BR112015016299B1 - POLYMER H-NOX PROTEIN, ITS PRODUCTION METHOD, PHARMACEUTICAL COMPOSITION COMPRISING THE SAME, RECOMBINANT NUCLEIC ACID, VECTOR, TRANSGENIC MICROORGANISM, AND KIT - Google Patents

Abstract

POLYMER H-NOX PROTEIN, PRODUCTION METHOD THEREOF, RECOMBINANT H-NOX PROTEIN, COMPOSITIONS, RECOMBINANT NUCLEIC ACID, VECTOR, AND CELL. The invention provides polymeric H-NOX proteins for the oxygen carrier with longer circulation half-life compared to monomeric H-NOX proteins. Polymeric H-NOX proteins extravasate into and preferentially accumulate in tumor tissue for sustainable oxygen transport. The invention also provides for the use of H-NOX proteins as radiosensitizers for the treatment of brain cancer.

Description

DECLARATION REGARDING RESEARCH OR DEVELOPMENT SPONSORED BY THE FEDERAL GOVERNMENT

[0001] This work was supported by Grant No. 2 R44 CA138006-02. The U.S. Government has rights in any patent issuance on this application.

TECHNICAL FIELD

[0002] This application concerns polymeric H-NOX proteins and methods of using them to transport oxygen. Polymeric H-NOX proteins provide a new therapeutic tool to deliver O2 to humans and, for veterinary purposes, animals.

BACKGROUND OF THE INVENTION

[0003] H-NOX proteins (named Heme-nitric oxide and Oxygen binding domain) are members of a family of highly conserved and well-characterized hemoproteins (Iyer, LM et al. (2003) BMC Genomics 4(1) :5; Karow, DS et al. (2004) Biochemistry 43(31): 10203-10211; Boon, EM et al. (2005) Nature Chem. Biol. 1:5359; Boon, EM et al. (2005) Curr Opin. Chem. Biol. 9(5):441-446; Boon, EM et al. (2005) J. Inorg. Biochem. 99(4):892-902; Cary, SP et al. (2005) Proc Natl Acad Sci USA 102(37):13064-9; Karow DS et al. (2005) Biochemistry 44(49):16266-74; Cary, SP et al. (2006) Trends Biochem Sci 31(4):231- 9; Boon, EM et al (2006) J Biol Chem 281(31):21892-902; Winger, JA et al (2007) J Biol Chem 282(2):897-907). H-NOX proteins are neutral nitric oxide, unlike previous hemoglobin-based oxygen transporters, H-NOX does not pass through circulating nitric oxide, and thus is not associated with hypertensive or renal side effects. The low intrinsic reactivity of NO (and the high stability of NO) make 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 pass endogenous NO through H-NOX proteins. Importantly, the presence of a distal tyrosine pocket in some H-NOX proteins (Pellicena, P. et al. (2004) Proc Natl. Acad Sci USA 101(35):12854-12859) is suggestive of high reactivity to undesirable NO, contraindicating use as a blood substitute. For example, by analogy, a Mycobacterium tuberculosis hemoglobin protein, with a structurally analogous distal tyrosine pocket, reacts extremely quickly with NO, and is used by Mycobacterium to effectively pass through and avoid defensive NO produced by an infected host (Ouellet, H. et al. (2002) Proc. Natl. Acad. Sci.U S A 99(9):5902-5907). However, it was surprisingly discovered that H-NOX proteins actually have a much lower reactivity to NO than that of hemoglobin which makes their use as a blood substitute possible.

[0004] It has been discovered that H-NOX proteins that bind NO but not O2 can be converted into H-NOX proteins that bind both NO and O2 by introducing a single amino acid mutation (see WO 2007/139791 and WO 2007/139767 ). Thus, the affinity of H-NOX proteins for O2 and NO and the ability of H-NOX proteins to discriminate between O2 and NO ligands can be altered by introducing one or more amino acid mutations, allowing the H-NOX proteins are adapted to bind O2 or NO with the desired affinities. Furthermore, mutations can be introduced to further alter the affinity for O2 and/or NO. The H-NOX protein family can therefore be manipulated to exhibit improved or optimal kinetic or thermodynamic properties for O2 transport. For example, mutant H-NOX proteins have been generated with altered dissociation constants and/or dissociation-to-O2 binding ratios that improve the utility of H-NOX proteins for a variety of clinical and industrial applications. The ability to tune H-NOX proteins to bind and transport O2 is a therapeutic avenue that addresses and overcomes the central flaws of current O2 transporters.

[0005] H-NOX proteins are relatively small in size and can be filtered by the kidneys resulting in a short circulation half-life. What is needed for certain therapeutic uses is an H-NOX with a longer circulation half-life that can bind and transport O2 and/or NO to distal tissues for sufficient periods of time. Here they are provided as polymeric H-NOX proteins with the longest circulation half-life. Furthermore, H-NOX proteins extravasate into tumors where they accumulate at different rates. Polymeric H-NOX proteins are tuned to transport oxygen through normoxic regions of tumors and release oxygen deep into hypoxic zones within tumors. This combination of characteristics represents a significant advance in the use of oxygen transporters as modifiers of the hypoxic niches of tumors to increase the effectiveness of radiotherapy, chemotherapy and other anticancer treatments dependent on the oxygenation of tumor cells.

[0006] All references mentioned herein, including patent applications and publications, are incorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

[0007] In some aspects, the invention provides polymeric H-NOX protein characterized by the fact that it encompasses two or more than two H-NOX domains. In some embodiments, the two or more than two H-NOX domains are homologous H-NOX domains. In other embodiments, the H-NOX domains are heterologous H-NOX domains. In some embodiments, the polymeric H-NOX protein is a dimer, a trimer, a tetramer, or a pentamer. In some embodiments, the H-NOX domains are covalently linked.

[0008] In some embodiments of the invention, the polymeric H-NOX protein is characterized by the fact that it encompasses monomers, wherein the monomers encompass 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 C-terminus of the H-NOX domain is covalently linked to the polymerization domain. In other embodiments, the N-terminus of the H-NOX domain is covalently linked to the polymerization domain. In some embodiments, the monomers associate to form a polymeric H-NOX protein.

[0009] In some embodiments of the invention, the polymeric H-NOX protein is a trimeric H-NOX protein. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses one or more of one trimerization domain. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses three monomers, wherein the monomers encompass an H-NOX domain and a trimerization domain. In some embodiments, the trimerization domain is a bacteriophage T4 trimerization domain. In some embodiments, the trimerization domain is a foldon domain. In some embodiments, the foldon domain is characterized by the fact that it encompasses the amino acid sequence of SEQ ID NO:4. In some embodiments, the H-NOX domain is covalently linked to the trimerization domain. In some embodiments, the C-terminus of the H-NOX domain is covalently linked to the N-terminus of the trimerization domain. In other embodiments, the terminus of the H-NOX domains is covalently linked to the N-terminus of the trimerization domain.

[00010] In some embodiments of any of the above embodiments, the polymeric H-NOX protein does not encompass a guanylyl cyclase domain.

[00011] In some embodiments of the above modality, the polymeric H-NOX protein is characterized by the fact that it encompasses at least one marker. In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses at least one His6 tag.

[00012] In some embodiments of any of the above embodiments, the linker amino acids are located between the H-NOX domain and/or the polymerization domain and/or the label. In some embodiments, the linker amino acid is a Gly-Ser-Gly sequence of an Arg-Gly-Ser sequence.

[00013] In some embodiments of any of the above embodiments, at least one of the H-NOX domains is an H-NOX domain from Thermoanaerobacter tengcongensis, an H-NOX domain from L. pneumophilia 2, an H-NOX domain from Homo sapiens β1, an H-NOX domain from Canis lupus, an H-NOX domain from Rattus norvegicus β1, an H-NOX domain from Drosophila melangaster β1, an H-NOX domain from D. melangaster CG14885-PA, an H-NOX domain from Caenorhabdis elegans GCY-35, a Nostoc punctiforme H-NOX domain, a Caulobacter crescentus H-NOX domain, a Shewanella oneidensis H-NOX domain, or a Cperdidoridium acetobutilicum H-NOX domain. In some embodiments, the H-NOX domain corresponds to the T. tengcongensis H-NOX domain set forth in SEQ ID NO:2.

[00014] In some embodiments of any of the above embodiments, at least one of the H-NOX domains is characterized by the fact that it encompasses one or more of a distal pocket mutation. In some embodiments, the distal pocket mutation is an amino acid substitution at a site corresponding to L144 of H-NOX of T. tengcongensis. In some embodiments, at least one of the H-NOX domains is a T. tengcongensis H-NOX domain and at least one of the T. tengcongensis H-NOX domains is characterized by the fact that it encompasses an amino acid substitution at position 144. In In some embodiments, the amino acid substitution at position 144 is an L144F substitution. In some embodiments, at least two of the H-NOX domains are T. tengcongensis H-NOX domains and at least two of the T. tengcongensis H-NOX domains are characterized by the fact that they encompass an amino acid substitution at position 144. In some embodiments, the amino acid substitution of at least one of T. tengcongensis at position 144 is an L144F substitution. In some embodiments, at least one of the H-NOX domains is characterized by the fact that it encompasses at least two distal pocket mutations. In some embodiments, at least two distal pocket mutations are amino acid substitutions at sites corresponding to W9 and L144 of T. tengcongensis H-NOX. In some embodiments, at least one of the H-NOX domains is a T. tengcongensis H-NOX domain and at least one of the T. tengcongensis H-NOX domains is characterized by the fact that it encompasses 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 a L144F substitution.

[00015] In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses three wild types of T. tengcongensis H-NOX domains, each of the H-NOX domains is covalently linked to its C-terminus to the N-terminal of a bacteriophage T4 foldon domain through a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminal of the foldon domain through an Arg-Gly-Ser amino acid linker.

[00016] In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses three L144F H-NOX domains from T. tengcongensis, each of the H-NOX domains is covalently linked to its C-terminus to the N- terminal of a bacteriophage T4 foldon domain via a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminus of the foldon domain via an Arg-Gly-Ser amino acid linker.

[00017] In some embodiments of any of the above embodiments, the O2 dissociation constant of the polymeric H-NOX protein is within 2 orders of magnitude of that of hemoglobin, and in which the NO reactivity of the H-NOX protein is at least 10 times lower than hemoglobin. In some embodiments, the O2 dissociation constant of the polymeric H-NOX protein is between approximately 1 nM and approximately 1000 nM at 20 ° C. In other embodiments, the O2 dissociation constant of the polymeric H-NOX protein is between approximately 1 μM and approximately 10 μM at 20 ° C. In still other embodiments, the O2 dedissociation constant of the H-NOX protein is between approximately 10 μM and approximately 50 μM at 20 ° C. In some embodiments, the NO reactivity of the polymeric H-NOX protein is less than approximately 700 s-1 at 20 °C. In some embodiments, the NO reactivity of the polymeric H-NOX protein is at least 100 times lower than that of hemoglobin. In additional embodiments, the NO reactivity of the polymeric H-NOX protein is at least 1,000 times lower than that of hemoglobin. In some embodiments, the oxygen koff of the polymeric H-NOX protein is less than or equal to approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the polymeric H-NOX protein is between approximately 0.21 s-1 and approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the H-NOX protein is between approximately 1.35 s-1 and approximately 2.9 s-1 at 20 °C. In some embodiments, the heme autoxidation rate of the polymeric H-NOX protein is less than approximately 1 h-1 at 37 °C.

[00018] In some embodiments of the above embodiment, the polymeric H-NOX protein is greater than 50 kDal, greater than 100 kDal, or greater than 150 kDal. In some embodiments, the polymeric H-NOX protein preferentially accumulates in one or more than one tissue in a mammal compared to a corresponding monomeric H-NOX protein comprising a single H-NOX domain upon administration of the H-NOX protein. NOX to the animal. In some embodiments, the polymeric H-NOX protein persists in a mammal for 1, 2, 3, 4, 6, 12, or 24 hours after administration of the H-NOX protein to the mammal. In some embodiments, less than 10% of the polymeric H-NOX is eliminated by the mammal through the kidneys within less than approximately 1 hour, 2 hours or 3 hours after administration of the H-NOX protein to the mammal.

[00019] In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses the amino acid sequence established 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.

[00020] In some aspects, the invention provides a recombinant H-NOX protein characterized by the fact that it encompasses 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 C-terminus of the H-NOX domain is linked to the polymerization domain. In other embodiments, the N-terminus of the H-NOX domain is linked to the polymerization domain. In some embodiments, the H-NOX domain is linked to the N-terminus of the polymerization domain. In some other embodiments, the H-NOX domain is linked to the C-terminus of the polymerization domain. In some embodiments, the polymerization domain is a trimerization domain. In additional embodiments, the trimerization domain is a bacteriophage T4 trimerization domain. In still other embodiments, the trimerization domain is a foldon domain. In some embodiments, the foldon domain is characterized by the fact that it encompasses SEQ ID NO:4.

[00021] In some embodiments of the above embodiment, the recombinant H-NOX protein does not encompass a guanylyl cyclase domain.

[00022] In some embodiments of the above modality, the recombinant H-NOX protein is characterized by the fact that it encompasses a marker. In some embodiments, the recombinant H-NOX protein is characterized by the fact that it encompasses a His6 tag.

[00023] In some embodiments of the above aspect, the linker amino acids are located between the H-NOX domain and/or the polymerization domain and/or the label. In some embodiments, the linker amino acid is a Gly-Ser-Gly sequence of an Arg-Gly-Ser sequence.

[00024] In some embodiments of the above embodiments, the H-NOX domain is an H-NOX domain from Thermoanaerobacter tengcongensis, an H-NOX domain from L. pneumophilia 2, an H-NOX domain from Homo sapiens β1, an H- NOX from Canis lupus, an H-NOX domain from Rattus norvegicus β1, an H-NOX domain from Drosophila melangaster β1, an H-NOX domain from D. melangaster CG14885-PA, an H-NOX domain from Caenorhabdis elegans GCY-35, an H-NOX domain from Nostoc punctiforme, H-NOX domain from Caulobacter crescentus, an H-NOX domain from Shewanella oneidensis, or H-NOX domain from Cperdidoridium acetobutilicum. In some embodiments, the H-NOX domain corresponds to the T. tengcongensis H-NOX domain set forth in SEQ ID NO:2.

[00025] In some embodiments of the above embodiments, the H-NOX domain is characterized by the fact that it encompasses one or more of a distal pocket mutation. In some embodiments, the distal pocket mutation is an amino acid substitution at a site corresponding to L144 of H-NOX of T. tengcongensis. In some embodiments, at least one of the H-NOX domains is a T. tengcongensis H-NOX domain and at least one of the T. tengcongensis H-NOX domains is characterized by the fact that it encompasses an amino acid substitution at position 144. In In some embodiments, the amino acid substitution at position 144 is an L144F substitution. In some embodiments, at least one of the H-NOX domains is characterized by the fact that it encompasses at least two distal pocket mutations. In some embodiments, at least two distal pocket mutations are amino acid substitutions at sites corresponding to W9 and L144 of T. tengcongensis H-NOX. In some embodiments, at least one of the H-NOX domains is a T. tengcongensis H-NOX domain and at least one of the T. tengcongensis H-NOX domains is characterized by the fact that it encompasses 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 a L144F substitution.

[00026] In some embodiments, the recombinant H-NOX protein is characterized by the fact that it encompasses a wild type of the T. tengcongensis H-NOX domain covalently linked to its C-terminus to the N-terminus of a bacteriophage foldon domain T4 through a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminus of the foldon domain via an Arg-Gly-Ser amino acid linker.

[00027] In some embodiments, the recombinant H-NOX protein is characterized by the fact that it encompasses an H-NOX domain from L144F of T. tengcongensis covalently linked to its C-terminus to the N-terminus of a foldon domain of bacteriophage T4 through a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminus of the foldon domain via an Arg-Gly-Ser amino acid linker.

[00028] In some embodiments of the above embodiments, in which the O2 dissociation constant of the recombinant H-NOX protein is within 2 orders of magnitude of that of hemoglobin, and in which the NO reactivity of the H-NOX protein is at least 10 times lower than that of hemoglobin. In some embodiments, in which the O2 dissociation constant of the recombinant H-NOX protein is between approximately 1 nM and approximately 1000 nM at 20 ° C. In other embodiments, in which the O2 dissociation constant of the recombinant H-NOX protein is between approximately 1 μM and approximately 10 μM at 20 ° C. In still other embodiments, the O2 dissociation constant of the H-NOX protein is between approximately 10 μM and approximately 50 μM at 20 ° C. In some embodiments, a NO reactivity of the recombinant H-NOX protein is less than approximately 700 s-1 at 20 °C. In some embodiments, the NO reactivity of the recombinant H-NOX protein is at least 100 times lower than that of hemoglobin. In additional embodiments, the NO reactivity of the recombinant H-NOX protein is at least 1,000 times lower than that of hemoglobin. In some embodiments, the oxygen koff of the recombinant H-NOX protein is less than or equal to approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the recombinant H-NOX protein is between approximately 0.21 s-1 and approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the H-NOX protein is between approximately 1.35 s-1 and approximately 2.9 s-1 at 20 °C. In some embodiments, the heme autoxidation rate of the recombinant H-NOX protein is less than approximately 1 h-1 at 37 °C.

[00029] In some embodiments of the above aspect, the recombinant H-NOX protein is characterized by the fact that it encompasses the amino acid sequence established 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.

[00030] In some aspects, the invention provides a pharmaceutical composition characterized by the fact that it encompasses the polymeric H-NOX protein that encompasses two or more than two H-NOX domains. In some embodiments, the pharmaceutical composition is characterized by the fact that it encompasses the polymeric H-NOX protein of any of the above embodiments. In some embodiments, the pharmaceutical composition is further characterized by the fact that it comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile. In some embodiments, the pharmaceutical composition is essentially endotoxin-free. In some embodiments, the recombinant H-NOX protein is characterized by the fact that it encompasses the amino acid sequence established 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.

[00031] In some embodiments, the pharmaceutical composition is characterized by the fact that it encompasses the polymeric H-NOX protein is characterized by the fact that it encompasses three wild types of the H-NOX domains of T. tengcongensis, each of the H-NOX domains is linked covalently to its C-terminus to the N-terminus of a bacteriophage T4 foldon domain via a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminus of the foldon domain via an Arg-Gly-Ser amino acid linker.

[00032] In some embodiments, the pharmaceutical composition is characterized by the fact that it encompasses the polymeric H-NOX protein that encompasses three L144F H-NOX domains from T. tengcongensis, each of the H-NOX domains is covalently linked to its C -terminal to the N-terminal of a bacteriophage T4 foldon domain via a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminus of the foldon domain via an Arg-Gly-Ser amino acid linker.

[00033] In some aspects, the invention provides a pharmaceutical composition characterized by the fact that it encompasses a recombinant H-NOX protein that encompasses an H-NOX domain and a polymerization domain. In some embodiments, the pharmaceutical composition is characterized by the fact that it encompasses a recombinant H-NOX protein that encompasses an H-NOX domain and a polymerization domain of any of the above embodiments. In some embodiments, the pharmaceutical composition is further characterized by the fact that it comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is sterile. In some embodiments, the pharmaceutical composition is essentially endotoxin-free. In some embodiments, the recombinant H-NOX protein is characterized by the fact that it encompasses 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.

