high magnification shows weak diffusion outside
vessels in healthy brain.
[0266] To compare penetration and retention times between T.
tengcongensis L144F H-NOX monomer and T. tengcongensis L144F H-NOX
trimer, mice bearing orthotopic brain tumors were injected via tail
vein with either 750 mg/kg Alexa-647 labeled H-NOX monomer or 750
mg/kg Alexa-647 labeled H-NOX trimer and subjected to
bioluminescence imaging at various time points. Alexa-647 labeled
H-NOX proteins were generated to confirm fluorescence excitation
and emission spectra of fluorescently labeled H-NOX proteins as
follows.
[0267] Purified protein, H-NOX monomer protein, H-NOX trimer, or
BSA (Sigma, used as a control), was thawed on ice and buffer
exchanged into endotoxin-free Labeling Buffer (50 mM HEPES, 50 mM
NaCl, pH 8.0) using endotoxin-free dialysis cassettes (Pierce
Slide-A-Lyzer, 7 kDa MWCO). Protein concentration after dialysis
into Labeling Buffer was determined by UV-vis spectroscopy. Alexa
647 dye (Alexa Fluor.RTM. 647 carboxylic acid, succinimidyl ester,
Invitrogen # A-20006) was prepared immediately before addition to
the labeling reactions. Dye was warmed to room temperature and then
dissolved in DMSO at a final concentration of 10 mg/mL. The mixture
was vortexed for 10 seconds and then dye was added to each labeling
reaction. Labeling reactions used a range of protein:dye ratios to
control the extent of Alexa labeling. Reactions consisted of
protein (in Labeling Buffer) and dye for a final DMSO concentration
of 5-10%. Reactions were incubated for 1 hour at room temperature
(protected from light) with moderate shaking. After the reaction,
free Alexa dye was removed by extensive dialysis into
endotoxin-free formulation buffer (30 mM Triethanolamine, 50 mM
NaCl, pH 7.4) using endotoxin-free dialysis cassettes (Pierce
Slide-A-Lyzer, 7 kDa MWCO cutoff).
[0268] After dialysis into formulation buffer, the protein
concentration and extent of labeling was determined by UV-vis
spectroscopy using the intrinsic absorbance of H-NOX (at 280 and
415 nm) and Alexa dye (653 nm) to determine the molar ratio of dye
to protein after labeling. Fluorescence of the labeled protein was
analyzed by excitation at 647 nm to collect an emission spectrum.
The emission spectrum of the labeled protein was consistent with
published data and Invitrogen data. Labeled protein was further
analyzed by size exclusion chromatography to ensure that labeling
did not affect the oligomerization state of the protein. Final
endotoxin contamination in the labeled protein was determined using
the Charles River LAL Gel Clot assay (0.03 EU/mL sensitivity).
[0269] For bioluminescence imaging, mice were anesthetized by IP
injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and then
injected by IP with 33.3 mg of D-luciferin (potassium salt, Gold
Biotechnology, St. Louis, Mo., USA) dissolved in sterile saline.
Tumor bioluminescence was determined 10 minutes after luciferin
injection, using the IVIS Lumina System (Caliper Life Sciences,
Alameda, Calif., USA) and LivingImage software, as the sum of
photon counts per second in regions of interest defined by a lower
threshold value of 25% of peak pixel intensity. Imaging acquisition
was non-invasive, and animal body temperature was maintained using
a heated imaging platform. For BT-12 mice treated with H-NOX
monomer or H-NOX trimer, imaging was performed at 0, 0.5, 1, 2, and
4 hrs post injection. For GBM-41 mice treated with H-NOX monomers
or H-NOX trimers, imaging was performed at 0, 0.5, 1, 2, 4, and 6
hrs post injection. For U251 mice treated with H-NOX monomers or
H-NOX trimers, imaging was performed at 0, 0.5, 1, 2, 4, 6, and 72
hrs post injection. Tumor bioluminescence has previously been shown
to be directly proportional to tumor volume in mice bearing
orthotopic GB xenografts. See Moeller, B J et al., (2007) Cancer
Metastasis Rev, 26:241-248, which is incorporated herein in its
entirety by reference. Comparison of H-NOX monomer and trimer
biodistribution demonstrated that in the BT-12 mouse model of
glioblastoma, both L144F H-NOX monomer (FIGS. 9A and 23A) and L144F
H-NOX trimers (FIGS. 9B and 23A) penetrated brain tumors. H-NOX
trimer had a significantly longer retention time in tumors as
compared to H-NOX monomers. Whereas HNOX monomer was largely
eliminated from tumors by 2 hours (FIGS. 9A and 23A), H-NOX trimer
continued to accumulate in tumors for several hours (FIGS. 9B and
23A). H-NOX intracranial localization was confirmed by ex vivo
imaging of brain tissue isolated from mice 30 and 60 minutes post
injection with the H-NOX monomer (FIGS. 10A, 21C and 7B) and 60
minutes post injection with the H-NOX trimer (FIGS. 10C and 21D).