[00034] In some aspects, the invention provides a method of transporting O2 to a brain tumor in an individual with brain cancer characterized by the fact that it encompasses administering an effective dose of an H-NOX protein to an individual. In some embodiments, administration of the H-NOX protein is used in combination with radiation therapy or chemotherapy.

[00035] In some aspects, the invention provides a method of treating brain cancer in an individual with brain cancer characterized in that it encompasses administering an effective dose of an H-NOX protein to an individual, and administering a dose effective radiation to an individual.

[00036] In some aspects, the invention provides a method of reducing the growth of a brain tumor in an individual with brain cancer characterized by the fact that it encompasses administering an effective dose of an H-NOX protein to an individual, and administering an effective dose of radiation to an individual.

[00037] In some embodiments of the above aspects, radiation or chemotherapy is administered to an individual 1, 2, 3, 4, 5 or 6 hours after H-NOX is administered. In some embodiments, the radiation is X-radiation. In some embodiments, the X-radiation is administered from approximately 0.5 gray to approximately 75 gray. In some embodiments, administration of the H-NOX protein and/or administration of radiation is repeated. In some embodiments, administration is repeated two, three, or four times. In some embodiments, administration is repeated after one week, two weeks, three weeks, or four weeks.

[00038] In some embodiments of the above aspects, 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.

[00039] In some embodiments of the above aspects, administration of the H-NOX protein and radiation is used in combination with other therapies.

[00040] In some embodiments of the above aspects, the H-NOX protein is the H-NOX of T. tengcongensis, the L. pneumophilia 2 H-NOX, the H. sapiens β1, the R. norvegicus β1, the D. melangaster β1, D. melangaster CG14885-PA, C. elegans GCY-35, N. punctiforme H-NOX, C. crescentus H-NOX, S. oneidensis H-NOX, or C. acetobutilicum H-NOX. In some aspects, the H-NOX protein is characterized by the fact that it encompasses an H-NOX domain of the corresponding T. tengcongensis H-NOX domain set forth in SEQ ID NO:2. In some embodiments, H-NOX is characterized by the fact that it encompasses one or more of a distal pocket mutation. In some embodiments, the distal pocket mutation is an amino acid substitution at a site corresponding to L144 of H-NOX of T. tengcongensis. In some embodiments, the H-NOX is the H-NOX of T. tengcongensis characterized by the fact that it encompasses an amino acid substitution at position 144. In some embodiments, the amino acid substitution at position 144 is an L144F substitution. In some embodiments, H-NOX is characterized by the fact that it encompasses at least two distal pocket mutations. In some embodiments, at least two distal pocket mutations are amino acid substitutions at sites corresponding to W9 and L144 of T. tengcongensis H-NOX. In some embodiments, H-NOX is H-NOX from T. tengcongensis characterized by the fact that it encompasses amino acid substitutions at positions 9 and 144. In some embodiments, the amino acid substitution at position 9 is a W9F substitution and the substitution amino acid at position 144 is an L144F substitution.

[00041] In some embodiments of the above aspects, the polymeric H-NOX protein does not encompass a guanylyl cyclase domain.

[00042] In some embodiments of the above aspects, the H-NOX protein is characterized by the fact that it encompasses a marker. In some aspects, the tag is a His6 tag.

[00043] In some embodiments of the above aspects, the O2 dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of hemoglobin, and in which the NO reactivity of the H-NOX protein is at least 10 times lower than hemoglobin. In some embodiments, the O2 dissociation constant of the polymeric H-NOX protein is between approximately 1 nM and approximately 1000 nM at 20 ° C. In other embodiments, the O2 dissociation constant of the H-NOX protein is between approximately 1 μM and approximately 10 μM at 20 °C. In still other embodiments, the O2 dissociation constant of the H-NOX protein is between approximately 10 μM and approximately 50 μM at 20 ° C. In some embodiments, the NO reactivity of the H-NOX protein is less than approximately 700 s-1 at 20 °C. In some embodiments, the NO reactivity of the H-NOX protein is at least 100 times lower than that of hemoglobin. In additional embodiments, the NO reactivity of the H-NOX protein is at least 1,000 times lower than that of hemoglobin. In some embodiments, the oxygen koff of the H-NOX protein is less than or equal to approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the H-NOX protein is between approximately 0.21 s-1 and approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the H-NOX protein is between approximately 1.35 s-1 and approximately 2.9 s-1 at 20 °C. In some embodiments, the rate of heme autoxidation of the H-NOX protein is less than approximately 1 h-1 at 37 °C.

[00044] In some aspects, the invention provides a method for transporting oxygen to an individual in need of it, said method is characterized by the fact that it encompasses administering to an individual an effective dose of the polymeric H-NOX protein. In some embodiments, administration of the H-NOX protein is used in combination with radiation therapy or chemotherapy.

[00045] In some aspects, the invention provides a method for treating cancer in an individual in need thereof characterized by the fact that it encompasses administering an effective dose of polymeric H-NOX protein to an individual, and administering a effective dose of radiation to an individual.

[00046] In some aspects, the invention provides a method for reducing tumor growth in an individual in need thereof characterized by the fact that it encompasses administering an effective dose of an H-NOX protein to an individual, and administering an effective dose of radiation to an individual.

[00047] In some embodiments of the above aspects, radiation or chemotherapy is administered to an individual 1, 2, 3, 4, 5 or 6 hours after H-NOX is administered. In some embodiments, the radiation is X-radiation. In some embodiments, the X-radiation is administered from approximately 0.5 gray to approximately 75 gray. In some embodiments, administration of the H-NOX protein and/or administration of radiation is repeated. In some embodiments, administration is repeated two, three, or four times. In some embodiments, administration is repeated after one week, two weeks, three weeks, or four weeks.

[00048] In some embodiments of the above embodiment, the cancer is brain cancer, lung cancer, colorectal cancer, or skin cancer. In some embodiments, the individual is a mammal. In further embodiments, the mammal is a human. In other embodiments, the mammal is a pet, a laboratory research animal, or a farm animal. In still other 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.

[00049] In some embodiments of the above aspects, administration of the H-NOX protein and radiation is used in combination with other therapies.

[00050] In some embodiments of the above aspects, the polymeric H-NOX protein is characterized by the fact that it encompasses two or more than two H-NOX domains. In some embodiments, the two or more than two H-NOX domains are homologous H-NOX domains. In other embodiments, the H-NOX domains are heterologous H-NOX domains.

[00051] In some embodiments of the above aspects, the polymeric H-NOX protein is a dimer, a trimer, a tetramer, or a pentamer. In some embodiments, the H-NOX domains are covalently linked.

[00052] In some embodiments of the above aspects, the polymeric H-NOX protein is characterized by the fact that it encompasses monomers, in which the monomers encompass 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 C-terminus of the H-NOX domain is covalently linked to the polymerization domain. In other embodiments, the N-terminus of the H-NOX domain is covalently linked to the polymerization domain. In some embodiments, monomers associate to form a polymeric H-NOX protein.

[00053] In some embodiments of the above aspects, the polymeric H-NOX protein is a trimeric H-NOX protein. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses one or more of one trimerization domain. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses three monomers, wherein the monomers encompass an H-NOX domain and a trimerization domain. In some embodiments, the trimerization domain is a bacteriophage T4 trimerization domain. In some embodiments, the trimerization domain is a foldon domain. In some embodiments, the foldon domain is characterized by the fact that it encompasses the amino acid sequence of SEQ ID NO:4. In some embodiments, the H-NOX domain is covalently linked to the trimerization domain. In other embodiments, the C-terminus of the H-NOX domain is covalently linked to the N-terminus of the trimerization domain. In some embodiments, the N-terminus of the H-NOX domain is covalently linked to the N-terminus of the trimerization domain.

[00054] In some embodiments of the above aspects, a tag is covalently linked to the C-terminus of the trimerization domain. In some embodiments, a His6 tag is covalently linked to the C-terminus of the trimerization domain.

[00055] In some embodiments of the above aspects, the linker amino acids are located between the H-NOX domain and/or the polymerization domain and/or the label. In some embodiments, the linker amino acid is a Gly-Ser-Gly sequence of an Arg-Gly-Ser sequence.

[00056] In some embodiments of the above aspects, the polymeric H-NOX protein does not encompass a guanylyl cyclase domain.

[00057] In some embodiments of the above aspects, at least one of the H-NOX domains is a T. tengcongensis H-NOX domain, an L. pneumophilia 2 H-NOX domain, an H. sapiens β1 H-NOX domain , an H-NOX domain from C. lupus, an H-NOX domain from R. norvegicus β1, an H-NOX domain from D. melangaster β1, an H-NOX domain from D. melangaster CG14885-PA, an H- NOX from C. elegans GCY-35, an H-NOX domain from N. punctiforme, H-NOX domain from C. crescentus, an H-NOX dS domain. oneidensis, or H-NOX domain of C. acetobutilicum. In some embodiments, the H-NOX domain corresponds to the T. tengcongensis H-NOX domain set forth in SEQ ID NO:2. In some embodiments of the above aspects, the H-NOX protein is T. tengcongensis H-NOX, L. pneumophilia 2 H-NOX, H. sapiens β1, R. norvegicus β1, D. melangaster β1, D. melangaster CG14885-PA, C. elegans GCY-35, N. punctiforme H-NOX, C. crescentus H-NOX, S. oneidensis H-NOX, or C. acetobutilicum H-NOX. In some embodiments, the H-NOX protein is characterized by the fact that it encompasses an H-NOX domain of the corresponding T. tengcongensis H-NOX domain set forth in SEQ ID NO:2. In some embodiments, H-NOX is characterized by the fact that it encompasses one or more of a distal pocket mutation. In some embodiments, the distal pocket mutation is an amino acid substitution at a site corresponding to L144 of H-NOX of T. tengcongensis. In some embodiments, the H-NOX is the H-NOX of T. tengcongensis characterized by the fact that it encompasses an amino acid substitution at position 144. In some embodiments, the amino acid substitution at position 144 is an L144F substitution. In some embodiments, H-NOX is characterized by the fact that it encompasses at least two distal pocket mutations. In some embodiments, at least two distal pocket mutations are amino acid substitutions at sites corresponding to W9 and L144 of T. tengcongensis H-NOX. In some embodiments, H-NOX is H-NOX from T. tengcongensis characterized by the fact that it encompasses amino acid substitutions at positions 9 and 144. In some embodiments, the amino acid substitution at position 9 is a W9F substitution and the substitution amino acid at position 144 is an L144F substitution.

[00058] In some embodiments, the polymeric H-NOX protein of the methods is characterized by the fact that it encompasses three wild types of T. tengcongensis H-NOX domains, each of the H-NOX domains is covalently linked to its C- terminal to the N-terminus of a bacteriophage T4 foldon domain via a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminus of the foldon domain via an Arg-Gly-Ser amino acid linker.

[00059] In some embodiments, the polymeric H-NOX protein of the method is characterized by the fact that it encompasses three L144F H-NOX domains from T. tengcongensis, each of the H-NOX domains is covalently linked to its C-terminus to the N-terminus of a bacteriophage T4 foldon domain via a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminus of the foldon domain via an Arg-Gly-Ser amino acid linker.

[00060] In some embodiments of the above aspects, the O2 dissociation constant of the H-NOX protein is within 2 orders of magnitude that of hemoglobin, and in which the NO reactivity of the H-NOX protein is at least 10 times more lower than hemoglobin. In some embodiments, the O2 dissociation constant of the polymeric H-NOX protein is between approximately 1 nM and approximately 1000 nM at 20 ° C. In other embodiments, the O2 dissociation constant of the H-NOX protein is between approximately 1 μM and approximately 10 μM at 20 °C. In still other embodiments, the O2 dissociation constant of the H-NOX protein is between approximately 10 μM and approximately 50 μM at 20 ° C. In some embodiments, the NO reactivity of the H-NOX protein is less than approximately 700 s-1 at 20 °C. In some embodiments, the NO reactivity of the H-NOX protein is at least 100 times lower than that of hemoglobin. In additional embodiments, the NO reactivity of the H-NOX protein is at least 1,000 times lower than that of hemoglobin. In some embodiments, the oxygen koff of the H-NOX protein is less than or equal to approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the H-NOX protein is between approximately 0.21 s-1 and approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the H-NOX protein is between approximately 1.35 s-1 and approximately 2.9 s-1 at 20 °C. In some embodiments, the rate of heme autoxidation of the H-NOX protein is less than approximately 1 h-1 at 37 °C.

[00061] In some embodiments of the above aspects, the polymeric H-NOX protein is greater than 50 kDal, greater than 100 kDal, or greater than 150 kDal. In some embodiments, the polymeric H-NOX protein preferentially accumulates in one or more tissues in a mammal compared to a corresponding monomeric H-NOX protein comprising a single H-NOX domain following administration of the H-NOX protein. NOX to the animal. In some embodiments, the polymeric H-NOX protein persists in a mammal for 1, 2, 3, 4, 6, 12, or 24 hours after administration of the H-NOX protein to the mammal. In some embodiments, less than 10% of the polymeric H-NOX is eliminated by the mammal through the kidneys within less than approximately 1 hour, 2 hours or 3 hours after administration of the H-NOX protein to the mammal.

[00062] In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses the amino acid sequence established 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.

[00063] In some aspects, the invention provides recombinant nucleic acid encoding the polymeric H-NOX protein of any of the embodiments described herein. In some embodiments, the nucleic acid is in a vector. The invention also provides a cell characterized by the fact that it encompasses a nucleic acid or a vector encoding the polymeric H-NOX protein or monomeric H-NOX subunit described herein. In some embodiments, the nucleic acid is characterized by the fact that it encompasses the nucleic acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:25 or SEQ ID NO:27.

[00064] In some aspects, the invention provides a method of producing a polymeric H-NOX protein characterized by the fact that it encompasses the cultivation of a cell characterized by the fact that it comprises a nucleic acid encoding the polymeric H-NOS protein or subunit Monomeric H-NOX under the appropriate conditions for the production of polymeric H-NOX protein. In additional embodiments, the method is characterized by the fact that it includes a H-NOX protein purification step.

[00065] In some aspects, the invention provides kits characterized by the fact that they encompass the polymeric H-NOX protein that encompasses two or more than two H-NOX domains. In some embodiments, the kits further include instructions for using the polymeric H-NOX protein. In some embodiments, the two or more than two H-NOX domains are homologous H-NOX domains. In other embodiments, the H-NOX domains are heterologous H-NOX domains. In some embodiments, the polymeric H-NOX protein is a dimer, a trimer, a tetramer, or a pentamer. In some embodiments, the H-NOX domains are covalently linked.

[00066] In some embodiments of the invention, the polymeric H-NOX protein of the kit is characterized by the fact that it encompasses monomers, in which the monomers encompass 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 C-terminus of the H-NOX domain is covalently linked to the polymerization domain. In other embodiments, the N-terminus of the H-NOX domain is covalently linked to the polymerization domain. In some embodiments, monomers associate to form a polymeric H-NOX protein.

[00067] In some embodiments of the invention, the polymeric H-NOX protein of the kit is a trimeric H-NOX protein. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses one or more of one trimerization domain. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses three monomers, wherein the monomers encompass an H-NOX domain and a trimerization domain. In some embodiments, the trimerization domain is a bacteriophage T4 trimerization domain. In some embodiments, the trimerization domain is a foldon domain. In some embodiments, the foldon domain is characterized by the fact that it encompasses the amino acid sequence of SEQ ID NO:4. In some embodiments, the H-NOX domain is covalently linked to the trimerization domain. In some embodiments, the C-terminus of the H-NOX domain is covalently linked to the N-terminus of the trimerization domain. In other embodiments, the terminus of the H-NOX domains is covalently linked to the N-terminus of the trimerization domain.

[00068] In some embodiments of any of the above embodiments, the polymeric H-NOX protein of the kit does not encompass a guanylyl cyclase domain.

[00069] In some embodiments of the above modality, the polymeric H-NOX protein of the kit is characterized by the fact that it encompasses at least one marker. In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses at least one His6 tag.

[00070] In some embodiments of any of the above embodiments, the linker amino acids are located between the H-NOX domain and/or the polymerization domain and/or the label. In some embodiments, the linker amino acid is a Gly-Ser-Gly sequence of an Arg-Gly-Ser sequence.

[00071] In some embodiments of any of the above embodiments, at least one of the H-NOX domain of the kit is an H-NOX domain from Thermoanaerobacter tengcongensis, an H-NOX domain from L. pneumophilia 2, an H-NOX domain from Homo sapiens β1, a Canis lupus H-NOX domain, a Rattus norvegicus β1 H-NOX domain, a Drosophila melangaster β1 H-NOX domain, a D. melangaster CG14885-P H-NOX domain, a H-domain -NOX from Caenorhabdis elegans GCY-35, an H-NOX domain from Nostoc punctiforme, H-NOX domain from Caulobacter crescentus, an H-NOX domain from Shewanella oneidensis, or H-NOX domain from Cperdidoridium acetobutilicum. In some embodiments, the H-NOX domain corresponds to the T. tengcongensis H-NOX domain set forth in SEQ ID NO:2.

[00072] In some embodiments of any of the above embodiments, at least one of the H-NOX domains of the kit is characterized by the fact that it encompasses one or more of a distal pocket mutation. In some embodiments, the distal pocket mutation is an amino acid substitution at a site corresponding to L144 of H-NOX of T. tengcongensis. In some embodiments, at least one of the H-NOX domains is a T. tengcongensis H-NOX domain and at least one of the T. tengcongensis H-NOX domains is characterized by the fact that it encompasses an amino acid substitution at position 144. In some embodiments, at least one of the H-NOX domains is characterized by the fact that it encompasses an amino acid substitution at position 144. embodiments, the amino acid substitution at position 144 is an L144F substitution. In some embodiments, at least two of the H-NOX domains are T. tengcongensis H-NOX domains and at least two of the T. tengcongensis H-NOX domains are characterized by the fact that they encompass an amino acid substitution at position 144. In some embodiments, The amino acid substitution in at least one of the T. tengcongensis at position 144 is an L144F substitution. In some embodiments, at least one of the H-NOX domains is characterized by the fact that it encompasses at least two distal pocket mutations. In some embodiments, at least two distal pocket mutations are amino acid substitutions at sites corresponding to W9 and L144 of T. tengcongensis H-NOX. In some embodiments, at least one of the H-NOX domains is a T. tengcongensis H-NOX domain and at least one of the T. tengcongensis H-NOX domains is characterized by the fact that it encompasses amino acid substitutions at positions 9 and 144. In In some embodiments, the amino acid substitution at position 9 is a W9F substitution and the amino acid substitution at position 144 is a L144F substitution.

[00073] In some embodiments, the polymeric H-NOX protein of the kit is characterized by the fact that it encompasses three wild types of T. tengcongensis H-NOX domains, each of the H-NOX domains is covalently linked to its C- terminal to the N-terminus of a bacteriophage T4 foldon domain via a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminus of the foldon domain via an Arg-Gly-Ser amino acid linker.

[00074] In some embodiments, the polymeric H-NOX protein of the kit is characterized by the fact that it encompasses three L144F H-NOX domains from T. tengcongensis, each of the H-NOX domains is covalently linked to its C-terminal to the N-terminus of a bacteriophage T4 foldon domain via a Gly-Ser-Gly amino acid linker. In some embodiments, a His6 tag is linked to the C-terminus of the foldon domain via an Arg-Gly-Ser amino acid linker.

[00075] In some embodiments of any of the above embodiments, the O2 dissociation constant of the kit's polymeric H-NOX protein is within 2 orders of magnitude that of hemoglobin, and in which the NO reactivity of the H-NOX protein is at least 10 times lower than that of hemoglobin. In some embodiments, the O2 dissociation constant of the polymeric H-NOX protein is between approximately 1 nM and approximately 1000 nM at 20 ° C. In other embodiments, the O2 dissociation constant of the polymeric H-NOX protein is between approximately 1 μM and approximately 10 μM at 20 ° C. In still other embodiments, the O2 dissociation constant of the H-NOX protein is between approximately 10 μM and approximately 50 μM at 20 ° C. In some embodiments, the NO reactivity of the polymeric H-NOX protein is less than approximately 700 s-1 at 20 °C. In some embodiments, the NO reactivity of the polymeric H-NOX protein is at least 100 times lower than that of hemoglobin. In additional embodiments, the NO reactivity of the polymeric H-NOX protein is at least 1,000 times lower than that of hemoglobin. In some embodiments, the oxygen koff of the polymeric H-NOX protein is less than or equal to approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the polymeric H-NOX protein is between approximately 0.21 s-1 and approximately 0.65 s-1 at 20 °C. In some embodiments, the oxygen koff of the H-NOX protein is between approximately 1.35 s-1 and approximately 2.9 s-1 at 20 °C. In some embodiments, the heme autoxidation rate of the polymeric H-NOX protein is less than approximately 1 h-1 at 37 °C.

[00076] In some embodiments of the above embodiment, the polymeric H-NOX protein of the kit is greater than 50 kDal, greater than 100 kDal, or greater than 150 kDal. In some embodiments, the polymeric H-NOX protein preferentially accumulates in one or more tissues in a mammal compared to a corresponding monomeric H-NOX protein comprising a single H-NOX domain following administration of the H-NOX protein. NOX to the animal. In some embodiments, the polymeric H-NOX protein persists in a mammal for 1, 2, 3, 4, 6, 12, or 24 hours after administration of the H-NOX protein to the mammal. In some embodiments, less than 10% of the polymeric H-NOX is eliminated by the mammal through the kidneys within less than approximately 1 hour, 2 hours or 3 hours after administration of the H-NOX protein to the mammal.

[00077] In some embodiments, the kit is characterized by the fact that it encompasses any of the polymeric H-NOX proteins as described here. In some embodiments, the kit is characterized by the fact that it encompasses any of the monomeric H-NOX subunits as described herein. In some embodiments, the polymeric H-NOX protein of the kit is characterized by the fact that it encompasses 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.

[00078] In some aspects, the invention provides an article of manufacture characterized by the fact that it encompasses the polymeric H-NOX protein as described herein. In some embodiments, the article of manufacture is characterized by comprising an H-NOX protein and a pocket. In some embodiments, the bag is an IV bag. In some embodiments, the H-NOX protein of the article of manufacture is to transport O2 to an individual in need thereof. In some embodiments, the individual has a brain tumor. In some embodiments, the brain tumor is glioblastoma. In some embodiments, the polymeric H-NOX protein is used in conjunction with radiation therapy.

[00079] In some aspects, the invention provides a unit dose of polymeric H-NOX protein as described herein. In some embodiments, the H-NOX protein of the dose unit is to transport O2 to an individual in need thereof. In some embodiments, the individual has a brain tumor. In some embodiments, the brain tumor is glioblastoma. In some embodiments, the polymeric H-NOX protein is used in conjunction with radiation therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[00080] Figure 1 shows the nucleic acid (SEQ ID NO:3) and amino acid sequence (SEQ ID NO:4) of the fibritin foldon domain of bacteriophage T4.

[00081] Figure 2A shows the nucleic acid (SEQ ID NO:5) and amino acid sequence (SEQ ID NO:6) of the fibritin foldon domain of bacteriophage T4 fused to the C-terminus of the Thermoanaerobacter tengcongensis L144F H-NOX sequence and which includes the His6 tag. Figure 2B shows the nucleic acid (SEQ ID NO:7) and amino acid sequence (SEQ ID NO:8) of the L144F H-NOX-foldon monomer without a His6 tag.

[00082] Figure 3A shows an alignment of the DNA sequence of the Thermoanaerobacter tengcongensis H-NOX-foldon His6 wild-type chimeric protein (top; SEQ ID NO: 9) and the sequencing data of clone 3I-A (bottom; SEQ ID NO:5) which encodes the H-NOX variant L144F with the fused foldon and His6 sequences. The L144F substitution and the Xho I and Hind III restriction sites used for the fusion are highlighted. Figure 3B shows the amino acid sequence of the wild type of Thermoanaerobacter tengcongensis H-NOX-foldon His6 monomer (SEQ ID NO:10). Figure 3C shows the nucleic acid (SEQ ID NO:11) and amino acid sequence (SEQ ID NO:12) of the wild-type H-NOX-foldon monomer without a His6 tag. Figure 3D shows the nucleic acid (SEQ ID NO:25) and amino acid (SEQ ID NO:26) from Canis lupus H-NOX (1-385) fused to the C-terminus of the bacteriophage T4 foldon domain. Figure 3E shows the nucleic acid (SEQ ID NO:27) and amino acid (SEQ ID NO:28) from Canis lupus H-NOX (1-194) fused to the C-terminus of the bacteriophage T4 foldon domain.

[00083] Figure 4 shows SDS-PAGE gel steps in the initial purification of the H-NOX-foldon fusion protein. The Scale is the Novex Sharp Protein Standard (Invitrogen, Gr e Isl e, NY) with 3.5 kDa b and runs to the bottom of the gel. Standard ranges are known amounts of His6-tagged monomeric H-NOX protein (23 kDa). Induction of the H-NOX-foldon fusion can be seen by comparing lanes 4 and 5 (the fusion monomer has a molecular weight of 26.7 kDa). The double b can be seen in lanes 11, 13, 14, and 15 as a result of insufficient DTT in the SDS-PAGE buffer sample for this amount of protein. Track 1: scale; Lane 2: 0.5 μg standard; Lane 3: 1.0 μg standard; Band 4: pre-induced; Lane 5: harvested supernatant; Lane 6: harvested bead: Lane 7: after lysis: Lane 8; after centrifugation: Lane 9: after bead centrifugation; Track 10: NiNTA flow; Track 11: NiNTA group; Lane 12: incorrect NiNTA group; Lane 13: pre-DEAE sample; Track 14; Post-DEAE group; Track 15: final product.

[00084] Figure 5 shows an SDS PAGE gel of steps in the expression and purification of the H-NOX-foldon fusion protein. The H-NOX-foldon fusion protein is >95% pure after purification. The relative mobility on the SDS-PAGE gel is consistent with the 26.7 kDa monomer. Lane 1: Precision Plus Dual Color Protein Markers; Track 2: Pre-induced; Band 3: Induced; Lane 4: After lysis; Range 5: Post-heated; Lane 6: Post-centrifuged; Band 7: Ni column flow; Lane 8: Ni wash column; Track 9: After-Ni column; Lane 10: Pre-DEAE spine; Track 11: After DEAE column.

[00085] Figure 6 is a graph showing LC-MS data unrolled from H-NOX-foldon fusion protein analysis. The final mass is consistent with the predicted molecular mass of 26.677 AMU for one H-NOX-foldon monomer unit.

[00086] Figure 7 shows an analytical size chromatogram of H-NOX-foldon protein exclusion chromatography. The chromatogram follows three wavelengths (254, 280, and 418 nm) to monitor both a protein and heme constituents. The H-NOX-foldon Protein elutes at 14.24 mL retention volume and an estimated molecular size of 75.6 kDa (similar to the 80.0 kDa predicted for an H-NOX-foldon trimer).

[00087] Figure 8 shows spectroscopic analysis of the H-NOX-foldon protein. Foldon domain fusion does not interfere with the tertiary structure of the H-NOX domain, self-coordinating porphyrin IX binding, or oxygen binding to the porphyrin. Characteristic spectral peaks including the Soret peak (415 nm), and the α/β peaks (550600 nm) are all preserved between an H-NOX monomer and the H-NOX Protein-foldon fusion.

[00088] Figure 9 is a model of the trimerized H-NOX protein with the trimerized foldon domain in the center. Cofactor porphyrin IX and bound oxygen are shown with spheres.

[00089] Figure 10 shows H-NOX trimer plasma profiles after intravenous bolus (100 mg/kg) in two different rats.

[00090] Figure 11 shows IgG and IgM antibodies are produced in response to the dosage of H-NOX trimer (50mg/kg) in rats. IgG or IgM antibodies in plasma (diluted 1:10,000) from mice (curves for individual mice are shown) dosed intravenously with 50mg/kg H-NOX trimer on Days 1, 3, 5, and 22. Plasma samples were examined in the ELISA assay in triplicate. Average, +/- SEM.

[00091] Figure 12 shows that H-NOX monomer and H-NOX trimer is distributed and retained in mice bearing HCT-116 colon-derived tumors. A) Immunohistochemical 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 eliminated in 60 minutes. B) Quantification of H-NOX Protein by staining intensity in HCT-116 tumor sections. N= 6, all as groups. Mean values +/- SEM. C) Biodistribution of H-NOX in syngeneic RIF1 sarcoma tumors.

[00092] Figure 13 shows that H-NOX monomer and H-NOX trimer reduced tumor hypoxia in mice bearing HCT-116 colon-derived tumors. A) Representative tumor section of 125 mm3 tumor isolated from mice treated with vehicle, H-NOX monomer, or H-NOX trimer. B) Quantification of an antipimonidazole antibody (Hypoxia Test-1) intensity in tumor sections. N= 6, all as groups. Mean values +/- SEM. * indicates hypoxia throughout the tumor, ** indicates no hypoxia in the tumor.

[00093] Figure 14 shows tumor penetration and oxygenation by H-NOX monomer in mice bearing HCT-116 colon-derived tumors. A) Tumor sections stained with an anti-H-NOX protein antibody. B) Tumor sections stained with Hypoxia Test-1. C) Quantification of Hypoxia Test-1 as a function of distance from vasculature in tumors from six mice per group. * indicates hypoxia throughout the tumor, ¥ indicates no hypoxia within the tumor.

[00094] Figure 15 shows that H-NOX trimer was distributed and retained in mice carrying a syngeneic RIF-1 sarcoma tumor. Immunofluorescence images of representative section of an isolated 400 mm3 tumor from a mouse 120 minutes after administration of A) 750 mg/kg H-NOX trimer or C) buffer, and of isolated 800 mm3 tumor from a mouse 120 minutes after administration of B) 750 mg/kg H-NOX trimer or D) buffer. H-NOX protein staining was done with anti-H-NOX antibody. Panels E and F show tumor oxygenation by H-NOX trimer in mice bearing syngeneic RIF-1 sarcoma tumor. E) Tumor sections stained with an anti-pimonidazole antibody two hours after administration of H-NOX or buffer control. Photo of the entire tumor is shown. F) Tumor sections stained with anti-pimonidazole antibody (Hypoxia Test-1) and anti-CD31 antibody (BD Bioscience) two hours after administration of H-NOX or buffer control. High magnification photo is shown. G) Biodistribution of H-NOX in syngeneic RIF1 sarcoma tumors. Two hours after intravenous injection, H-NOX trimer diffuses from the vasculature into the tumor tissue. Immunohistochemical staining of tumor sections with H-NOX antibody and CD31 antibody (vascularization marker, BD Bioscience). No fluorescent staining is detected in the buffer-injected mice.

[00095] Figure 16 shows that the H-NOX trimer penetrated tumor in mice bearing sarcoma-derived tumor and reduced tumor hypoxia. A) Membrane Western blot was probed with an anti-H-NOX antibody for detection of H-NOX trimer, with Hypoxia Test-1 for detection of proteins associated with hypoxia, or with an anti-actin antibody for assessment of total protein levels. B) Quantification of pimonidazole staining intensity in tumor sections. C) Quantification of the intensity of anti-HIF-1α staining in tumor sections.

[00096] Figure 17 is a panel of immunohistochemistry images showing tumor penetration by the H-NOX trimer and reduced brain tumor hypoxia in mice bearing U251 orthotopic brain tumors. A) Staining of H-NOX trimer with an anti-H-NOX antibody in a U251 tumor two hours after administration with H-NOX trimer or saline (control). B) Hypoxia-1 test staining in U251 tumors two hours after administration with H-NOX trimer or saline (control). Enlarged images of a portion of the tumors are shown.

[00097] Figure 18 shows tumor penetration by the H-NOX trimer and reduced brain tumor hypoxia in mice bearing U251 orthotopic brain tumors. A) Immunofluorescence images of hypoxia-1 test staining in U251 tumors two hours after administration with H-NOX trimer (right of panels) or saline (buffer, left of panels). B) Quantification of Hypoxia-1 test staining from immunofluorescence images (H-NOX trimer - right of panels or saline - left of panels). C) Images of HIF-1α immunofluorescence staining in U251 tumor two hours after administration with H-NOX trimer or saline (buffer). D) Quantification of HIF-1α staining from immunofluorescence images.

[00098] Figure 19 shows the biodistribution of H-NOX trimer in orthotopic U251 brain tumor and in healthy brain. A) Staining of H-NOX trimer with an anti-H-NOX antibody in a U251 tumor two hours after administration with H-NOX trimer. B) DAPI nuclear staining in U251 tumors showing tumor location in the brain. C) and D) Enlarged images of a portion of the tumor from A) show a diffusion pattern of H-NOX within the tumor and a restricted vascular pattern outside the tumor. E) Staining of the H-NOX trimer with an anti-H-NOX antibody and vascularization staining with an anti-CD31 antibody (BD Bioscience) in healthy mouse brain.

[00099] Figure 20 shows real-time fluorescent images of H-NOX monomer or H-NOX trimer in mouse orthotopic U251 glioblastoma tumors. A) H-NOX monomer was eliminated for two hours. B) 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.

[000100] Figure 21 shows ex vivo fluorescence images of H-NOX monomer or H-NOX trimer in mouse orthotopic BT-12 glioblastoma tumors. BT-12 brain tumor carriers were resected A) 30 minutes after administration of 750 mg/kg H-NOX monomer, B) 60 minutes after administration of 750 mg/kg H-NOX monomer, C) 60 minutes after administration of 750 mg/kg H-NOX trimer, or D) 60 minutes after vehicle administration.

[000101] Figure 22 shows real-time fluorescence images of H-NOX monomer in mouse orthotopic U251 glioblastoma tumors. The image was acquired at A) 30 minutes, B) 60 minutes, C) 120 minutes, and D) 240 minutes after administration of the H-NOX monomer.

[000102] Figure 23 shows real-time fluorescence images of H-NOX trimer in mouse orthotopic U251 glioblastoma tumors. The image was acquired at A) 30 minutes, B) 60 minutes, C) 120 minutes, and D) 240 minutes after H-NOX trimer administration. Arrows indicate areas of fluorescence; asterisks indicate peak level of fluorescence intensity.

[000103] Figure 24 shows real-time fluorescence images of H-NOX monomer in mouse orthotopic U251 glioblastoma tumors. Accumulation of H-NOX monomer in the kidney at A) 30 minutes and B) 60 minutes after administration of H-NOX monomer.

[000104] Figure 25 shows real-time fluorescence images of H-NOX trimer in mouse orthotopic GBM-43 glioblastoma spinal and intracranial tumors. Distribution of H-NOX trimer in the spine A) before administration of the H-NOX trimer and B) 0.5 hour, C) 1 hour, D) 2 hours, E) 4 hours, and F) 6 hours after administration of the H-NOX trimer.

[000105] Figure 26 shows real-time fluorescence images of H-NOX trimer in mouse orthotopic U251 glioblastoma intracranial tumors. The top panel shows the distribution of H-NOX trimer in the brain before administration of the H-NOX trimer (0 min) and at 30 min, 1 hour, 2 hours, 4 hours, 6 hours, and 72 hours after administration of the H-NOX trimer. NOX trimer. The panel at the bottom shows the distribution of H-NOX monomer.

[000106] Figure 27 shows real-time bioluminescence images of H-NOX trimer in mouse orthotopic U251 glioblastoma spinal and intracranial tumors. Distribution of H-NOX trimer A) before administration of the H-NOX trimer and at B) 30 min, C) 1 hour, D) 2 hours, E) 4 hours, and F) 6 hours after administration of the H-NOX trimer at a dose of 295 mg/kg.

[000107] Figure 28 shows real-time fluorescence images of H-NOX trimer in mouse orthotopic U251 glioblastoma tumors. Distribution of H-NOX trimer A) before administration of the H-NOX trimer and at B) 30 min, C) 1 hour, D) 2 hours, E) 4 hours, and F) 6 hours after administration of the H-NOX trimer at a dose of 30 mg/kg.

[000108] Figure 29 shows real-time fluorescence images of variant L144F H-NOX trimer distribution in an orthotopic U251 mouse glioblastoma model that contains small intracranial tumors. Distribution of H-NOX trimer L144F variant A) before administration of the H-NOX trimer and at B) 30 min, C) 1 hour, D) 2 hours, E) 4 hours, and F) 6 hours after administration of H- NOX trimer variant L144F at a dose of 30 mg/kg. Small tumors were 1000 times smaller than larger tumors as determined by bioluminescence qualification (BLI).

[000109] Figure 30 shows fluorescence images of H-NOX trimer distribution. Ex vivo fluorescence imaging of mouse orthotopic GBM43 glioblastoma model administered A) 30 mg/kg H-NOX trimer or B) 750 mg/kg H-NOX trimer. Real-time bioluminescence imaging in an orthotopic U251 mouse glioblastoma model containing C) large intracranial tumors or D) small intracranial tumors following administration of 295 mg/kg H-NOX trimer.

[000110] Figure 31 shows real-time fluorescence images of H-NOX trimer distribution in two mouse orthotopic glioblastoma tumor models (U251 and GBM43) and an atypical teratoid / rhabdoid tumor model (AT/RT) . Images were taken 60 minutes after administration of the H-NOX trimer and the color scale for each image was optimized.

[000111] Figure 32 shows ex vivo fluorescence images of H-NOX protein distribution in a hemisphere tumor carrier from three mouse models of orthotopic glioblastoma tumors. A) Distribution of H-NOX trimer 60 minutes after administration in a GBM43 orthotopic glioblastoma mouse model, B) Distribution of H-NOX trimer 6 days after administration in an orthotopic U251 glioblastoma mouse model, C) H-NOX monomer distribution 30 minutes after administration in a BT-12 atypical teratoid/rhabdoid tumor (AT/RT) mouse model, D) Distribution of H-NOX trimer 60 minutes after administration in an orthotopic BT-12 AT/RT mouse model, and E) lack of H-NOX protein signal 30 minutes after vehicle administration in an orthotopic AT/RT BT-12 mouse model.

[000112] Figure 33 is an immunofluorescence image showing escape of H-NOX trimer from the vasculature and diffusion through a U251 brain tumor in an orthotopic glioblastoma tumor mouse model. Tumor sections were stained with an anti-H-NOX antibody (the top panel) and an anti-CD31 (vasculature) antibody (the bottom panel).

[000113] Figure 34 shows H-NOX trimer ELISA assay in healthy mouse brain. A) ELISA assay in brain after intravenous injection of H-NOX trimer (750 mg/kg). B) ELISA assay in brain after intravenous injection of H-NOX trimer (200 mg/kg). C) Brain/plasma ratio of H-NOX trimer (750 mg/kg). D) Brain/plasma ratio of H-NOX trimer (200 mg/kg). Brain and plasma were collected at 30, 60, 90, and 120 min after H-NOX trimer administration. N= 3, all as groups. Mean values +/- SEM.