Further visualization by bioluminescence at 30, 60, 120, and 240
minutes post injection demonstrated that the H-NOX monomer (FIG.
11) and H-NOX trimer (FIG. 12) also localized to metastatic
colonies in the spinal column Whereas H-NOX monomer substantially
accumulated in the spinal column at 30 minutes (FIG. 11A) as
compared to H-NOX trimer (FIG. 12A), it was largely eliminated by 2
hours (FIG. 11B-D) while the H-NOX trimer continued to accumulate
in the spinal column for several hours (FIG. 12B-D). By using a
smaller amount of labeled protein and increasing the signal
intensity, it was revealed that H-NOX monomer accumulated in the
kidneys over time suggesting a route of elimination (FIG. 13).
[0270] The accumulation of L144F H-NOX trimers in the brain and
spinal column was confirmed in the GBM-43 (FIG. 14) and U251 mouse
models (FIGS. 15 and 23B). Localization of L144F H-NOX trimers was
further investigated in U251 mice that were injected with a higher
H-NOX trimer dose of 295 mg/kg and a lower dose of 30 mg/kg.
Bioluminescence imaging at 0, 0.5, 1, 2, 4, and 6 hr post-injection
demonstrated that the L144F H-NOX trimer accumulated in brain
tumors of the mice at both the high and low concentrations of H-NOX
trimer administration (FIGS. 16 and 17, respectively). In
comparison, localization of an H-NOX trimer assembled from a H-NOX
monomer L144F variant did not accumulate in small brain tumors as
evidenced by bioluminescence images 0, 0.5, 1, 2, 4, and 6 hr
post-injection with 30 mg/kg (FIG. 18). Ex vivo bioluminescence
imaging of isolated brain from mice treated with 30 mg/kg L144F
H-NOX trimer (FIG. 19A) or 750 mg/kg L144F trimer (FIG. 19B) showed
that the amount of H-NOX protein in a single dose had little effect
on H-NOX localization to intracranial tumors. Furthermore,
real-time bioluminescence imaging of mice bearing large (FIG. 19C)
or small tumors (FIG. 19D) showed that after administration of 295
mg/kg of L144F H-NOX trimer, the trimer distributed to intracranial
tumors regardless of tumor size (FIG. 19B). Real-time and ex vivo
bioluminescence imaging of three mouse models of glioblastoma, GBM,
U251, and BT-12, demonstrated that L144F H-NOX trimer distributed
to intracranial tumors and spinal tumors in all three models (FIGS.
20, 21 and 23A and B) Immunofluorescence imaging of a tumor section
stained with antibodies to H-NOX protein and the vasculature showed
that L144F H-NOX trimer left the vasculature and diffused
throughout the brain tumor (FIG. 22). Overall, these data
identified H-NOX proteins with clinically relevant tumor
biodistribution profiles.
[0271] Mice were sacrificed and the whole extracted brain analyzed
by imaging ex vivo, confirming localization of the protein to the
tumor bearing part of the brain. More detailed examination by
immunohistochemical analysis revealed different pattern of protein
localization between normal and tumor brain tissue (FIG. 23C).
While protein was detectable in blood vessels throughout the brain,
it extravasated and penetrated deep into tissue only in tumor areas
diffusing far from the blood vessels identified with CD31 marker
(FIG. 23D). Similar results have been obtained in other brain
orthotopic models including human patient-derived tumors GBM 43,
GBM 39, GBM 6 and immunocompetent mouse glioblastoma GL261, as well
as in subcutaneous xenograft HCT116 and immunocompetent RIF-1
models (data not shown).
[0272] To verify the partition of H-NOX trimers between plasma and
brain, L144F trimer was tested using a group of three female FVB
mice (FIG. 24). Candidate H-NOX trimers were injected at time 0 at
a dose of 200 and 750 mg/kg by intravenous bolus injection into the
tail vein. At 30 min, 1 hr, 1.5 hr and 2 hr post injection of the
candidate H-NOX trimer or buffer control, mice were sacrificed.
About one ml of blood was collected by intracardiac puncture and
brain were harvested. Collected blood was processed for plasma and
brain samples were lyzed to extract proteins. Plasma and brain were
subsequently analyzed for the presence of H-NOX trimer using an
ELISA assay with a polyclonal antibody against the H-NOX
protein.
Example 3
11-NOX Trimers Enhanced Effects of Radiation in In Vivo Mouse
Models of Glioblastoma
[0273] To determine if oxygenation of hypoxic tumors due to H-NOX
penetration could enhance radiation-induced tumor killing, studies
were conducted in groups of 10 athymic U251 mice bearing
intracranial glioblastoma tumors to evaluate the effects of
radiation therapy (RT) in the presence of H-NOX trimer. Mice were
treated with three fractions of radiation therapy at 2 Gy per
fraction on days 15, 17, and 20 post-tumor implantation either with
or without administration of 750 mg/kg Alexa-647 labeled T.
tengcongensis L144F H-NOX trimer delivered by intravenous
injection. Mice were monitored up to day 29 and subjected to
bioluminescence imaging at days 15, 17, 20, 22, 24, and 29. Mean
bioluminescence imaging (BLI) scores determined for each treatment
group demonstrated that multiple doses of L144F H-NOX trimer
resulted in statistically significant delays in tumor growth (FIGS.
25A and 25B) and despite the aggressive and mildly hypoxic nature
of the treated U251 orthotopic tumors, animal survival was also
significantly enhanced in L144F H-NOX treated groups (FIG. 25C).
Tumors were also harvested for immunohistochemistry staining and
analysis.
[0274] The effect of T. tengcongensis L144F H-NOX trimer on
radiation therapy of human glioblastoma was further investigated in
two mouse models bearing intracranial glioblastoma tumors, U251 and
GBM43. In one study, groups of 10 female athymic U251 mice bearing
intracranial glioblastoma tumors were treated with either 1)
treatment buffer alone, 2) treatment buffer in combination with a
single dose of 2 Gy radiation (irradiator set up=0.81; dose rate of
Cesium irradiator was 247 CGy/min), 3) 750 mg/kg L144F H-NOX trimer
by IV alone, or 4) L144F H-NOX trimer in combination with a single
dose of 2 Gy radiation (irradiator set up=0.81; dose rate of Cesium
irradiator was 247 CGy/min) Mice receiving the combination
treatment were irradiated 2 hours post L144F H-NOX trimer delivery
at the supratentorial portion of the brain. Treatment for all mice
began 14 days after intracranial injection of mice with
3.0.times.10.sup.5 U251 cells. It was found that animal survival
increased in cohorts receiving the combination treatment of L144F
H-NOX trimer and 2 Gy radiation (FIG. 26A). In another study,
groups of 10 GBM43 mice bearing intracranial glioblastoma tumors
were treated with either 1) 2 Gy radiation therapy; 2) 4 Gy
radiation therapy; 3) 8 Gy radiation therapy; 4) 2 cycles of 4 Gy
radiation therapy; 5) 4 Gy radiation therapy in combination with
L144F H-NOX trimer; or 6) treatment buffer. Mice receiving the
combination treatment were irradiated 1 to 1.5 hours post H-NOX
trimer delivery and mice receiving multiple doses of RT had
administration of RT separated by 4 days. Radiation treatment was
administered at the supratentorial portion of the brain for all RT
groups. Treatment for all mice began 7 days post-tumor
implantation. It was found that animal survival in cohorts
receiving the combination treatment of L144F H-NOX trimer and 4 Gy
radiation was similar to animal survival in cohorts receiving 4 Gy
treatment alone (FIG. 26B).