[000114] Figure 35 is a series of graphs showing that H-NOX trimer sensitized intracranial xenografts to fractionated radiation therapy in a mouse human U251 glioblastoma model. A) Bioluminescence Mean Qualifications Image (BLI) +/- SEM of mice in both treatment groups, as well as a control group of treated humans (no H-NOX, no RT). N= 9, all as groups. B) Individual BLI ratings for the RT and RT + H-NOX trimer groups on day 29 (table in A). The line shows group mean, +\- SEM. The BLI qualifications of RT + H-NOX trimer mice were significantly lower than those of mice treated with RT alone (p = 0.039, Studant's t-test). C) The H-NOX trimer group showed significantly better survival, as compared to mice that received only radiotherapy (p = 0.025, Log-Rank Test).

[000115] Figure 36 is a series of graphs showing that H-NOX trimer sensitized intracranial xenografts to two-fractionated radiation therapy in a mouse model of human glioblastoma. A) Percent survival in an orthotopic U251 mouse glioblastoma model administered 2 Gy of 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 buffer treatment (TB). Log-Rank Values: 2 Gy compared to 2 Gy + L144F Trimer (p = 0.158), 2 Gy compared to TB (p=0.0612), and L144F Trimer compared to TB (p=0.326). B) Survival percentage 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 therapy radiation therapy (4 Gy x 2), 4 Gy radiation therapy in combination with H-NOX trimer (4 Gy + H-NOX), or buffer treatment (untreated). Log-Rank Values: 4 Gy compared to 4 Gy + H-NOX (p = 0.597), 4 Gy compared to 4 Gy x 2 (p = 0.038), and 4 Gy x 2 compared to 4 Gy + H -NOX (p=0.111).

[000116] Figure 37 shows the nucleic acid and amino acid sequence of the H-NOX proteins. A. Wild type Thermoanaerobacter tengcongensis H-NOX (SEQ ID NOs:1 and 2). B. Wild type Legionella pneumophilia Orf2 H-NOX (SEQ ID NOs:13 and 14). C. Wild type Legionella pneumophilia Orf1 H-NOX (SEQ ID NOs:15 and 16). D. Homo sapiens β1 (1-385) H-NOX (SEQ ID NOs:17 and 18). E. Homo sapiens β2 (1-217) H-NOX (SEQ ID NOs:19 and 20). F. Rattus norvegicus β1 H-NOX (SEQ ID NOs:21 and 22). G. Rattus norvegicus β2 H-NOX (SEQ ID NOs:23 and 24).

DETAILED DESCRIPTION OF THE INVENTION

[000117] The present invention is based in part on the surprising discovery that polymeric H-NOX proteins preferentially extravasate and accumulate in tissues such as the brain, thus contributing to a longer oxygenation window and a longer circulation half-life compared to with monomeric H-NOX proteins. A trimeric H-NOX protein characterized by the fact that it encompasses three H-NOX domains from Thermoanaerobacter tengcongensis and encompasses a L144F mutation has been shown to be useful for transporting oxygen to hypoxic tumor tissue, such as glioblastoma tumor tissue, thereby enhancing therapy. of cancer radiation. Accordingly, the present invention provides oxygen carrier proteins, compositions, kits and methods; for example, as an adjuvant for radiation therapy.

Definitions

[000118] Unless otherwise defined, the meanings of all technical and scientific terms used herein are those commonly understood by those skilled in the art to which this invention belongs. One skilled in the art will also appreciate that any methods and materials similar or equivalent to those described herein may also be used to practice or test the invention.

[000119] For the use described herein, unless clearly indicated otherwise, the use of the terms "a", "a," and a/o refers to one or more than one.

[000120] 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 multiple dependent claims, the use of "or" again refers to more than one preceding independent or dependent claim.

[000121] Reference to "approximately" a value or parameter mentioned herein includes (and describes) embodiments that are directed to this value or parameter per se. For example, the description that refers to "approximately X" includes the description of "X."

[000122] It is understood that the aspects and embodiments of the invention described herein include "characterized by the fact that it encompasses," "encompassing," and "essentially encompassing" aspects and embodiments.

[000123] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum extent. Such amino acid residue polymers may contain natural or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and amino acid residue polymers. Both full-length proteins and fragments thereof are covered 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 that is characterized by the fact that it includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, provided that the protein maintains the desired activity. These modifications may be deliberate, such as through site-specific mutagenesis, or they may be accidental, such as through host mutations that produce the proteins or errors due to PCR amplification. As used herein, the protein may include two or more than two subunits, covalently or non-covalently associated; for example, the protein may include two or more associated monomers.

[000124] The terms "nucleic acid molecule", "nucleic acid" and "polynucleotide" can be used interchangeably, and refer to a nucleotide polymer. Such nucleotide polymers 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 comprising the nucleic acid or polynucleotide molecule.

[000125] As used herein, an "H-NOX Protein" means the protein that has an H-NOX domain (named Heme-nitric oxide and Oxygen Binding Domain). An H-NOX protein may or may not contain one or more of one of the other domains in addition to the H-NOX domain. In some examples, an H-NOX protein does not encompass a guanylyl cyclase domain. An H-NOX protein may or may not encompass a polymerization domain.

[000126] As used herein, the "polymeric H-NOX protein" is an H-NOX protein characterized by the fact that it encompasses two or more than two H-NOX domains. H-NOX domains can be associated covalently or non-covalently.

[000127] As used herein, an "H-NOX domain" is the entire portion of the protein that binds nitric oxide and/or oxygen through heme. The H-NOX domain can encompass heme or can be found as an apolipoprotein that is capable of heme binding. In some examples, an H-NOX domain is characterized by the fact that it 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 approximately 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.

[000128] 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 can promote an association of monomeric H-NOX domains to generate a polymeric H-NOX protein. An example of a polymerization domain is the foldon domain of bacteriophage T4, which promotes the formation of trimeric polypeptides. Other examples of domain polymerizations include, but are not limited to, Arc, POZ, coiled-coil domains (which includes GCN4, leucine zippers, Velcro), uteroglobin, collagen, 3-stranded coiled-coil domains (matrilin-1), thrombosporins , TRPV1-C, P53, Mnt, avidin, streptavidin, Bcr-Abl, COMP, verotoxin B subunit, CamKII, RCK, and N-Ethylmachoimide-sensitive fusion protein domains, STM3548, KaiC, TyrR, Hcp1, CcmK4, GP41, anthrax protective antigen, aerolysin, α-hemolysin, C4b-binding protein, Mi-CK, arylsurfatase A, and viral capsid proteins.

[000129] As used herein, an "amino acid sequence linker" or an "amino acid sequence spacer" is a short polypeptide sequence that can be used to link two domains of the protein. In some embodiments, the amino acid sequence linker is one, two, three, four, five, six, seven, eight, nine, ten, or more than ten amino acids in length. Example amino acid sequence linker includes, but is not limited to, a Gly-Ser-Gly sequence and an Arg-Gly-Ser sequence.

[000130] As used herein, a "His6 tag" refers to a peptide characterized by the fact that it encompasses six His residues attached to a polypeptide. A His6 tag can be used to facilitate protein purification; for example, using specific chromatography for the His6 tag. Following purification, the His6 tag can be cleaved using an exopeptidase.

[000131] The term "substantially similar" or "substantially the same," as used herein, denotes a sufficiently high degree of similarity between two or more of two numerical values such that one skilled in the art would consider the difference between the two or more than two values of little or no biological and/or statistical significance within the context of the biological characteristic measured by the mentioned value. In some embodiments, the two or more of two substantially similar values differ by no more than approximately any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[000132] The phrase "substantially reduced," or "substantially different," as used herein, denotes a sufficiently high degree of difference between two numerical values such that one skilled in the art would consider the difference between the two values to be statistical significance within the context of the biological characteristic measured by the aforementioned values. In some embodiments, the two substantially different numerical values differ by more than approximately any one of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80% , or 90%. In some embodiments, the two substantially different numerical values differ by approximately 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%.

[000133] A "native sequence" polypeptide is characterized by the fact that it encompasses a polypeptide that has the same amino acid sequence as a polypeptide found in nature. Thus, a native polypeptide sequence can have the naturally occurring polypeptide amino acid sequence of any organism. Such native polypeptide sequence may be isolated from nature or may 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., a sequence of extracellular domains), naturally occurring variant forms (e.g., alternatively spliced forms), and variant alleles of the naturally occurring polypeptide.

[000134] A "variant" polypeptide means a biologically active polypeptide that has at least approximately 80% of the amino acid sequence identical to the native sequence of the polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative substitutions as part of sequence identity. Such variants include, for example, polypeptides in which one or more amino acid residues are added, or deleted, at the V or C terminus of the polypeptide. In some embodiments, a variant will have at least approximately any one of 80%, 90%, or 95% identical amino acid sequence to the native sequence of the polypeptide. In some embodiments, the variant will have approximately any one of 80%-90%, 90%-95%, or 95%-99% amino acid sequence identical to the native sequence of the polypeptide.

[000135] As used herein, a "mutant protein" means a protein with one or more mutations compared to a protein that occurs in nature. In one embodiment, the mutant protein has a sequence that differs from all proteins occurring in nature. In various embodiments, the amino acid sequence of the mutant protein is at least approximately any one of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97, 98, 99, or 99 .5% identical to the corresponding region of the protein that occurs in nature. In some embodiments, the amino acid sequence of the mutant protein is at least approximately 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% identical to the corresponding region of the protein that occurs in nature. In some embodiments, the mutant protein is a protein fragment that contains at least approximately any one of 25, 50, 75, 100, 150, 200, 300, or 400 contiguous amino acids of the full-length protein. In some embodiments, the mutant protein is a protein fragment that contains approximately any one of 25-50, 50-75, 75-100, 100-150, 150-200, 200-300, or 300-400 contiguous amino acids of the protein. full extension. Sequence identity can be measured, for example, using sequence analysis software with specified default parameters thereof (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software program compares similar sequences by assigning degrees of homology to various amino acid substitutions, deletions, and other modifications.

[000136] As used herein, "mutation" means a change in a reference nucleic acid or amino acid sequence that occurs in nature. Example of nucleic acid mutations include insertion, deletion, frameshift mutation, silent mutation, nonsense mutation, or "sense" mutation. In some embodiments, the nucleic acid mutation is not a silent mutation. Example of protein mutation includes the insertion of one or more than one 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 of an amino acids (e.g., deletion of N-terminal, C-terminal, and/or internal residues, such as deletion of at least approximately any one of 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, or more amino acids or the deletion of approximately any of 5-10, 10-15, 15-25, 25-50, 50-75, 75100, 100-150, 150-200, 200-300, or 300-400 amino acids), substitution of one or more amino acids (e.g., substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), or combinations of two or more than two of the above. The nomenclature used that refers 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 the tyrosine has been replaced by a leucine at residue number 140. Likewise, an H-NOX protein variant can be referred to by the amino acid variations of the H-NOX protein. For example, a T. tengcongensis H-NOX protein Y140L 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 H-NOX protein W9F /Y140L refers to the 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.

[000137] An "evolutionarily conserved mutation" is the replacement of an amino acid in a protein with an amino acid in the corresponding position of other proteins in the same protein family.

[000138] As used herein, "derived from" refers to the source of the protein into which one or more mutations are introduced. For example, a protein that is "derived from a mammalian protein" refers to a protein of interest that results from the introduction of one or more mutations into the wild-type sequence (i.e., the sequence that occurs in nature). ) the mammalian protein.

[000139] As used herein, "Percentage (%) amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as a percentage of amino acid residues in a candidate sequence that are identical to those amino acid residues in a specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of 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 several ways that are within the state of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGNTM software ( DNASTAR). Those skilled in the art can determine appropriate parameters for measuring alignment, which include any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.

[000140] As used herein, "koff" refers to the rate of dissociation, such as the rate of release of O2 or NO from a protein. A lower numerical value of koff indicates a slower dissociation rate.

[000141] As used herein, "kon" refers to a rate of association, such as the rate of binding of O2 or NO to a protein. A lower numerical value of kon indicates a slower association rate.

[000142] As used herein, "dissociation constant" refers to the "kinetic dissociation constant" or a "calculated dissociation constant". A "kinetic dissociation constant" or "KD" is a ratio of the dissociated kinetic rate (koff) over the associated kinetic rate (kon), such as a KD value determined as an absolute value using standard methods (e.g., standard spectroscopic, stopped flow, or flash photolysis methods) which includes methods known to those skilled in the art and/or described herein. "Calculated dissociation constant" or "calculated KD" refers to an approximation of the kinetic dissociation constant based on the measured koff. The value for kon is derived through the correlation between kinetic KD and koff as described here.

[000143] As used herein, "oxygen affinity" is a qualitative term that refers to the strength of the oxygen binding to the heme portion of the protein. This affinity is affected by both koff and kon for oxygen. A numerically lower oxygen KD value means a higher affinity.

[000144] As used herein, "NO affinity" is a qualitative term that refers to the strength of the NO binding of a protein (such as binding to the heme group or to an oxygen bound to the heme group associated with the protein) . This affinity is affected by both koff and kon for NO. A numerically lower NO KD value means a higher affinity.

[000145] As used herein, "NO Stability" refers to the stability or resistance of the protein to oxidation by NO in the presence of oxygen. For example, the ability of the protein not to be oxidized when bound to NO in the presence of oxygen is indicative of the NO stability of the protein. In some embodiments, less than approximately any one of 50, 40, 30, 10, or 5% of an H-NOX protein is oxidized after incubation for approximately any one of 1.2, 4, 6, 8, 10, 15, or 20 hours at 20°C.

[000146] As used herein, "NO reactivity" refers to the rate at which the iron in the heme-binding protein is oxidized by NO in the presence of oxygen. A lower numerical value for NO reactivity in units of s-1 indicates a lower NO reactivity.

[000147] As used herein, an "autoxidation rate" refers to the rate at which the iron in the heme of the heme-binding protein is autoxidized. A lower numerical value of the autoxidation rate in units of s-1 indicates a lower autoxidation rate.

[000148] The term "vector" is used to describe a polynucleotide that can be structured to contain a cloned polynucleotide or polynucleotides that can be propagated in the 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 expression of the polypeptide of interest, and/or one or more of a selectable genetic marker (such as, for example, antibiotic resistance genes and genes that can be used in colorimetric assays, for example, β-galactosidase). The term "expression vector" refers to the vector that is used to express the polypeptide of interest in the host cell.

[000149] The "host cell" refers to a cell that can be or has been a recipient of the isolated vector or polynucleotide. Host cells can be prokaryotic cells or eukaryotic cells. Example of eukaryotic cells include mammalian cells, such as primates or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Example of prokaryotic cells include bacterial cells; for example, E. coli cells.

[000150] 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 generally 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 the 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 an "isolated" 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 DNA polynucleotide) in which it is typically found in nature, or is separated by minus some of the components of the cell in which it was produced, for example, in the case of RNA polynucleotide. Thus, a DNA polynucleotide that is contained in the vector within the host cell may be referred to as "isolated".

[000151] The terms "individual" or "subject" are used interchangeably here to refer to an animal; for example, a mammal. In some treatment modalities and methods, mammals include, but are not limited to, humans, rodents, monkeys, felines, canines, equines, bovines, porcines, sheep, goats, the mammal is a laboratory animal, the mammal is a farm animal, the mammal is a sport animal, and the mammal is a pet, are provided. In some examples, an "individual" or "subject" refers to an individual or subject who requires treatment for a disease or disorder.

[000152] The "disease" or "disorder" as used herein refers to the condition where treatment is necessary.

[000153] The term "cancer" refers to malignant proliferative disorder associated with uncontrolled cell proliferation, unrestricted cell growth and increased cell death from apoptosis.

[000154] The term "tumor" here is used to refer to the group of cells that exhibit abnormally high levels of proliferation and growth. A tumor can be benign, premalignant, or malignant; Malignant tumor cells are cancerous. Tumor cells can be solid tumor cells or leukemic tumor cells. The term "tumor growth" herein is used to refer to the proliferation or growth by a cell or cells enclosing a tumor that leads to a corresponding increase in the size of the tumor.

[000155] As used herein, "treatment" is an approach to obtaining beneficial or desired clinical results. "Treatment" as used herein encompasses any administration or application of therapy for disease in a mammal, which includes a human. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, any one or more of: relief of one or more symptoms, lessening the extent of disease, preventing or delaying the spread (e.g., (e.g., metastasis, e.g., metastasis to the lungs or lymph nodes) of disease, preventing or postponing the recurrence of disease, postponing or slowing the progress of disease, improving disease status, inhibiting disease or disease progression , inhibiting or slowing down the disease or its progression, stopping its development, and remission (whether partial or total). Also encompassed by "treatment" is the reduction of the pathological consequence of the proliferative disease. The methods of the invention contemplate any one or more of these aspects of treatment.

[000156] In the context of cancer, the term "treatment" is characterized by the fact that it includes any or all of: inhibiting growth of tumor cells or cancer cells, inhibiting replication of tumor cells or cancer cells, reducing overall tumor burden and improving one or more symptoms associated with the disease.

[000157] The terms "inhibition" or "inhibit" refer to a decrease or cessation of any phenotypic characteristics or the decrease or cessation in the incidence, degree, or probability of that characteristic. "Reduce" or "inhibit" is to diminish, reduce, or stop an activity, function, and/or quantity when compared to a reference. In certain embodiments, "reduce" or "inhibit" is meant as the ability to cause an overall decrease of 20% or greater. In other embodiments, "reduce" or "inhibit" is meant as the ability to cause an overall decrease of 50% or greater. In still other embodiments, by "reduce" or "inhibit" is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or 99%.

[000158] As used herein, "postpone the development of the disease" means to defer, hide, slow down, delay, stabilize, suppress and/or postpone the development of the disease (such as cancer). This postponement can be of varying length of time, depending on the history of the disease and/or the individual being treated. As is evident to one skilled in the art, a sufficient or significant delay may, in fact, encompass prevention of the individual not developing the disease. For example, a late stage of cancer, such as development of metastasis, may be delayed.

[000159] The term "reference" as used herein refers to any sample, standard, or level that is used for comparison purposes. The reference can be obtained from a healthy and/or non-diseased sample. In some examples, the reference may be obtained from an untreated sample. In some examples, the reference is obtained from a non-diseased or untreated sample of the individual subject. In some examples, the reference is obtained from one or more healthy individuals other than the subject or patient.

[000160] "Prevention," as used herein, is characterized by the fact that it includes the provision of prophylaxis with respect to the occurrence or recurrence of the disease in the subject who may be predisposed to the disease but has not yet been diagnosed with the disease.

[000161] An "effective dose" of an agent refers to an effective amount, in the dosages and for the period of time necessary, to achieve the desired therapeutic or prophylactic result.

[000162] A "therapeutically effective dose" of the substance/molecule of the invention, agonist or antagonist may vary according to factors such as the state of the disease, 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. The therapeutically effective dose is also one at which any toxic or harmful effects of the substance/molecule, agonist or antagonist are offset by the therapeutically beneficial effects. The therapeutically effective dose can be delivered in one or more than one administration.

[000163] A "prophylactically effective dose" refers to an effective amount, in the dosages and for the period 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 early stage of disease, the prophylactically effective dose will be lower than the therapeutically effective dose.