Example 4
Single Dose of Trimeric H-NOX Reduces Tumor Hypoxia Both in the
Tumor Core and at the Invasive Edges
[0275] Mice bearing orthotopic glioblastoma U251 tumors were
treated with either trimeric Tt L144F H-NOX (OMX-4.80) or buffer
alone (FIGS. 27A and 27D, top panels) via tail vein bolus
injection. Prior to euthanasia, mice were intraperitoneally
injected with the hypoxia marker pimonidazole. Tumors were
harvested 2 hr-30 hr after H-NOX administration, and assayed by
immunohistochemistry for pimonidazole (Hypoxyprobe-1 mAb) and total
cell nuclear (DAPI) staining in FIG. 27A and FIG. 27B, or for
HIF1.alpha. and tumor cell marker (HLA) in FIG. 27C and FIG. 27D.
Representative tumor sections from mice treated with buffer or
H-NOX are shown in FIG. 27a. Hypoxia staining (pimonidazole) is
shown in green and total cell nuclear staining (DAPI) is shown in
dark blue. Only 2 h and 24 h time points are shown. Quantification
of pimonidazole staining intensity in tumor sections is shown in
FIG. 27B. Tumors treated with OMX-4.80 show hypoxia reduction at
4-24 hr. Quantification of HIF1.alpha. staining intensity in tumor
sections is shown in FIG. 27C. Tumors treated with H-NOX exhibit
hypoxia reduction at 16-24 hr. This suggests an oxygenation window
between 2 and 30 hours post H-NOX injection. Representative tumor
sections from mice treated with buffer or H-NOX are shown in FIG.
27D. Hypoxia staining (HIF1.alpha.) is shown in green and staining
of human tumor cells in red (HLA). Only 2 h and 24 h time points
are shown.
[0276] To investigate reduction of hypoxia at the invasive edge of
the tumor, mice bearing orthotopic glioblastoma U251 tumors were
treated with either buffer control (FIGS. 28A, and 28C) or trimeric
Tt L144F H-NOX, OMX-4.80 (FIGS. 28B, 28D, 28E, and 28F), via tail
vein injection and received an injection of hypoxia marker,
pimonidazole, one hour prior to sacrifice. Brains containing tumors
were extracted and subjected to immunohistochemical analysis using
anti-pimonidazole (FIGS. 28A and 28B), HIF1.alpha. (FIGS. 28C and
28D), or OMX-4.80 antibodies (FIGS. 28E and 28F). Slides were
counterstained with DNA marker (DAPI, blue labeled nuclei) and
images merged (FIGS. 28A-28D, and 28F). Invasive edges (white
arrows) of the non-treated tumors tend to exhibit higher level of
hypoxia, as determined by both external (pimo) and cellular
(HIF1.alpha.) hypoxia markers. Upon trimeric Tt L144F H-NOX
treatment, hypoxia is significantly reduced throughout tumor tissue
(see FIG. 28), including the invasive edges (FIGS. 28B and 28D)
where OMX-4.80 can be readily detected (FIGS. 28E and 28F).
[0277] The ability of the trimeric Tt L144F H-NOX to oxygenate
hypoxic tumor areas in orthotopic GBM models using two markers of
hypoxia--the external hypoxia marker, pimonidazole, an analog of
the clinically relevant PET hypoxia marker-.sup.18F-FMISO, (FIGS.
27A and 28A,B), and hypoxia inducible factor, HIF1.alpha. (FIGS.
27B and 28C,D). This data demonstrates that single dose of H-NOX
administered i.v. induces a greater than 50% decrease in expression
of both hypoxia markers and that this effect is maintained for over
20 hr (FIG. 27). Furthermore, H-NOX can reach and efficiently
oxygenate invasive edges of the tumor (FIG. 28) which express
hypoxia-related aggressive tumor phenotypes associated with therapy
resistance and poor patient outcomes.
Example 5
Treatment with Trimeric H-NOX Enhances Efficacy of a Single Dose of
Radiation
[0278] Trimeric H-NOX sensitizes human GBM intracranial xenografts
and syngeneic RIF1 tumors to radiation therapy. Athymic mice
bearing orthotopic U251 (GBM) xenografts were treated with 10 Gy RT
either with or without pre-treatment with trimeric Tt L144F H-NOX
(650 mg/kg, IV) or with 15 Gy alone (FIG. 29A-D). Treatment was
administered on day 14 post-tumor implantation. Syngeneic RIF1
tumors, 250-300 mm.sup.3 size, were treated with 15 Gy RT either
with or without pre-treatment with trimeric Tt L144F H-NOX (750
mg/kg, IV) or with 25 Gy alone (FIG. 29D). FIG. 29A shows mean
bioluminescence imaging (BLI) scores.+-.SEM from mice bearing human
GBM intracranial xenografts and treated with trimeric Tt L144F
H-NOX and 10 gray radiation or buffer and 10 gray radiation, as
well as an untreated (buffer, no RT) control group. The BLI scores
of the H-NOX+RT mice are significantly lower than those from mice
treated with RT alone (p=0.015, Student's t-test). FIG. 29B shows
that trimeric Tt L144F H-NOX significantly enhanced survival, as
compared to mice that received only radiotherapy (p<0.05,
logrank test). FIG. 29C shows tumor growth delay in the H-NOX+10 Gy
group was not statistically different from 15 Gy alone group. Tumor
volume measurements in RIF1 tumors showed 6.4 days growth delay at
4.times. volume in trimeric Tt L144F H-NOX+15 Gy group relative to
15 Gy alone, an equivalent to 25 Gy RT. Unlike, trimeric Tt L144F
H-NOX, treatment with an inactive form of the H-NOX protein that
does not release oxygen, did not cause any radiation enhancement as
compared to radiation alone (FIG. 29D).
[0279] A single dose of trimeric H-NOX, administered 24 hr prior to
radiation, delayed tumor growth 2.7 fold and increased survival 48%
of mice bearing 14 d-old orthotopic GBM tumors (FIG. 29). This
effect was equivalent to increasing the RT dose by 50% resulting in
OER of 1.5 (FIG. 29C). Similar results were obtained in syngeneic
subcutenous tumor model with OER of 1.7. Importantly, inactive form
of the trimeric H-NOX protein that cannot release oxygen under
hypoxic tumor conditions, did not affect the tumor growth (FIG.
29D).
Example 6
Targeting Hypoxia in Glioblastoma Multiforme with H-NOX
[0280] Brain tumors were collected from mice bearing intracranial
orthotopic glioblastoma multiforme tumors (U251, GBM6, GBM39, GBM43
and GL261). Hypoxia was evaluated by immunohistochemistry of
endogenous markers: the transcription factor HIF-1.alpha. (FIG.
31B) and by the exogenous clinically relevant hypoxia marker,
pimonidazole (FIG. 31A). Prior to euthanasia, mice were
intraperitoneally injected with the hypoxia marker pimonidazole.
Tumors were harvested and assayed by immunohistochemistry for
pimonidazole (Hypoxyprobe-1 polyclonal Ab). Pimonidazole staining
reveals distinct patterns of hypoxia in each GBM models (FIG. 31A
and Table 2). Other tumors were harvested and assayed by
immunohistochemistry for HIF-1.alpha.. HIF-1.alpha. staining
reveals distinct patterns of hypoxia in each GBM models. Table 2
shows a comparison of hypoxic levels between GBM models as measured
by pimonidazole staining or HIF-1.alpha. staining ("+", weak
staining; "++", moderate staining; "+++", strong staining) is
determined by visual scoring of the % of total tumor area
exhibiting pimonidazole immunoreactivity.