[000164] The terms "pharmaceutical formulation" and "pharmaceutical composition" refer to the preparation which is in such a form as to allow the biological activity of the active ingredient(s) to be effective, and which does not contain, furthermore, components that are impermissibly toxic to the subject to which the formulation would be administered. Such formulations can be sterile and essentially free of endotoxins.

[000165] A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid, or liquid filling, diluent, encapsulating material, auxiliary formulation, or carrier conventional in the prior art for use with the therapeutic agent that together encompass the "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 in the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.

[000166] A "sterile" formulation is aseptic or essentially free of live microorganisms and their spores.

[000167] Administration "in combination with" one or more of an additional therapeutic agent includes simultaneous (concurrent) and consecutive or sequential administration in any order.

[000168] The term "concurrently" here is used to refer to the administration of two or more than two therapeutic agents, where at least part of the administration overlaps in time or where the administration of a therapeutic agent falls within a short period of time. time relative to the administration of another therapeutic agent. For example, two or more of two therapeutic agents are administered with a time separation of no more than approximately 60 minutes, such as no more than approximately any one of 30, 15, 10, 5, or 1 minutes.

[000169] The term "sequentially" here is used to refer to the administration of two or more of two therapeutic agents where the administration of one or more of one agent continues after discontinuing the administration of one or more of one agent. For example, administration of two or more than two therapeutic agents are administered with a time separation of more than approximately 15 minutes, such as approximately any one of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days , 3 days, 1 week, 2 weeks, or 1 month.

[000170] As used herein, "in conjunction with" refers to the administration of a treatment modality, in addition, to other treatment modalities. As such, "in conjunction with" refers to the administration of a treatment modality before, during, or after the administration of other treatment modalities to an individual.

[000171] The term "package leaflet" is used to refer to instructions usually included in commercial packaging of therapeutic products, which contain information about indications, use, dosage, administration, combination therapy, contraindications and/or warnings relating to use of such therapeutic products.

[000172] An "article of manufacture" is any manufacture (e.g., a package or container) or kit characterized by the fact that it comprises at least one reagent, e.g., a medicine for treating the disease or disorder (e.g., cancer) , or a test to specifically detect a biomarker described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit to carry out the methods described herein.

H-NOX proteins Overview of the H-NOX Protein Family

[000173] 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 Heme-nitric oxide and Oxygen-binding domain). An H-NOX protein may or may not contain one or more of one of the other domains in addition to the H-NOX domain. H-NOX proteins are members of the highly conserved and well-characterized hemoprotein family (Iyer, L. M. et al. (February 3, 2003). BMC Genomics 4(1):5; Karow, D. S. et al. (August 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, MA 02111-1307, USA). In some embodiments, an H-NOX protein has, or is predicted to have, a secondary structure that is characterized by the fact that it includes six alpha helices, followed by two beta strands, followed by one alpha helix, followed by two beta strands. -ribbons. An H-NOX protein can be an apoprotein that is capable of heme binding or a holoprotein with bound heme. An H-NOX protein can covalently or non-covalently bind a heme group. Some H-NOX proteins bind NO but not O2, and others bind both NO and O2. The facultative aerobic H-NOX domains that have been isolated bind NO but not O2. The H-NOX proteins of the aerobic prokaryotic obligates, C. elegans, and D. melanogaster bind NO and O2. Mammals have two H-NOX proteins: β1 and β2. 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 approximately any one of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 , 95, 97, 98, 99, or 99.5% identical to the corresponding region of the naturally occurring entire Thermoanaerobacter tengcongensis H-NOX protein (e.g., SEQ ID NO:2) or a naturally occurring sGC protein (e.g. example, the naturally occurring sGC β1 protein). In some embodiments, the H-NOX domain of an H-NOX protein or the entire H-NOX protein is at least approximately any one 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 the corresponding region of the entire Thermoanaerobacter tengcongensis H-NOX protein that occurs naturally (e.g., SEQ ID NO:2) or the naturally occurring sGC protein (e.g., the naturally occurring sGC β1 protein). As discussed herein, an H-NOX protein may optionally contain one or more of a mutation relative to the corresponding naturally occurring H-NOX protein. In some embodiments, the H-NOX protein is characterized by including one or more domains in addition to the H-NOX domain. In particular embodiments, the H-NOX protein is characterized by the fact that it includes one or more of a domain or the entire sequence of other proteins. For example, the H-NOX protein may be the fusion protein that is characterized by the fact that it includes an H-NOX domain and part or all of other proteins, 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 encompass a guanylyl cyclase domain. In some embodiments, the H-NOX protein is characterized by the fact that it encompasses a marker; for example, a His6 tag.

Polymeric H-NOX proteins

[000174] In some aspects, the invention provides polymeric H-NOX proteins characterized by the fact that they encompass two or more than two H-NOX domains. The two or more H-NOX domains can be covalently linked or non-covalently 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 octamer, a nanomer, or a decamer. In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses homologous H-NOX domains. In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses heterologous H-NOX domains; for example, the H-NOX domain may be characterized by the fact that it encompasses amino acid variants from particular H-NOX domain species or may encompass H-NOX domains from different species. In some embodiments, at least one of the H-NOX domains of the polymeric H-NOX protein is characterized by the fact that it encompasses the mutation corresponding to a T. tengcongensis H-NOX L144F Mutation. In some embodiments, at least one of the H-NOX domains of the polymeric H-NOX protein is characterized by the fact that it encompasses the mutation corresponding to a W9F/L144F mutation of T. tengcongensis H-NOX. In some embodiments, the polymeric H-NOX proteins encompass 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 is characterized by the fact that it encompasses at least one trimerization domain. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses three T. tengcongensis H-NOX domains. In some embodiments of the trimeric H-NOX domain it is characterized by the fact that it encompasses three H-NOX domains of T. tengcongensis L144F. In some embodiments, the trimeric H-NOX domain is characterized by the fact that it encompasses three T. tengcongensis W9F/L144F H-NOX domains.

[000175] In some aspects of the invention, the polymeric H-NOX protein is characterized by the fact that it comprises two or more associated monomers. Monomers can be covalently linked or non-covalently linked. In some embodiments, monomeric subunits of the polymeric H-NOX protein are produced where the monomeric subunits associate in vitro or in vivo to form a polymeric H-NOX protein. In some embodiments, the monomers encompass 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 the polymeric H-NOX protein is characterized by the fact that it encompasses the corresponding mutation for a T. tengcongensis H-NOX L144F mutation. In some embodiments, at least one of the monomeric subunits of the polymeric H-NOX protein is characterized by the fact that it encompasses the corresponding mutation for a T. tengcongensis H-NOX W9F/L144F mutation. 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 is characterized by the fact that it encompasses an H-NOX domain and a bacteriophage T4 foldon domain. In some embodiments, the monomer of a trimeric H-NOX protein is characterized by comprising a T. tengcongensis H-NOX domain and a foldon domain. In some embodiments, the monomer of a trimeric H-NOX protein is characterized by comprising a T. tengcongensis L144F H-NOX domain and a foldon domain. In some embodiments, the monomer of a trimeric H-NOX protein is characterized by comprising a T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some embodiments, the H-NOX protein trimer is characterized by the fact that it encompasses three monomers, each of the monomers characterized by the fact that it encompasses 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 ligand. In some embodiments, at least one H-NOX domain is characterized by the fact that it encompasses a marker. In some embodiments, at least one H-NOX domain is characterized by encompassing a His6 tag. In some embodiments, the His6 tag is linked to the foldon domain with a linker amino acid; for example, an Arg-Gly-Ser ligand. In some embodiments, all H-NOX domains encompass a His6 tag. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses 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.

[000176] The example H-NOX domain of T. tengcongensis is approximately 26.7 kDal. In some embodiments, the polymeric H-NOX protein has an atomic mass greater than any of approximately 50 kDal, 75 kDal, 100 kDal, 125 kDal, to approximately 150 kDal.

[000177] The invention provides polymeric H-NOX proteins that show greater accumulation in one or more than one tissue in an individual compared to a corresponding monomeric H-NOX protein characterized by the fact that it encompasses a single H-NOX domain after administration of the protein H-NOX to an individual. The corresponding H-NOX protein refers to the monomeric form of the H-NOX protein characterized by the fact that it encompasses at least one of the H-NOX domains of the polymeric H-NOX protein. Tissues of preferential accumulation of polymeric H-NOX include, but are not limited to, tumors and tissue with damaged vasculature. In some embodiments, the polymeric H-NOX protein persists in a mammal at least approximately 1, 2, 3, 4, 6, 12 or 24 hours after administration of the H-NOX protein to an individual. In some embodiments, the polymeric H-NOX protein persists in a mammal for approximately 1-2, 2-3, 3-4, 4-6, 6-12, or 12-24 hours after administration of the H-NOX protein to a individual In some embodiments, less than approximately 10% of the polymeric H-NOX is eliminated by the mammal through the kidneys within less than approximately 1 hour, 2 hours or 3 hours after administration of the H-NOX protein to an individual.

Sources of H-NOX proteins and H-NOX domains

[000178] 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 the polymeric H-NOX protein is the protein or domain of a mammal (e.g., a primate (e.g., human, chimpanzee, gorilla, ape, lemur, etc.) .), a bovine, an equine, a porcine, a canine, or a feline), an insect, a yeast, or a bacterium or is derived from such a protein. Example of the mammalian H-NOX protein includes wild-type human and rat soluble guanylate cyclase (such as, the β1 subunit). Examples of the H-NOX proteins include a wild-type H-NOX protein from mammals, for example, 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 from D. melanogaster, A. gambiae, and M. sexta; Examples of non-mammalian wild-type O2-binding H-NOX proteins include wild-type H-NOX proteins from 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 wild-type prokaryotic H-NOX proteins include T. tengcongensis, V. cholera, V. fischerii, N. punctiforme, D. desulfuricans, L. pneumophila 1, L. pneumophila 2, and C. acetobutilicum.

[000179] NCBI Registry Numbers for examples of H-NOX proteins include the following: Homo sapiens β1 [gi:2746083], Rattus norvegicus β1 [gi:27127318], Drosophila melangaster β1 [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_272624 ],Cperdidoridium acetobutilicum [gi:15896488], and Thermoanaerobacter tengcongensis [gi:20807169]. Canis lupus H-NOX is provided by GenBank entry DQ008576. Nucleic acid and amino acid sequence of example H-NOX proteins and domains are provided in Drawing 37.

[000180] Example H-NOX Protein also includes the following H-NOX Proteins that are listed by their gene name, followed by their species abbreviations and Genbank Identifiers (such as the following protein sequence available as Can 21, 2006 ; Can 22, 2006; Can 21, 2007; or Can 22, 2007, all of which are incorporated herein by reference in their entireties): Npun5905_Npu_23129606, alr2278_Ana_17229770, SO2144_Sone_24373702, Mdeg1343_Mde_2302 7521, VCA0720_Vch_15601476,CC2992_Ccr_16127222, Rsph2043_Rhsp_22958463 (gi:46192757), Mmc10739_Mcsp_22999020 , Tar4_Tte_20807169,Ddes2822_Dde_23475919,CAC3243_Cac_15896 488 ,gcy-31_Ce_17568389, CG14885_Dm_24647455,GUCY1B3_Hs_4504215, HpGCS-beta1_Hpul _14245738, Gycbeta100B_Dm_24651577, CG4154_Dm_24646993 (gi:NP_650424.2, gi:62484298), gcy-32_Ce_13539160,gcy-36_Ce_17568391 (gi:32566352, gi :86564713 ), gcy-35_Ce-17507861 (gi:71990146), gcy-37_Ce_17540904 (gi:71985505), GCY1a3_Hs_20535603, GCY1a2-Hs_899477, or GYCa-99B_Dm_729270 (gi:6806773 8) (Lakshminarayan et al. (2003). BMG Genomics 4:5-13). Species abbreviations used in these names include Ana - Anabaena Sp; Ccr - Caulobacter crescentus; Cac - Cperdidoridium acetobutilicum; 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 - Hecamundongosntrotus pulcherrimus; Hs - Homo sapiens.

[000181] Other examples of H-NOX proteins include the following H-NOX proteins that are listed by their organismal names and Pfam Database record number (such as the following protein sequence available May 21, 2006; 22 May 22, 2006; May 17, 2007; May 21, 2007; or May 22, 2007, which are incorporated herein in their entirety by reference): 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 Q75WF0_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 Q6 INK9_XENLA, Homo sapiens Q5T8J7_HUMAN, Homo sapiens GCYA2_HUMAN, Homo sapiens GCYB2_HUMAN, Homo sapiens GCYB1_HUMAN, Gorilla gorilla Q9N193_9PRIM, 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 mu sculus 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 norve gicus 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 O77106_MANSE, Manduca sexta O76340_MANSE, Apis mellifera Q5UAF0_APIME, Apis mellifera Q5FAN0_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 GCY8E_DROME, Drosophila melanogaster GCYDA_DROME, Droso phila 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_9BACT, Psychroflexus torquis ATCC 700755 Q1VQE5_9FLAO, marine gamma proteobacterium HTCC2207 Q1YPJ5_9GAMM, marine gamma proteobacterium HTCC2207 Q1YTK4_9GAMM, Caulobacter crescentus Q9A451_CAUCR, Acidiphilium cryptum JF-5 Q2DG60_ACICY, Rhodobacter sphaeroides Q3J0U9_RHOS4, Silicibacter pomeroyi Q5LPV1_SILPO, Paracoccus denitrificans PD1222, Q3PC67_PARDE, Silicibacter sp TM1040 Q3QNY2_9RHOB, Jannaschia sp Q28ML8_JANSC, Magnetococcus sp MC-1 Q3XT27_9PROT, Legionella pneumophila Q5WXP0_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_9VIBR, Vibrio vulnificus Q8DAE2_VIBVU, Vibrio alginolyticus 12G01 Q1VCP6_VIBAL, Vibrio sp. 0_9GAMM, Oceanobacter sp RED65 Q1N035_9GAMM, Desulfovibrio desulfuricans Q310U7_DESDG, Halothermothrix orenii H 168 Q2AIW5_9FIRM, Thermoanaerobacter tengcongensis Q8RBX6_THETN, Caldicellulosiruptor saccharolyticus DSM 8903 Q2ZH17_CALSA, Clostridium acetobutylicum Q97E73_CLOAB, Alkaliphilus metalliredigenes QYMF Q3C763_9CLOT, 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 that belong to an H-NOX protein family using the Pfam database as described here.

[000182] Furthermore, 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 further identify H-NOX proteins and nucleic acids based on the similarities of their primary, secondary and/or predicted structure of the protein to which they are derived. the well-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. protein alignments and, Copyright (C) 1996-2006 The Pfam Consortium; GNU LGPL Free Software Foundation, Inc., 59 Temple Place - Suite 330, Boston, MA 02111-1307, USA). Standard databases such as a swissprot-trembl database (on the internet 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 effective secondary and/or tertiary structure of an H-NOX protein can be determined using standard methods.

[000183] In some embodiments, the H-NOX domain has the same amino acid in the corresponding position as any of the following distal pocket residues in H-NOX T. tengcongensis: Thr4, Ile5, Thr8, Trp9, Trp67, Asn74, Ile75, Phe78, Phe82, Tyr140, Leu144, or any combination of two or more of the above. In some embodiments, the H-NOX domain has a proline or an arginine at the position corresponding to Pro115 or Arg135 of T. tengcongensis H-NOX, respectively, based on the sequence alignment of its amino acid sequence. In some embodiments, the H-NOX domain has a histidine that corresponds to His105 of R. norvegicus β1 H-NOX. In some embodiments, the H-NOX domain has or is predicted to have a secondary structure that is characterized by the fact that it 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.

[000184] If desired, the 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 O2 transporter can be tested to determine that the H-NOX domain binds O2 using standard methods such as those described herein. If desired, one or more of the mutations described here can be introduced into the H-NOX domain to optimize its characteristics as an O2 transporter. For example, one or more of a mutation may be introduced to alter its O2 dissociation constant, oxygen koff, heme autoxidation rate, NO reactivity, NO stability, or any combination of two or more of the foregoing. Technical standards such as those described here can be used to measure these parameters.

Mutant H-NOX proteins

[000185] As further discussion mentioned herein, an H-NOX protein or an H-NOX domain of the polymeric H-NOX protein may contain one or more of a mutation, such as a mutation that alters the O2 dissociation constant, the koff by oxygen, the rate of heme autoxidation, a reactivity to NO, a/o NO Stability, or any combination of two or more of the foregoing compared to the corresponding wild-type protein. In some embodiments, the invention provides polymeric H-NOX protein characterized by the fact that it encompasses one or more of an H-NOX domain that may contain one or more of a mutation, such as a mutation that alters the O2 dissociation constant, the koff for oxygen, the rate of heme autoxidation, the reactivity to NO, the stability of NO, or any combination of two or more of the foregoing compared to that of the corresponding wild-type protein. Panels of developed H-NOX Domains can be generated by random mutagenesis followed by empirical selection for required or desired dissociation constants, dissociation rate, NO reactivity, stability, physio-compatibility, or any combination of two or more of two of the above in view of the teachings provided here using techniques as described herein and, furthermore, as is known to those skilled in the art. 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 sequences of wild-type and mutant H-NOX proteins) or evolutionarily conserved residues identified from sequence alignments (see, e.g. , 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 sequences of wild-type and mutant H-NOX proteins).

[000186] In some embodiments of the invention, the mutant H-NOX protein or mutant H-NOX domain of the polymeric H-NOX protein has a sequence that differs from all H-NOX proteins or domains that occur in nature. In various embodiments, the amino acid sequence of the mutant protein is at least approximately any one of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97, 98, 99, or 99 .5% identical to the corresponding region of a naturally occurring H-NOX protein. In various embodiments, the amino acid sequence of the mutant protein is approximately 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 the corresponding region of a naturally occurring H-NOX protein. In some embodiments, the mutant protein is a protein fragment that contains at least approximately any one of 25, 50, 75, 100, 150, 200, 300, or 400 contiguous amino acids of the full-length protein. In some embodiments, the mutant protein is a protein fragment that contains 25-50, 5075, 75-100, 100-150, 150-200, 200-300, or 300-400 contiguous amino acids of the full-length protein. Sequence identity can be measured, for example, using sequence analysis software with specified default parameters thereof (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software program compares similar sequences by assigning degrees of homology to various amino acid substitutions, deletions, and other modifications.

[000187] In some embodiments of the invention, the mutant H-NOX protein or the mutant H-NOX domain of the polymeric H-NOX protein is characterized by the fact that it encompasses the insertion of one or more than one amino acid (for example, the /o 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 is characterized by the fact that it encompasses the deletion of one or more than one amino acid (e.g., the deletion of N-terminal, C-terminal, and/or internal residues, such as the deletion of at least approximately any of 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, or more amino acids or the 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 is characterized by the fact that it encompasses the substitution of one or more than one amino acid (e.g., s substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), or combinations of two or more of the aforementioned. In some embodiments, the mutant protein has at least one amino acid change compared to a naturally occurring protein. In some embodiments, the mutant nucleic acid sequence encodes a protein that has at least one amino acid change compared to a naturally occurring protein. In some embodiments, the nucleic acid is a non-degenerate version of a naturally occurring nucleic acid that encodes the protein with an amino acid sequence identical to a naturally occurring protein.