TABLE-US-00003 TABLE 2 Tumor hypoxia in four models of glioblastoma
GBM model Pimonidazole HIF-1.alpha. GL261 +++ + U251 ++ +++ GBM43
+/++ ++ GBM6 + +
[0281] As described in Example 2, all hypoxia markers exhibited
high signal in poorly vascularized areas of U251 and GL261, as
determined by costaining with CD31 endothelial marker. In contrast,
GBM6, GBM43 and GBM39 tumors showed significant range in hypoxia
marker signal intensity between samples, indicating heterogeneity
in extent of hypoxia within and between GBM models. When H-NOX was
administered to mice bearing intracranial U251 tumors, H-NOX
significantly decreased HIF-1.alpha. stabilization and pimonidazole
accumulation, demonstrating that H-NOX increases tumor oxygenation.
In addition, H-NOX treated tumors exhibited fewer Ki67+ cells,
suggesting that H-NOX-mediated acute re-oxygenation inhibits tumor
cell proliferation.
[0282] FIG. 32A shows a schematic representation of quantitative
oxygen dependencies for OMX-4.80, OMX-1.80, bioreductive activation
of imaging agents (pimonidazole), and biological responses to
hypoxia (HIF-1.alpha.). Three commonly used units for oxygen
concentration are shown on the x axis.
[0283] Mice bearing orthotopic glioblastoma U251 tumors treated
with either polymeric OMX-4.80 (750 mg/kg) or buffer alone via tail
vein bolus injection in Example 4 were used to compare hypoxia
measured by pimonidazole and measured by HIF-1.alpha. in response
to H-NOX treatment. As described in Example 4, prior to euthanasia,
mice were intraperitoneally injected with the hypoxia marker
pimonidazole. Tumors were harvested 2 hours-30 hours after OMX-4.80
administration, and assayed by immunohistochemistry for
pimonidazole (Hypoxyprobe-1 polyclonal Ab) and HIF-1.alpha..
Quantification of immunohistochemical signal for pimonidazole and
HIF-1.alpha. was performed with ImageJ. As shown in FIG. 32B,
HIF-1.alpha. levels correlate significantly with pimonidazole
levels (Spearman's correlation coefficient; r=0.6468, p<0.0001).
The solid line represents the linear regression curve of the best
fit.
Sequence CWU 1
1
281555DNAThermoanaerobacter 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
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Ser Arg Leu Lys Val Arg Ile 165 170 175 Lys Phe Lys Asn Pro Val Phe
Glu 180 381DNAArtificial SequenceSynthetic Construct 3ggttatattc
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ctaccttttt a 81427PRTArtificial SequenceSynthetic Construct 4Gly
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15 Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 20 25
5686DNAArtificial SequenceSynthetic Construct 5atgaagggga
caatcgtcgg gacatggata aagaccctga gggaccttta cgggaatgat 60gtggttgatg
aatctttaaa aagtgtgggt tgggaaccag atagggtaat tacacctctg
120gaggatattg atgacgatga ggttaggaga atttttgcta aggtgagtga
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tcaccatcac cattga 6866229PRTArtificial SequenceSynthetic Construct
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
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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 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 Tyr Lys
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Arg Asp Gly Gln Ala Tyr Val Arg 195 200 205 Lys Asp Gly Glu Trp Val
Leu Leu Ser Thr Phe Leu Arg Gly Ser His 210 215 220 His His His His
His225 7663DNAArtificial SequenceSynthetic Construct 7atgaagggga
caatcgtcgg gacatggata aagaccctga gggaccttta cgggaatgat 60gtggttgatg
aatctttaaa aagtgtgggt tgggaaccag atagggtaat tacacctctg
120gaggatattg atgacgatga ggttaggaga atttttgcta aggtgagtga