[000188] In some embodiments, the mutation in the H-NOX protein or the H-NOX domain of the polymeric H-NOX protein is an evolutionary conserved mutation (also denoted as class I mutations). Examples, class I mutations are listed in Table 1A. In Table 1A, mutations are numbered/annotated according to human β1 H-NOX sequences, but are analogous to all H-NOX sequences. Thus, the corresponding position in any other H-NOX proteins may be mutated to the indicated residue. For example, Phe4 of human β1 H-NOX can undergo a tyrosine mutation since other H-NOX proteins have a tyrosine at this position. The corresponding phenylalanine residue can be mutated to tyrosine in any other H-NOX proteins. In particular embodiments, one or more of a mutation is confined to evolutionarily conserved residues. In some embodiments, one or more of a mutation 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 selection for NO/O2 dissociation constants, NO reactivity, stability, and physiocompatibility in view of the teachings provided here. Table 1A. Example of Class I H-NOX mutations targeting evolutionarily conserved residues

[000189] In some embodiments, the mutation is a distal pocket mutation, such as mutation of the residue in alpha helix A, D, E, or G (Pellicena, P. et al. (August 31, 2004). Proc Natl Acad Sci USA 101(35):12854-12859). Example distal pocket mutations (also denoted as class II mutations) are listed in Table 1B. In Table 1B, mutations are numbered/annotated according to the human β1 H-NOX sequence, but are analogous for all H-NOX sequences. Because multiple substitutions provide viable mutations at each recited residue, the residue at each indicated position can be modified to any other naturally occurring or non-naturally occurring amino acid (denoted as "X"). Such mutations can produce H-NOX proteins with a variety of desired affinity, stability, and reactivity characteristics. Table 1B. Example of Class II H-NOX mutations targeting distal pocket residues

[000190] In particular embodiments, the mutation is the distal pocket heme mutation. As described here, a crucial determining molecule that prevents the O2-in NO-binding of members of the H-NOX family is the absence of the H-bond donor in the distal heme pocket. Accordingly, in some embodiments, the mutation alters the H-bond between the H-NOX domain and the linkers within the distal pocket. In some embodiments, the mutation alters a distal pocket H-bond and/or transmits reduced O2 binding ligand relative to the corresponding wild-type H-NOX domain. Example distal pocket residues include Thr4, Ile5, Thr8, Trp9, Trp67, Asn74, Ile75, Phe78, Phe82, Tyr140, and Leu144 of T. tengcongensis H-NOX and the corresponding residue in any other H-NOX proteins. In some embodiments, the H-NOX protein or H-NOX domain of the polymeric H-NOX protein is characterized by the fact that it encompasses one or more of a distal pocket mutation. In some embodiments, the H-NOX protein or H-NOX domain of the polymeric H-NOX protein is characterized by the fact that it encompasses one, two, three, four, five, six, seven, eight, nine, ten or more than ten mutations. of distal pouch. In some embodiments, the distal pocket mutation corresponds to the T. tengcongensis H-NOX L144F mutation. In some embodiments, the distal pocket mutation is the T. tengcongensis H-NOX L144F mutation. In some embodiments, H-NOX protein or the H-NOX domain of the polymeric H-NOX protein is characterized by the fact that it encompasses two distal pocket mutations. In some embodiments, the H-NOX protein or H-NOX domain of the polymeric H-NOX protein corresponds to the T. tengcongensis H-NOX W9F/ L144F mutation. In some embodiments, the H-NOX protein or H-NOX domain of the polymeric H-NOX protein is a T. tengcongensis H-NOX W9F/ L144F mutation.

[000191] Residues that are not in the distal pocket can also affect the three-dimensional structure of the heme group; this changing structure affects the binding of O2 and NO to iron in the heme group. Accordingly, in some embodiments, the H-NOX protein or H-NOX domain of the polymeric H-NOX protein has one or more mutations outside the distal pocket. Examples of residues that can be mutated but are not in the distal pocket include Pro115 and Arg135 of H-NOX from T. tengcongensis. In some embodiments, the mutation is in the proximal pocket which is characterized by the fact that it includes His105 as a residue that binds heme irons.

[000192] In some embodiments when two or more than two mutations are present; at least one mutation is in the distal pocket, and at least one mutation is outside the distal pocket (e.g., the mutation in the proximal pocket). In some embodiments, all mutations are in the distal pocket.

[000193] To reduce the immunogenicity of H-NOX protein or H-NOX domains derived from sources other than humans, the 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 may 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 of a surface amino acid may be combined with mutation of two or more of two distal pocket residues, mutation of one or more of a residue outside the distal pocket (e.g., mutation in the pocket proximal), or combinations of two or more of two of the aforementioned.

[000194] The invention also relates to any combination of mutation described herein, such as double, triple, or multiple higher mutations. For example, combinations of any of the mutations described here can be made in the same H-NOX protein. It is noted that mutations in equivalent positions in other mammalian or non-mammalian H-NOX proteins are also encompassed by this invention. Examples of mutant H-NOX proteins or mutant H-NOX domains encompass one or more of a mutation that imparts altered O2 or NO binding relative to the corresponding wild-type H-NOX domain and are operative as a physiologically compatible O2 gas transporter in the blood. of mammal.

[000195] The residue number for the mutation indicates the position in the sequence of the particular H-NOX protein being described. For example, T. tengcongensis I5A refers to the substitution of isoleucine for alanine at the fifth position in H-NOX of T. tengcongensis. The same isoleucine to alanine mutation can be made at 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 β1 amino acid sequence of mammalian H-NOX domains differs by at most two amino acids, mutations that produce the desirable mutant H-NOX proteins or H-NOX domains when introduced into wild-type mouse β1 H-NOX proteins are also expected. that produces the desirable mutant H-NOX proteins or H-NOX domains when introduced into wild-type β1 H-NOX proteins or H-NOX domains from other mammals, such as humans.

[000196] In some embodiments, the H-NOX protein is a trimer characterized by the fact that it encompasses three T. tengcongensis L144F H-NOX domains and three foldon domains. In some embodiments, the H-NOX protein is a trimer characterized by the fact that it encompasses three T. tengcongensis W9F/L144F H-NOX domains and three foldon domains. In some embodiments, the H-NOX protein is a trimer characterized by the fact that it encompasses three wild-type T. tengcongensis H-NOX domains and three foldon domains.

Modifications to H-NOX Proteins

[000197] Any of the wild types or mutant H-NOX proteins, which includes polymeric H-NOX proteins, can be modified and/or formulated using standard methods to improve therapeutic or industrial applications. For example, and particularly applied to engineered heterologous H-NOX proteins, a variety of methods are known in the art for isolating such immunosurveillance agents, which includes cross-linking, 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. (15 August 2004). Biochem J. 382(Pt 1):183-189, all of which are incorporated herein by reference in their integrity, particularly with regard to protein modification) as well as other techniques known to those skilled in the art. Fusing an H-NOX protein, which includes polymeric H-NOX protein, with a human protein such as human serum albumin can increase 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).

[000198] In some embodiments, the H-NOX protein is characterized by the fact that it encompasses one of more markers; e.g., to assist in the purification of H-NOX protein. Examples of tags include, but are not limited to, His6, FLAG, GST, and MBP. In some embodiments, the H-NOX protein is characterized by the fact that it encompasses one of more than one His6 tag. One or more than one His6 tag can be removed before using the polymeric H-NOX protein; e.g., by treatment with an exopeptidase. In some embodiments, the H-NOX protein is a trimer characterized by the fact that it encompasses three T. tengcongensis L144F H-NOX domains, three foldon domains, and three His6 tags. In some embodiments, the H-NOX protein is a trimer characterized by the fact that it encompasses three T. tengcongensis W9F/L144F H-NOX domains, three foldon domains, and three His6 tags. In some embodiments, the H-NOX protein is a trimer characterized by the fact that it encompasses three wild-type T. tengcongensis H-NOX domains, three foldon domains, and three His6 tags.

Polymerization domain

[000199] In some aspects, the invention provides polymeric H-NOX proteins characterized by the fact that they encompass two or more than two H-NOX domains and one or more than one polymerization domain. Polymerization domains are used to link two or more than two H-NOX domains to form a polymeric H-NOX protein. One or more than one polymerization domain can be used to produce dimers, trimers, tetramers, pentamers, etc. of H-NOX proteins. Polymerization domains are known in the art, such as: the bacteriophage T4 fibritin foldon, Arc, POZ, coiled-coil domains (which includes GCN4, leucine zippers, Velcro), uteroglobin, collagen, 3-braided coiled-coil domains (matrilin-1), thrombosporins, TRPV1-C, P53, Mnt, avidin, streptavidin, Bcr-Abl, COMP, verotoxin B subunit, CamKII, RCK, and N-Ethylmachoimide-sensitive fusion protein domains, STM3548, KaiC, TyrR, Hcp1, CcmK4, GP41, anthrax protective antigen, aerolysin, α-hemolysin, C4b-binding protein, Mi-CK, arylsurfatase A, and viral capsid proteins. The polymerization domains may or may not be 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 of 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 still 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.

[000200] Linkers can be used to join a polymerization domain to an H-NOX domain; for example, linker amino acids. In some embodiments, a linker comprising any 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 domain. -NOX. Examples of the linkers include, but are not limited to, Gly-Ser-Gly and Arg-Gly-Ser linkers.

Trimerization domain of Bacteriophage T4 fibritin

[000201] An example of a polymerization domain is the foldon domain of bacteriophage T4. The wac gene of bacteriophage T4 encodes the protein fibritin, a 486 amino acid protein with a trimerization domain at the C-terminus (residues 457-483) (Efimov, V. P. et al. (1994) J Mol Biol 242:470-486). The domain is capable of trimerizing 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 residue 27 trimerization domain, often referred to as a "foldon domain," has been used to construct chimeric trimers in numerous 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 example foldon domain sequence is shown in Figure 1 and provided by SEQ ID NO:4.

[000202] The isolated foldon domain folds into a single β-hairpin structure and trimerizes into a β-propeller structure involving three hairpins (Guthe, S. et al. (2004) J Mol Biol 337:905-915). The structure of the foldon domain has only 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 resolved by X-ray crystallography (Papanikolopoulou, K., et al., (2004) J Biol Chem 279:89918998; Stetefeld, J. et al. (2003) Structure 11:339-346; Yokoi, N. et al. (2010) Small 6: The foldon domain is very stable, capable of maintaining tertiary structure and oligomerization at >10% SDS, 6.0M guanidine hydrochloride, or 80°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.

[000203] 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 still 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.

[000204] In some embodiments, linkers are used to join a foldon domain to an H-NOX domain. In some embodiments, a linker comprising any 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 domain. -NOX. Examples of the 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 characterized by the fact that it spans from the N-terminus to the C-terminus: a T. tengcongensis H-NOX domain, a Gly-Ser-Gly linker amino acid, and a foldon domain. . In some embodiments, the invention provides a trimeric H-NOX protein characterized by the fact that it spans from the N-terminus to the C-terminus: a T. tengcongensis H-NOX domain, a Gly-Ser-Gly linker amino acid, a foldon domain, an Arg-Gly-Ser linker amino acid, and a His6 tag. In some embodiments, the T. tengcongensis H-NOX domain is characterized by the fact that it encompasses a L144F mutation. In some embodiments, the T. tengcongensis H-NOX domain is characterized by the fact that it encompasses 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

[000205] 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 characterized by the fact that they encompass an H-NOX domain as described herein and a polymerization domain. The H-NOX domain and the polymerization domain can be covalently linked or non-covalently linked. In some embodiments, a 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, an 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 still other embodiments, a 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, an 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 encompass a guanylyl cyclase domain.

[000206] In some embodiments, the monomeric H-NOX protein is characterized by the fact that it encompasses a wild-type H-NOX domain. In some embodiments of the invention, the monomeric H-NOX protein is characterized by the fact that it encompasses one or more mutations in the H-NOX domain. In some embodiments, one or more of a mutation alters the O2 dissociation constant, the oxygen koff, the rate of heme autooxidation, the reactivity to NO, the stability of NO, or any combination of two or more of two of the above in comparison with the corresponding wild-type H-NOX domain. In some embodiments, the mutation is a distal pocket mutation. In some embodiments, the mutation is characterized by the fact that it encompasses the mutation that is not in the distal pocket. In some embodiments, the distal pocket mutation corresponds to a T. tengcongensis L144 mutation (e.g., an L144F mutation). In some embodiments, the recombinant monomeric H-NOX protein is characterized by the fact that it encompasses two distal pocket mutations corresponding to a W9 and an L144 mutation of T. tengcongensis (e.g., a W9F/L144F mutation).

[000207] 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 is characterized by the fact that it encompasses 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, a C-terminus of the T. tengcongensis H-NOX Domain is covalently linked to the N-terminus of the foldon domain. In some embodiments, a 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 H-NOX domain of L144F. In some embodiments, the T. tengcongensis domain is an H-NOX domain of W9F/L144F. In some embodiments, the T. tengcongensis domain is the wild-type H-NOX domain.

[000208] In some embodiments, the H-NOX domain is covalently linked to the polymerization domain using an amino acid sequence linker. In some embodiments, the amino acid sequence linker is one, two, three, four, five, six, seven, eight, nine, ten, or more than ten amino acids in length. Example amino acid sequence linker includes, but is 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 characterized by the fact that it encompasses three H-NOX domains and three trimerization sequences in which the H-NOX domain is covalently linked to the trimerization domain through an amino acid sequence linker. In some embodiments, the monomeric H-NOX protein is characterized by the fact that it encompasses the following from the N-terminus to the C-terminus: a T. tengcongensis H-NOX Domain of L144F, a Gly-Ser-Gly amino acid sequence linker , and a foldon domain. In some embodiments, the monomeric H-NOX protein is characterized by the fact that it encompasses the following from the N-terminus to the C-terminus: a T. tengcongensis H-NOX Domain of W9F/L144F, a Gly-Ser amino acid sequence linker -Gly, and a foldon domain. In some embodiments, the monomeric H-NOX protein is characterized by comprising the following from the N-terminus to the C-terminus: a wild-type T. tengcongensis H-NOX domain, a linker of Gly-Ser-amino acid sequences, Gly, and a foldon domain.

[000209] In some embodiments, the recombinant monomeric H-NOX protein is characterized by the fact that it encompasses a marker; e.g., His6, FLAG, GST, or an MBP tag. In some embodiments, the recombinant monomeric H-NOX protein is characterized by the fact that it encompasses a His6 tag. In some embodiments, the recombinant monomeric H-NOX protein does not encompass a marker. In some embodiments, the tag (e.g., a His6 tag) is covalently linked to the polymerization domain using an amino acid sequence spacer. In some embodiments, the amino acid sequence linker is one, two, three, four, five, six, seven, eight, nine, ten, or more than ten amino acids in length. Example amino acid sequence linker includes but is 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 characterized by the fact that it encompasses three H-NOX domains, three trimerization sequences, and three His6 tags, to which the H-NOX domain is covalently linked. to the trimerization domain through an amino acid sequence linker and the trimerization domain is covalently linked to the His6 tag through an amino acid sequence linker. In some embodiments, the monomeric H-NOX protein is characterized by the fact that it encompasses the following from the N-terminus to the C-terminus: a T. tengcongensis H-NOX Domain of L144F, a Gly-Ser-Gly amino acid sequence linker , a foldon domain, an Arg-Gly-Ser sequence linker, and a His6 tag. In some embodiments, the monomeric H-NOX protein is characterized by the fact that it encompasses the following from the N-terminus to the C-terminus: a T. tengcongensis H-NOX Domain of W9F/L144F, a Gly-Ser amino acid sequence linker -Gly, a foldon domain, an Arg-Gly-Ser sequence linker, and a His6 tag. In some embodiments, the monomeric H-NOX protein is characterized by comprising the following from the N-terminus to the C-terminus: a wild-type T. tengcongensis H-NOX domain, a Gly-Ser-amino acid sequence linker Gly, a foldon domain, an Arg-Gly-Ser sequence linker, and a His6 tag.

[000210] In some embodiments, the recombinant monomeric H-NOX protein is characterized by the fact that it encompasses 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

[000211] As described herein, a large number of diverse mutant H-NOX proteins have been generated, which include polymeric H-NOX proteins, providing variations in NO and O2 dissociation constants, O2 koff, NO reactivity, and stability. To provide operative gas transporters in the blood, H-NOX proteins can be used to functionally replace or supplement endogenous O2 transporters such as hemoglobin. In some embodiments, H-NOX proteins, such as polymeric H-NOX proteins, are used to transport O2 to hypoxic tumor tissue (e.g., a glioblastoma) as an adjuvant for radiation therapy or chemotherapy. Accordingly, in some embodiments, an H-NOX protein has a similar or better O2 association rate, O2 dissociation rate, O2 binding dissociation constant, NO stability, NO reactivity, autooxidation rate, plasma retention time, or any combination of two or more of two of the above compared to an endogenous O2 transporter 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 characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis H-NOX domain of L144F and a foldon domain. In some embodiments, the polymeric H-NOX protein is a trimeric H-NOX protein characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses 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 characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis L144F H-NOX domain and a foldon domain.

[000212] In various embodiments, the koff for O2 for an H-NOX protein, which includes the polymeric H-NOX protein, is between approximately 0.01 to approximately 200 s-1 at 20 ° C, such as approximately 0.1 to approximately 200 s-1, approximately 0.1 to 100 s-1, approximately 1.0 to approximately 16.0 s-1, approximately 1.35 to approximately 23.4 s-1, approximately 1.34 to approximately 18 s-1, approximately 1 .35 to approximately 14.5 s-1, approximately 0.21 to approximately 23, 4 s-1, approximately 1.35 to approximately 2.9 s-1, approximately 2 to approximately 3 s-1, approximately 5 to approximately 15 s-1, or approximately 0.1 to approximately 1 s-1. In some embodiments, the H-NOX protein has an oxygen koff that is less than or equal to approximately 0.65 s-1 at 20°C (such as between approximately 0.21 s-1 to approximately 0.65 s-1 at 20°C).

[000213] In various embodiments, the kon for O2 is an H-NOX protein, which includes the polymeric H-NOX protein, is between approximately 0.14 to approximately 60 μM-1s-1 at 20 °C, such as approximately 6 to approximately 60 μM-1s-1, approximately 6 to 12 μM-1s-1, approximately 15 to approximately 60 μM-1s-1, approximately 5 to approximately 18μM-s-1, or approximately 6 to approximately 15 μM-1s- 1.

[000214] In various embodiments, the kinetic or calculated KD for O2 binding by an H-NOX protein, which includes the polymeric H-NOX protein, is between approximately 1 nM to 1 mM, approximately 1 μM to approximately 10 μM, or approximately 10 μM to approximately 50 μM. In some embodiments, the calculated KD for O2 binding is anywhere from approximately 2 nM to approximately 2 μM, approximately 2 μM to approximately 1 mM, approximately 100 nM to approximately 1 μM, approximately 9 μM to approximately 50 μM, approximately 100 μM to approximately 1 mM, approximately 50 nM to approximately 10 μM, approximately 2 nM to approximately 50 μM, approximately 100 nM to approximately 1.9 μM, approximately 150 nM to approximately 1 μM, or approximately 100 nM to approximately 255 nM, approximately 20 nM to approximately 2 μM, 20 nM to approximately 75 nM, approximately 1 μM to approximately 2 μM, approximately 2 μM to approximately 10 μM, approximately 2 μM to approximately 9 μM, or approximately 100 nM to 500 nM at 20 °C. In some embodiments, the kinetic or calculated KD for O2 binding is less than approximately any one of 100 nM, 80 nM, 50 nM, 30 nM, 25 nM, 20 nM, or 10 nM at 20 °C.

[000215] In various embodiments, the kinetic or calculated KD for O2 binding by an H-NOX protein, which includes the polymeric H-NOX protein, is within approximately 0.01 to approximately 100 times that of hemoglobin under the same conditions (such as at 20 °C), such as between approximately 0.1 to approximately 10 times or between approximately 0.5 to approximately 2 times that of hemoglobin under the same conditions (such as at 20 °C). In various embodiments, the kinetic or calculated KD for NO binding by an H-NOX protein is within approximately 0.01 to approximately 100 times that of hemoglobin under the same conditions (such as at 20 °C), such as between approximately 0.1 to approximately 10 times or between approximately 0.5 to approximately 2 times that of hemoglobin under the same conditions (such as at 20 °C).

[000216] In some embodiments, less than approximately any one of 50, 40, 30, 10, or 5% of an H-NOX protein, which includes the polymeric H-NOX protein, is oxidized after incubation for approximately any one of 1 , 2, 4, 6, 8, 10, 15, or 20 hours at 20 °C.

[000217] In various embodiments, a NO reactivity of an H-NOX protein, which includes the polymeric H-NOX protein, is less than approximately 700 s-1 at 20 °C, such as less than approximately 600 s-1, 500 s-1, 400 s-1, 300 s-1, 200 s-1, 100 s-1, 75 s-1, 50 s-1, 25 s-1, 20 s-1, 10 s-1, 50 s-1, 3 s-1, 2 s-1, 1.8 s-1, 1.5 s-1, 1.2 s-1, 1.0 s-1, 0.8 s-1, 0.7 s- 1 , or 0.6 s-1 at 20 °C. In various embodiments, a NO reactivity of an H-NOX protein is between approximately 0.1 to approximately 600 s-1 at 20 °C, such as between approximately 0.5 to approximately 400 s-1, approximately 0.5 to approximately 100 s-1, approximately 0.5 to approximately 50 s-1, approximately 0.5 to approximately 10 s-1, approximately 1 to approximately 5 s-1, or approximately 0.5 to approximately 2.1 s-1 to 20 °C. In various embodiments, the reactivity of an H-NOX protein is at least approximately 10, 100, 1,000, or 10,000 times lower than that of hemoglobin under the same conditions, such as at 20 ° C.

[000218] In various embodiments, the rate of heme autooxidation of an H-NOX protein, which includes the polymeric H-NOX protein, is less than approximately 1.0 h-1 at 37 ° C, such as less than approximately any of 0.9 h-1, 0.8 h-1, 0.7 h-1, 0.6 h-1, 0.5 h-1, 0.4 h-1, 0.3 h -1, 0.2 h-1, 0.1 h-1, or 0.05 h-1 at 37 C. In various embodiments, the rate of heme autoxidation of an H-NOX protein is between approximately 0.006 to approximately 5.0 h-1 at 37°C, such as approximately 0.006 to approximately 1.0 h-1, 0.006 to approximately 0.9 h-1, or approximately 0.06 to approximately 0.5 h-1 at 37° W.

[000219] In various embodiments, the mutant H-NOX protein, which includes the polymeric H-NOX protein, has (a) an O2 or NO dissociation constant, association rate (kon for O2 or NO), or rate of dissociation (koff for O2 or NO) within 2 orders of magnitude that of hemoglobin, (b) has a weaker NO affinity (e.g., at least approximately 10 times, 100 times, or 1000 times weaker) than the of sGC β1, respectively, (c) a reactivity to NO with bound O2 at least 1000 times lower than hemoglobin, (d) an in vivo plasma retention time at least 2, 10, 100, or 1000 times higher than that of hemoglobin, or (e) any combination of two or more of two of the above.

[000220] Suitable examples of O2 transporters provide dissociation constants within two orders of magnitude that of hemoglobin, i.e. between approximately 0.01 and 100 times, such as between approximately 0.1 and 10 times, or between approximately 0.5 and 2 times that of hemoglobin. A variety of established techniques can be used to quantify dissociation constants, such as the techniques described here (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), V eegriff, K. D. et al. (August 15, 2004). Biochem J. 382(Pt 1):183-189, all of which are incorporated herein by reference in their entirety, particularly with regard to measurement of dissociation constants), as well as those known to those skilled in the art. Example O2 transporters provide low or minimize the NO reactivity of the H-NOX protein with bound O2, such as a lower NO reactivity than that of hemoglobin. In some embodiments, the reactivity to NO is much lower, such as at least approximately 10, 100, 1,000, or 10,000-fold lower than that of hemoglobin. A variety of established techniques can 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. (August 15, 2004). Biochem J. 382(Pt 1):183-189, all of which are incorporated herein by reference in their entirety, particularly with regard to measuring reactivity to NO) as well as those known to those skilled in the art. Because H-NOX from wild-type T. tengcongensis has such lower NO reactivity, other wild-type H-NOX proteins and mutant H-NOX proteins may have similar low NO reactivity. For example, H-NOX from T. tengcongensis Y140H has a reactivity to NO similar to that of wild-type T. tengcongensis H-NOX.

[000221] Furthermore, suitable O2 transporters provide high or maximized stability, particularly in vivo stability. A variety of stability metrics can 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 can be used to quantify stability, such as the techniques described here (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 by those experienced in the technique. For in vivo stability in plasma, blood, or tissue, example stability metrics include retention time, release rate, and half-life. H-NOX proteins from thermophilic organisms are expected to be stable at high temperatures. In various embodiments, plasma retention times are at least approximately 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 those skilled in the art, hemoglobin-based blood substitutes are limited by the rapid release of cell-free hemoglobin from the plasma due to the presence of hemoglobin receptors that remove hemoglobin-free cells from the plasma. Since there are no receptors for H-NOX proteins in plasma, wild-type and mutant H-NOX proteins are expected to have longer plasma retention times than hemoglobin. If desired, plasma retention time can be increased by PEGylation or cross-linking of an H-NOX protein or by fusing an H-NOX protein with other proteins using standard methods (such as those described herein and those known to those skilled in the art). .

[000222] In various embodiments, the H-NOX protein, which includes the polymeric H-NOX protein, has an O2 dissociation constant between approximately 1 nM to approximately 1 mM at 20 ° C and an NO reactivity of at least approximately 10 times lower than that of hemoglobin under the same conditions, such as at 20°C. In some embodiments, the H-NOX protein has an O2 dissociation constant between approximately 1 nM to approximately 1 mM at 20°C and a NO reactivity of less than approximately 700 s-1 at 20°C (e.g., lower at approximately 600 s-1, 500 s-1, 100 s-1, 20 s-1, or 1.8 s-1 at 20 °C). In some embodiments, the H-NOX protein has an O2 dissociation constant within 2 orders of magnitude that of hemoglobin and an NO reactivity at least approximately 10 times lower than that of hemoglobin under the same conditions, such as at 20°C. In some embodiments, the H-NOX protein has an oxygen koff of between approximately 0.01 to approximately 200 s-1 at 20°C and a NO reactivity at least approximately 10 times lower than that of hemoglobin under the same conditions. , such as at 20 °C. In some embodiments, the H-NOX protein has a koff per oxygen that is less than approximately 0.65 s-1 at 20°C (such as between approximately 0.21 s-1 to approximately 0.64 s-1 at 20°C). C) and a reactivity to NO at least approximately 10 times lower than that of hemoglobin under the same conditions, such as at 20 °C. In some embodiments of the invention, an O2 dissociation constant of the H-NOX protein is between approximately 1 nM to approximately 1 μM (1000 nM), approximately 1 μM to approximately 10 μM, or approximately 10 μM to approximately 50 μM. In particular embodiments, an O2 dissociation constant of the H-NOX protein is between approximately 2 nM to approximately 50 μM, approximately 50 nM to approximately 10 μM, approximately 100 nM to approximately 1.9 μM, approximately 150 nM to approximately 1 μM , or approximately 100 nM to approximately 255 nM at 20 °C. In various embodiments, the O2 dissociation constant of the H-NOX protein is less than approximately 80 nM at 20 °C, such as between approximately 20 nM to approximately 75 nM at 20 °C. In some embodiments, the NO reactivity of the H-NOX protein is at least approximately 100 times lower or approximately 1,000 times lower than that of hemoglobin under the same conditions, such as at 20 ° C. In some embodiments, a NO reactivity of the H-NOX protein is less than approximately 700 s-1 at 20 °C, such as less than approximately 600 s-1, 500 s-1, 400 s-1, 300 s-1 , 200 s-1, 100 s-1, 75 s- 1, 50 s-1, 25 s-1, 20 s-1, 10 s-1, 50 s-1, 3 s-1, 2 s-1 , 1.8 s-1, 1.5 s-1, 1.2 s-1, 1.0 s-1, 0.8 s-1, 0.7 s-1, or 0.6 s-1 at 20 °C. In some embodiments, the oxygen koff of the H-NOX protein is between 0.01 to 200 s-1 at 20 °C, such as approximately 0.1 to approximately 200 s-1, approximately 0.1 to 100 s-1, approximately 1.35 to approximately 23.4 s-1, approximately 1.34 to approximately 18 s-1, approximately 1.35 to approximately 14.5 s-1, approximately 0.21 to approximately 23.4 s-1, approximately 2 to approximately 3 s-1, approximately 5 to approximately 15 s-1, or approximately 0.1 to approximately 1 s-1. In some embodiments, the O2 dissociation constant of the H-NOX protein is between approximately 100 nM to approximately 1.9 μM at 20 °C, and the oxygen koff of the H-NOX protein is between approximately 1.35 s-1 to approximately 14.5 s-1 at 20 °C. In some embodiments, the rate of heme autooxidation of the H-NOX protein is less than approximately 1 h-1 at 37 °C, such as less than approximately any of 0.9 h-1, 0.8 h- 1, 0.7 h-1, 0.6 h-1, 0.5 h-1, 0.4 h-1, 0.3 h-1, 0.2 h-1, or 0.1 h- 1. In some embodiments, the oxygen koff of the H-NOX protein is between approximately 1.35 s-1 to approximately 14.5 s-1 at 20 °C, and the rate of heme autooxidation of the H-NOX protein is less than approximately 1 h-1 at 37 °C. In some embodiments, the oxygen koff of the H-NOX protein is between approximately 1.35 s-1 to approximately 14.5 s-1 at 20 °C, and the NO reactivity of the H-NOX protein is less than approximately 700 s-1 at 20 °C (e.g., less than approximately 600 s-1, 500 s-1, 100 s-1, 20 s-1, or 1.8 s-1 at 20 °C). In some embodiments, the heme autooxidation rate of the H-NOX protein is less than approximately 1 h-1 at 37 °C, and the NO reactivity of the H-NOX protein is less than approximately 700 s-1 at 20 °C (e.g., less than approximately 600 s-1, 500 s-1, 100 s-1, 20 s-1, or 1.8 s-1 at 20 °C).

[000223] In some embodiments, the viscosity of the H-NOX protein solution, which includes a polymeric H-NOX protein solution, is between 1 and 4 centipoise (cP). In some embodiments, the colloidal oncotic pressure of the H-NOX protein solution is between 20 and 50 mm Hg.

O2 and/or NO Binding Measurement

[000224] Those skilled in the art can easily determine the oxygen and nitric oxide binding characteristics of any H-NOX protein that includes a polymeric H-NOX protein such as a trimeric H-NOX protein by methods known in the art. technique and by, but not limited to, examples of methods described below.

KD: Kinetic: Ratio of kof to kon

[000225] The kinetic KD value is determined for wild-type and mutant H-NOX proteins, which include 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 measurement of O2 association rates, O2 dissociation rates, dissociation constants for O2 binding, auto -oxidation, and NO dissociation rates.

kon (O2 membership fee)

[000226] The association of O2 with heme is measured using flash photolysis at 20 °C. It is not possible to flash off the FeII-O2 complex as a result of the very rapid twinned recombination kinetics; Thus, the FeII-CO complex is subjected to flash photolysis with laser light at 560 nm (Hewlett-Packard, Palo Alto, CA), producing the 5-coordinate FeII intermediate, to which the O2 molecular bond is followed at various lengths. wave. Protein samples are made by anaerobic reduction with 10 mM dithionite, followed by desalting on a PD-10 column (Millipore, Inc., Billerica, MA). Samples are then diluted to 20 μM heme in 50 mM TEA, 50 mM NaCl, pH 7.5 buffer in the controlled atmosphere of the quartz cuvette, with a size of 100 μL to 1 mL and a path length of 1-cm. CO gas is circulated over the headspace of this cuvette for 10 minutes to form the FeII-CO complex, the formation of which is verified by UV visible spectroscopy (Soret maximum 423 nm). This sample is then used to measure CO rebinding kinetics after flash photolysis while still under 1 atmosphere of CO gas, or is opened and stirred in air for 30 minutes to fully oxygenate the buffer before flash photolysis to observe the rebinding events. O2-. The association of O2 with heme is monitored at multiple wavelengths versus time. These traces are fitted with a single exponential using Igor Pro software (Wavemetrics, Inc., Oswego, OR; 2005 latest version). This rate is independent of observation wavelength, but depends on O2 concentration. UV-visible spectroscopy is used to confirm all complexes and intermediates (Cary 3K, Varian, Inc. Palo Alto, CA). Transient absorption data are collected using instruments described in Dmochowski, I. J. et al. (August 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 data is digitized at 200 mega samples s-1.

koff (O2 dissociation rate)

[000227] To measure koff, FeII-θ2 protein complexes (5 μM heme) are diluted in 50 mM TEA, 50 mM NaCl, pH 7.5 anaerobic buffer, and are quickly mixed with an equal volume of the same buffer (anaerobic) containing various concentrations of dithionite and/or saturated CO gas. Data are obtained from a HI-TECH Scientific SF-61 stopped-flow spectrophotometer equipped with a Neslab RTE-100 constant temperature bath set at 20 °C (TGK Scientific LTD., Bradford On Avon, UK). Dissociation of O2 from heme is monitored as an increase in absorbance at 437 nm, a maximum in the FeII - FeII-O2 difference spectrum, or 425 nm, a maximum in the FeII - FeII-CO difference spectrum. The final traces are fitted 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 measured dissociation rates are independent of dithionite concentration and independent of saturated CO as a trap for the reduced species, with and without 10 mM dithionite present.

Kinetic KD

[000228] The kinetic KD is determined by calculating a ratio of koff to kon using the koff and kon measurements described above.

Calculated KD

[000229] To measure the calculated KD, the values for the koff and kinetic KD that are obtained as described above are represented graphically. The linear relationship between koff and kinetic KD is defined by the equation (y=mx+b). The koff values were then interpolated along the line to derive the calculated KD using Excel: MAC 2004 (Microsoft, Redmond, WA). In the absence of measured kon, this interpolation provides a way to relate koff to KD.

Auto-oxidation Rate

[000230] To measure the rate of auto-oxidation, protein samples are reduced anaerobically, then diluted to 5 μM heme in 50 mM TEA, 50 mM NaCl, pH 7.5 aerobic buffer. These samples are then incubated in the Cary 3E spectrophotometer equipped with a Neslab RTE-100 constant temperature bath set at 37 °C and scanned periodically (Cary 3E, Varian, Inc., Palo Alto, CA). The rate of autooxidation is determined from the difference between the maximum and minimum in the FeIII - FeII difference spectrum plotted against time and fitted with a single exponential using Excel: MAC 2004 (Microsoft, Redmond, WA).

Reaction rate with NO

[000231] Reactivity to NO is measured using purified proteins (H-NOX, polymeric H-NOX, Homo sapiens hemoglobin (Hs Hb) etc.) prepared at 2 μM in buffer A and NO prepared at 200 μM in Buffer A (Buffer A: 50 mM Hepes, pH 7.5, 50 mM NaCl). Data are obtained from a HI-TECH Scientific SF-61 stopped-flow spectrophotometer equipped with a Neslab RTE-100 constant temperature bath set at 20 °C (TGK Scientific LTD., Bradford On Avon, UK). The protein is quickly mixed with NO in a 1:1 ratio with an integration time of 0.00125 sec. The wavelengths of maximum change are fitted to a single exponential using the software that is part of the spectrometer, essentially measuring the limiting rate step of oxidation by NO. The end products of the reaction are ferric-NO for HNOX proteins and ferric-aquo for Hs Hb.

p50 measurements

[000232] 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 the HemOx analyzer. The measuring chamber starts at 0% oxygen and is slowly raised, gradually, until it reaches 100% oxygen. An in-chamber oxygen test measures % oxygen saturation. A second test (UV-Vis light) measures two absorption wavelengths, adjusted to the alpha and beta peaks of the UV-Vis spectrum of the hemoprotein (e.g., a protein such as H-NOX complexed with heme). These absorption peaks increase linearly as hemoproteins bind oxygen. The percentage 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.

[000233] Specifically, the HemOx analyzer (TCS Scientific Corporation, New Hope, PA) determines the oxyhemoprotein dissociation curve (ODC) by exposing 50 μL of blood or hemoprotein to a partial increase in oxygen pressure and deoxygenating it with nitrogen gas. A Clark oxygen electro will detect 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 shown on the y-axis. Blood samples are taken from the antemedial vein, anticoagulated with heparin, and kept at 4°C on wet ice until assayed. Fifty μL of whole blood is diluted in 5 μL of Hemox-solution, a buffer provided by the manufacturer that maintains the pH of the solution at a value of 7.4±0.01. The buffer sample is poured into a cuvette that is part of the HemOx analyzer and the temperature of the mixture is equilibrated and brought to 37°C; the sample is then oxygenated to 100% with air. After adjusting the O2 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 a point at which O2 saturation is 50% using the software that is part of the HemOx analyzer. The time required for a complete registration is approximately 30 minutes.

H-NOX nucleic acids

[000234] The invention also features nucleic acids encoding any of the mutant H-NOX proteins, polymeric H-NOX, or monomer subunits of the recombinant H-NOX protein as described herein.

[000235] In particular embodiments, the nucleic acid is characterized by the fact that it includes a segment of or the entire nucleic acid sequence of any of the nucleic acids encoding an H-NOX protein or an H-NOX domain. In some embodiments, the nucleic acid is characterized by the fact that it includes at least approximately 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, or more contiguous nucleotides of an H-NOX nucleic acid and contains a or more than one mutation (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations) compared to an H-NOX nucleic acid from which it was derived. In various embodiments, a mutant H-NOX nucleic acid contains fewer than approximately 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations compared to an H-NOX nucleic acid of which was derived. The invention also features degenerate variants of any nucleic acid encoding the mutant H-NOX protein.

[000236] In some embodiments, the nucleic acid is characterized by the fact that it includes nucleic acids that encode two or more than two H-NOX domains. In some embodiments, nucleic acids that include two or more than two H-NOX domains are linked in such a manner that the polymeric H-NOX protein is expressed from the nucleic acid. In additional embodiments, the nucleic acid is characterized by the fact that it includes nucleic acids that encode one or more than one polymerization domain. In some embodiments, nucleic acids that include the two or more of two H-NOX domains and one or more of one polymerization domain are linked in such a way that the polymeric H-NOX protein is expressed from the nucleic acid.

[000237] In some embodiments, the nucleic acid is characterized by the fact that it includes a segment or the entire nucleic acid sequence of any nucleic acid that encodes a polymerization domain. In some embodiments, the nucleic acid is characterized by the fact that it encompasses a nucleic acid that encodes 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 linked in such a manner that the polypeptide produced is a fusion protein characterized by the fact that it encompasses an H-NOX domain and a polymerization.