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ctagtgaatt ttttaatgat gatggatgag 300gtacacctac agcttaccaa
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420tttttagggt ttatagaggg tagttctaaa tttttcaagg aagaaatttc
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tcgtaaagat ggcgaatggg tattactttc taccttttta 660tga
6638220PRTArtificial SequenceSynthetic Construct 8Met 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
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Phe Ser Glu65 70 75 80 Trp Phe Pro Ser Tyr Phe Ala Gly Arg Arg Leu
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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
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 Tyr Lys Lys Asn Leu Glu Gly
Ser 180 185 190 Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala
Tyr Val Arg 195 200 205 Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe
Leu 210 215 220 91158DNAArtificial SequenceSynthetic Construct
9gcgcaacgca attaatgtaa gttagctcac tcattaggca ccccaggctt tacactttat
60gcttccggct cgtataatgt gtggaattgt gagcggataa caatttcaca caggaaacag
120gatcgatcca tcgatgagct tactccccat ccccctgttg acaattaatc
atcggctcgt 180ataatgtgtg gaattgtgag cggataacaa tttcacacag
gaaacaggat cagcttactc 240cccatccccc tgttgacaat taatcatcgg
ctcgtataat gtgtggaatt gtgagcggat 300aacaatttca cacaggaaac
aggatccatc gatgcttagg aggtcatatg aaggggacaa 360tcgtcgggac
atggataaag accctgaggg acctttacgg gaatgatgtg gttgatgaat
420ctttaaaaag tgtgggttgg gaaccagata gggtaattac acctctggag
gatattgatg 480acgatgaggt taggagaatt tttgctaagg tgagtgaaaa
aactggtaaa aatgtcaacg 540aaatatggag agaggtagga aggcagaaca
taaaaacttt cagcgaatgg tttccctcct 600attttgcagg gagaaggcta
gtgaattttt taatgatgat ggatgaggta cacctacagc 660ttaccaagat
gataaaagga gccactcctc caaggcttat tgcaaagcct gttgcaaaag
720atgccattga aatggagtac gtttctaaaa gaaagatgta cgattacttt
ttagggctta 780tagagggtag ttctaaattt ttcaaggaag aaatttcagt
ggaagaggtc gaaagaggcg 840aaaaagatgg cttttcaagg ctaaaagtca
ggataaaatt taaaaacccc gtttttgagt 900ataagaaaaa ctcgagggca
gcggcggtta tattcctgaa gctccaagag atgggcaggc 960ttacgttcgt
aaagatggcg aatgggtatt actttctacc tttttaaggg gtagtcacca
1020tcaccatcac cattgatcta gagtcgacct gcagcccaag cttatcgatg
ataagctgtc 1080aaacatgagc agatctgagc ccgcctaatg agcgggcttt
tttttcagat ctgcttgaag 1140acgaaagggc ctcgtgat
115810231PRTArtificial SequenceSynthetic Construct 10Met 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 Tyr Lys Lys Asn Leu Glu
Gly Gly 180 185 190 Ser Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly
Gln Ala Tyr Val 195 200 205 Arg Lys Asp Gly Glu Glu Trp Val Leu Leu
Ser Thr Phe Leu Arg Gly 210 215 220 Ser His His His His His His225
230 11669DNAArtificial SequenceSynthetic Construct 11atgaagggga
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 agtataagaa aaatctcgag
ggcagcggcg gttatattcc tgaagctcca 600agagatgggc aggcttacgt
tcgtaaagat ggcgaatggg tattactttc taccttttta 660aggggtagt
66912222PRTArtificial SequenceSynthetic Construct 12Met 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 Tyr Lys Lys Asn Leu Glu Gly
Gly 180 185 190 Ser Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln
Ala Tyr Val 195 200 205 Arg Lys Asp Gly Glu Glu Trp Val Leu Leu Ser
Thr Phe Leu 210 215 220 13564DNALegionella pneumophilia
13atgatgtcta 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
56414187PRTLegionella pneumophilia 14Met 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
15543DNALegionella pneumophilia 15atgaaaggta 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
54316180PRTLegionella pneumophilia 16Met 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
171158DNAHomo sapiens 17atgtacggat 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
115818385PRTHomo sapiens 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 19651DNAHomo sapiens
19atgtatggat 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 65120217PRTHomo sapiens 20Met
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 211158DNARattus norvegicus 21atgtacggtt 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 115822385PRTRattus norvegicus
22Met 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 232229DNARattus norvegicus 23atgtatggat 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
222924742PRTRattus norvegicus 24Met 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 251254DNAArtificial
SequenceSynthetic Construct 25atgtacggat tcgtgaatca cgcgctggag
ctgctggtga tccgcaacta cggccccgag 60gtgtgggaag acatcaaaaa agaggcacag
ttagatgaag aaggacaatt tcttgtcaga 120ataatatatg atgattccaa
aacttatgat ttggtggctg ctgcaagcaa agtcctcaat 180ctcaatgctg
gggaaatcct ccaaatgttc gggaagatgt tttttgtctt ttgccaagag
240tctggttatg atacaatctt gcgtgtcctg ggatctaatg tcagagaatt
tctacagaac 300cttgatgctc tacatgatca ccttgctacc atctacccag
gcatgcgtgc tccttcgttt 360aggtgcacgg atgcagaaaa gggcaaagga
ctcattctgc actactactc cgagagagag 420ggacttcagg atattgtcat
cggaatcatc aaaactgttg ctcaacaaat acatggcacc 480gaaatagaca
tgaaggttat tcagcaaaga aatgaagaat gtgatcatac tcaatttcta
540attgaggaaa aagagtcaaa agaagaagat ttttatgaag atcttgatag
atttgaagaa 600aatggtaccc aggaatcacg catcagccca tataccttct
gcaaagcttt tccttttcac 660ataatatttg accgggacct agtggttact
cagtgtggca acgccatata cagagtgctt 720ccccagctcc agcctgggaa
ctgcagccta ttgtctgtct tctctctcgt ccgtcctcat 780atcgacatta
gtttccatgg gatcctttca cacatcaata cggttttcgt attgagaagc
840aaggaaggat tgttggacgt agagaaattg gaatgtgagg atgagctgac
tggaactgag 900atcagctgct tacgtctcaa gggtcagatg atctacttac
ctgaagcaga tagcatcctt 960tttctgtgtt caccaagtgt gatgaatctg
gatgatctga caaggagagg tctgtatctg 1020agcgacatcc ccctgcacga
tgccacgcgt gacctcgttc ttttgggaga gcaattcaga 1080gaggaataca
aactgactca agaactggaa atcctcacag accggctgca gctcacgtta
1140agagccttgg aagatctcga gggcagcggc ggttatattc ctgaagctcc
aagagatggg 1200caggcttacg ttcgtaaaga tggcgaatgg gtattacttt
ctaccttttt atga 125426417PRTArtificial SequenceSynthetic Construct
26Met 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 Asp Leu Glu Gly Ser Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp
Gly385 390 395 400 Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu
Leu Ser Thr Phe 405 410 415 Leu 27681DNAArtificial
SequenceSynthetic Construct 27atgtacggat tcgtgaatca cgcgctggag
ctgctggtga tccgcaacta cggccccgag 60gtgtgggaag acatcaaaaa agaggcacag
ttagatgaag aaggacaatt tcttgtcaga 120ataatatatg atgattccaa
aacttatgat ttggtggctg ctgcaagcaa agtcctcaat 180ctcaatgctg
gggaaatcct ccaaatgttc gggaagatgt tttttgtctt ttgccaagag
240tctggttatg atacaatctt gcgtgtcctg ggatctaatg tcagagaatt
tctacagaac 300cttgatgctc tacatgatca ccttgctacc atctacccag
gcatgcgtgc tccttcgttt 360aggtgcacgg atgcagaaaa gggcaaagga
ctcattctgc actactactc cgagagagag 420ggacttcagg atattgtcat
cggaatcatc aaaactgttg ctcaacaaat acatggcacc 480gaaatagaca
tgaaggttat tcagcaaaga aatgaagaat gtgatcatac tcaatttcta
540attgaggaaa aagagtcaaa agaagaagat ttttatgaag atctcgaggg
cagcggcggt 600tatattcctg aagctccaag agatgggcag gcttacgttc
gtaaagatgg cgaatgggta 660ttactttcta cctttttatg a
68128226PRTArtificial SequenceSynthetic Construct 28Met 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 Glu Gly Ser Gly Gly Tyr Ile Pro Glu Ala
Pro Arg Asp 195 200 205 Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp
Val Leu Leu Ser Thr 210 215 220 Phe Leu225
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