[000238] In some embodiments, the nucleic acid is characterized by the fact that it encompasses nucleic acid that encodes one or more than one His6 tag. In some embodiments the additional nucleic acid encompasses nucleic acids encoding a sequence linker positioned between nucleic acids encoding the H-NOX domain, the polymerization domain and/or a His6 tag.

[000239] In some embodiments, the invention provides a nucleic acid that encodes 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.

[000240] In some embodiments, the invention provides nucleic acids that encode the following 5' to 3': a T. tengcongensis H-NOX Domain of L144F, a Gly-Ser-Gly amino acid sequence linker, and a foldon domain . In some embodiments, the invention provides nucleic acids that encode the following 5' to 3': a T. tengcongensis W9F/L144F H-NOX domain, a Gly-Ser-Gly amino acid sequence linker, and a foldon domain. In some embodiments, the invention provides nucleic acids that encode the following 5' to 3': a wild-type T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid sequence linker, and a foldon domain.

[000241] In some embodiments, the invention provides nucleic acids that encode the following 5' to 3': a T. tengcongensis H-NOX domain of L144F, a Gly-Ser-Gly amino acid sequence linker, a foldon domain, an Arg-Gly-Ser sequence linker, and a His6 tag. In some embodiments, the invention provides nucleic acids encoding the following 5' to 3': a T. tengcongensis W9F/L144F H-NOX domain, a Gly-Ser-Gly amino acid sequence linker, a foldon domain, a Arg-Gly-Ser sequence linker, and a His6 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 sequence linker, a foldon domain, a linker sequence Arg-Gly-Ser, and a His6 tag.

[000242] In some embodiments, the nucleic acid is characterized by the fact that it encompasses the nucleic acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:11.

[000243] The invention is also characterized by the fact that it includes a cell or population of cells that contain at least one nucleic acid that encodes the mutant H-NOX protein described here. Example cells include insect, plant, yeast, bacterial, and mammalian cells. These cells are useful for producing mutant H-NOX proteins using standard methods such as those described herein.

[000244] In some embodiments, the invention provides a cell characterized by the fact that it encompasses a nucleic acid that encodes 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 the H-NOX domain of wild-type T. thermoanaerobacter. In some embodiments, the H-NOX domain is a T. thermoanaerobacter L144F H-NOX domain. In some embodiments, the H-NOX domain is the H-NOX domain of T. thermoanaerobacter W9F/L144F. In some embodiments, the invention provides a cell characterized by the fact that it encompasses a nucleic acid that encompasses the nucleic acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO:11 .

H-NOX protein formulations

[000245] Any wild-type or mutant H-NOX protein, which includes polymeric H-NOX proteins, described herein can be used for the formulation of pharmaceutical or non-pharmaceutical compositions. In some embodiments, the formulations encompass a monomeric H-NOX protein characterized by the fact that it encompasses an H-NOX domain and a polymerization domain such that the monomeric H-NOX proteins associate in vitro or in vivo to produce an H-NOX protein. polymeric. As further discussed below, these formulations are useful in a variety of therapeutic and industrial applications.

[000246] In some embodiments, the pharmaceutical composition is characterized by the fact that it includes one or more of one wild type or the mutant H-NOX proteins described herein which includes 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 emulsions, and various types of dehydrating agents. humidification. Example of diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline. Compositions characterized by the fact that they encompass such carriers are formulated by 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, all of which are incorporated herein by reference in their entirety, particularly with respect to formulations). In some embodiments, the formulations are sterile. In some embodiments, the formulations are essentially endotoxin-free.

[000247] Although any suitable carrier known to those familiar with the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. The compositions may be formulated for any suitable manner of administration, which includes, for example, intravenous, intra-arterial, intravesicular, inhalation, intraperitoneal, intrapulmonary, intramuscular, subcutaneous, intratracheal, 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 saccharin, talc, cellulose, glucose, sucrose, or magnesium carbonate, may be employed. Biodegradable microspheres (e.g. polylactate polyglycolate) can also be used as carriers.

[000248] In some embodiments, pharmaceutical or non-pharmaceutical compositions include buffer (e.g., neutral buffered saline, phosphate buffered saline, etc.), 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 lyophilisate. H-NOX proteins can also be encapsulated within liposomes or nanoparticles using known technology. Other examples of formulations that can be used for H-NOX proteins are described by, e.g., U.S. Pat. US Nos. 6,974,795 and 6,432,918, all of which are incorporated herein by reference in their entirety, particularly with respect to protein formulations.

[000249] The compositions described herein can be administered as part of a sustainable release formulation (e.g., a formulation such as a capsule or sponge that produces a slow release of compound after administration). Such formulations can generally be prepared using known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired site. Sustainable-release formulations may contain an H-NOX protein dispersed in the carrier matrix and/or contained within a reservoir coated 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 the sustainable release formulation depends on the site of implantation, the rate and duration of expected release, and the nature of the condition being treated or prevented.

[000250] In some embodiments, the pharmaceutical composition contains an effective dose of wild-type or mutant H-NOX protein. In some embodiments, the pharmaceutical composition contains an effective dose of the polymeric H-NOX protein characterized by the fact that it encompasses two or more of two wild-type or mutant H-NOX domains. In some embodiments, the pharmaceutical composition contains an effective dose of 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 is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis L144F H-NOX domain and a foldon domain. In some embodiments, the formulation is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some embodiments, the formulation is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis H-NOX domain of L144F and a foldon domain.

[000251] An example dose of hemoglobin as a blood substitute is approximately 10 mg to approximately 5 grams or more of extracellular hemoglobin per kilogram of the patient's body weight. Thus, in some embodiments, an effective dose of an H-NOX protein for administration to a human is between a few grams to more than approximately 350 grams. Other examples of doses of an H-NOX protein include approximately any of 4.4, 5, 10, or 13 G/DL (where G/DL is the concentration of the H-NOX protein solution before infusion into the circulation) in an appropriate infusion rate, such as approximately 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 dose since the required effective dose could be achieved by the combined effect of the plurality of administrations. The selection of the amount of an H-NOX protein to include in a pharmaceutical composition depends on the dosage form used, the condition being treated, and the particular purpose to be achieved as determined by those generally skilled in the art. .

[000252] Example compositions include genetically developed recombinant H-NOX proteins, which can be isolated or purified, characterized by the fact that they encompass one or more of a mutation that collectively transmits altered O2 or NO binding relative to the corresponding H-NOX Protein wild type, and operative as a physiologically compatible gas transporter in mammalian blood. 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 characterized by the fact that it encompasses the wild-type or mutant H-NOX domain and a polymerization domain as described herein. In some embodiments, the composition is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it comprises three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis L144F H-NOX domain and a foldon domain. In some embodiments, the composition is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it comprises three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some embodiments, the composition is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it comprises three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis L144F H-NOX domain and a foldon domain.

[000253] To reduce or prevent an immune response in human subjects who are administered a pharmaceutical composition, H-NOX proteins or human domains (wild-type human proteins or human proteins into which one or more of a mutation has been introduced) or other proteins H-NOX or non-antigenic domains (e.g., the mammalian H-NOX protein) can be used. To reduce or eliminate the immunogenicity of H-NOX proteins derived from sources other than humans, the 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 may mutate to the corresponding amino acid in a human H-NOX protein.

Therapeutic applications of H-NOX proteins

[000254] Any of the wild-type or mutant H-NOX proteins, which include polymeric H-NOX proteins, or pharmaceutical compositions described herein can be used in therapeutic applications.

[000255] H-NOX proteins in particular, which include polymeric H-NOX proteins, can be selected for such applications based on the desired O2 association rate, O2 dissociation rate, O2 dissociation constant for binding, NO stability, NO reactivity, rate of autoxidation, 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, blood loss, or injuries. For example, an O2- bond of the H-NOX protein can be used in most situations where red blood or plasma cell expanders are currently used. Specifically, the H-NOX protein can be used as a red blood cell replacement for the treatment of trauma (e.g., battlefield, disaster relief, or accident), hemorrhage, hemorrhagic shock, surgery (e.g., abdominal aneurysm surgery, orthopedic surgery, such as hip replacement surgery, or any other surgery that produces high blood loss), hemodilution, blood extension uses (e.g., supplementing self-donation), and any other situation where volume of blood is lost or capacity to transport O2 is reduced. Examples of injury treatment applications include post-radiation injury repair (e.g., hyperbaric effect of oxygen), post-reconstructive surgery, diabetic ulcer repair, and burn injuries.

[000256] An oxygen bond of polymeric H-NOX can also be used to temporarily increase O2 transport during or after pre-donation of autologous blood before returning autologous blood to an individual (such as a replacement for blood that is removed during surgical procedures where the individual's blood is removed and saved for reinfusion at the end of surgery or during recovery). In some embodiments, the H-NOX proteins also function as a simple volume expander that provides oncotic pressure due to the presence of a large H-NOX protein molecule.

[000257] Because the distribution in the extracellular vasculature of the H-NOX protein is not limited by the size of the red blood cells, the polymeric H-NOX proteins of the present invention can be used to transport O2 to areas where red blood cells do not can arrive. These areas may include any tissue in areas that are located downstream of obstructions to red blood cell flow, such as areas downstream of one or more thrombus, sickle cell occlusions, arterial occlusions, peripheral vascular occlusions, balloons angioplastics, surgical instruments, tissues that are suffering from lack of oxygen or are hypoxic, and the like. Furthermore, all types of ischemic tissues can be treated using H-NOX proteins. Such ischemic tissues include, for example, perioperative ischemia, stroke, emergent stroke, transient ischemic attacks, myocardial stunning and hibernation, acute or unstable angina, emergent angina, and myocardial infarction (e.g., ST-segment elevation myocardial infarction ). Other examples of cardiovascular indications that can be treated using H-NOX proteins include cardioplegia and sickle cell anemia. Example indications include conditions of functional deficiency of hemoglobin such as where blood substitute or O2 transporter is indicated, which includes blood loss, hypoxia, etc.

[000258] H-NOX proteins, which include polymeric H-NOX proteins, can also be used as a complement to radiation or chemotherapy for the treatment of cancer. In some embodiments, an H-NOX protein is used as an adjuvant radiation therapy in solid tumors (e.g., individuals with poor pre-metastatic prognosis) or as an adjuvant PDT therapy in surface tumors (e.g., breast cancer). colon, lungs, or skin, or cancer on another inaccessible surface or location). H-NOX proteins can be used to treat anemia by providing the ability to transport oxygen to a patient who is suffering from anemia. Examples of neurological indications include ischemic stroke, traumatic brain injury, and spinal cord injury. The methods and compositions are applicable in both acute (rapid delivery of oxygen to tissues or a specific site, e.g., acute myocardial infarction, acute local or systemic tissue oxygenation, or blood transfusion) and chronic ( e.g., recovery after acute cardiac infarction).

[000259] In one particular aspect, the invention provides methods of using H-NOX proteins to transport O2 to brain tumors (e.g., glioblastoma). In some embodiments, administration of H-NOX is used as an adjunct to radiation therapy or chemotherapy. In some embodiments, the invention provides methods for treating brain cancer (e.g., glioblastoma) in an individual by administering an effective dose of an H-NOX protein and administering an effective dose of radiation to an individual. In some embodiments, the invention provides methods for reducing brain tumor growth (e.g., glioblastoma growth) in an individual by administering an effective dose of an H-NOX protein and administering an effective dose of radiation to an individual. . In some embodiments, the H-NOX protein is polymeric H-NOX protein (e.g., a trimeric H-NOX protein). In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses one or more of one H-NOX domain characterized by the fact that it encompasses the mutation in a position corresponding to L144 of H-NOX of T. tengcongensis. In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses one or more of one H-NOX domain characterized by the fact that it encompasses the mutation corresponding to the T. tengcongensis H-NOX L144F mutation. In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses one or more of one H-NOX Domain characterized by the fact that it encompasses the mutation in the positions corresponding to W9 and L144 of H-NOX of T. tengcongensis. In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses one or more of an H-NOX Domain characterized by the fact that it encompasses mutations corresponding to a T. tengcongensis H-NOX W9F/L144F Mutation. 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 is characterized by the fact that it encompasses a T. tengcongensis H-NOX Domain of L144F. In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses a W9F/L144F T. tengcongensis H-NOX domain and a foldon domain.

[000260] In various embodiments, the invention features a method of transporting O2 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 administration to an individual in need of the same wild-type or mutant H-NOX protein, which includes the polymeric H-NOX protein in an amount enough to transport O2 to an individual. In some embodiments, the invention provides methods of delivering or transporting blood gas to an individual such as a mammal, characterized by the fact that it encompasses the step of transportation (e.g., transfusion, etc.) to the individual's blood (e.g. ., a mammal) one or more of one of the H-NOX compositions. Methods for delivering O2 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 heme binding or is a holoprotein with bound heme. The H-NOX protein may or may not have heme bound prior to administration of the H-NOX protein to an individual. In some embodiments, O2 is linked to an H-NOX protein before being transported to an individual. In other embodiments, O2 is not bound to an H-NOX protein prior to administration of the protein to an individual, and the H-NOX protein transports O2 from one location in the individual to other locations in the individual.

[000261] Wild-type and mutant H-NOX proteins, which include polymeric H-NOX proteins, with a relatively low KD for O2 (such as less than approximately 80 nM or less than approximately 50 nM) are expected to be particularly useful to treat tissues with low oxygen tension (such as tumors, some lesions, or other areas where oxygen tension is very low, such as a p50 below 1 mm Hg). The high affinity of such H-NOX proteins for O2 may increase the length of time that O2 remains bound to an H-NOX protein, thereby reducing the amount of O2 that is released before the H-NOX protein reaches the tissue at hand. to be treated.

[000262] In some embodiments for direct transport of an O2-bound H-NOX protein to a particular site in the body (such as a glioblastoma), the koff for O2 is more important than the kD value because O2 is already bound to the protein (which makes kon less important) and oxygen needs to be released at or near a particular site in the body (at a rate influenced by koff). In some embodiments, the koff may also be important when H-NOX proteins are in the presence of red cells in the circulation, where they facilitate diffusion of O2 from the red cells, and perhaps prolonging the dilute ability of the red cells to transport O2 to additional points. non-vascularized.

[000263] In some embodiments for the H-NOX protein transporter that circulates in an individual's bloodstream, the H-NOX protein binds O2 in the lungs and releases O2 at one or more than one site in the body. For some of these applications, the kD value is more important than the koff since O2 binding is at or near equilibrium. In some extreme hemodilution embodiments, kD is more important than koff when the H-NOX protein is the main O2 transporter because an H-NOX protein will bind and release O2 continually as it travels through the circulation. Since hemoglobin has a p50 of 14 mm Hg, red blood cells (which act as capacitors) have a p50 of ~30 mm Hg, and HBOCs have been developed with ranges between 5 mm Hg and 90 mm Hg, the optimal range of KD for H-NOX proteins can therefore be between ~2 mm Hg to ~100 mm Hg for some applications.

[000264] Polymeric H-NOX proteins can also be used for imaging. In particular, imaging light (e.g., optical coherence tomography; see, e.g., Villard, J. W. (2002). Circulation 105:1843-1849, which is incorporated by reference in its entirety particularly with respect to tomography optical coherence) is overshadowed by erythrocytes. Perfusion with an H-NOX solution allows clearer images of the circulation and vessel walls because the H-NOX protein is much smaller than erythrocytes.

[000265] H-NOX proteins, which includes 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, intratracheal, or aerosol administration. ; by transdermal or mucous membrane absorption; or by injection (e.g., subcutaneous, intravenous, intra-arterial, intravesicular, or intramuscular injection). H-NOX proteins can 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 lesion, a tumor, a hypoxic tissue, or a hypoxic organ of an individual.

[000266] In some embodiments, a continuous sustainable release of the composition formulation is used. Administration of an H-NOX protein can occur, for example, over a period of seconds to hours depending on the end of administration. For example, as a vehicle that transports blood, an example of administration time is as quick as possible. Other time course examples include approximately any one of 10, 20, 30, 40, 60, 90, or 120 minutes. Example infusion rates for H-NOX solutions as blood replacements are approximately 30 mL/hour to approximately 13,260 mL/hour, such as approximately 100 mL/hour to approximately 3,000 mL/hour. An example of a total dose of H-NOX Protein is approximately 900 mg/kg administered over 20 minutes at 13,260 mL/hour. An example of a total dose of H-NOX protein for a pig is approximately 18.9 grams.

[000267] Example dosage 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 per day. The H-NOX protein can be administered, for example, over a period of a few days or weeks. In some embodiments, the H-NOX protein is administered for a longer period, such as a few months or years. The dosage frequency of the composition may be adjusted over the course of treatment based on the judgment of medical administration.

[000268] In some embodiments of the invention, the H-NOX protein (e.g., the polymeric H-NOX protein) is used as a complement to radiation therapy or chemotherapy. For example, for the treatment of glioblastoma. In some embodiments, H-NOX is administered to any individual at least 1, 2, 3, 4, 5 or 6 hours before administration of radiation or chemotherapy. In some embodiments, the radiation is X-irradiation. In some embodiments, the X-irradiation dose is any from approximately 0.5 gy to approximately 75 gy. In some embodiments, the cycle of H-NOX administration and radiation administration are repeated any one of one, two, three, four, five or six times. In some embodiments, the cycle of H-NOX administration and radiation administration are repeated after any one of approximately one week, two weeks, three weeks, four weeks, five weeks, or six weeks. In some embodiments, H-NOX administration therapy and radiation are used in conjunction with other therapies; for example, chemotherapy.

[000269] As highlighted above, the selection of dosage amounts for H-NOX proteins depends on the dosage form used, the frequency and number of administrations, the condition being treated, and the particular end to be achieved of according to the determination of specialized technicians in the field. In some embodiments, an effective dose of an H-NOX protein for administration to a human is between a few grams to more than 350 grams.

[000270] In some embodiments, two or more of two different H-NOX proteins are administered simultaneously, sequentially, or concurrently. In some embodiments, other compounds or therapies useful for transporting O2 are administered simultaneously, sequentially, or concurrently with the administration of one or more of an H-NOX protein.

[000271] Other examples of therapeutic applications for which H-NOX proteins can be used are described by, e.g., Pat. US Nos. 6,974,795 and 6,432,918, all of which are incorporated herein by reference in their entirety, particularly with respect to therapeutic applications for O2 transporters.

Kits with H-NOX Proteins

[000272] Articles of manufacture and kits that include any of the H-NOX proteins described herein that include polymeric H-NOX proteins, and suitable packaging are also provided. In some embodiments, the invention is characterized by the fact that it includes a kit with (i) the 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 O2 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 use of the kit for any of the industrial uses described herein (e.g., use of an H-NOX protein as a standard reference for analytical instrumentation requiring such a standard reference, enhancing cell growth in cell culture to maintain or increase O2 levels in vitro, addition of O2 to a solution, or removal of O2 from a solution).

[000273] In some embodiments, kits are provided for use in treating brain cancer (e.g., glioblastoma). In some embodiments, the kit is characterized by the fact that it encompasses a polymeric H-NOX protein. In some embodiments, the kit is characterized by the fact that it comprises an effective dose of the polymeric H-NOX protein characterized by the fact that it comprises two or more of two wild-type or mutant H-NOX domains. In some embodiments, the kit is characterized by the fact that it comprises an effective dose of the recombinant monomeric H-NOX protein characterized by the fact that it comprises the wild type or mutant H-NOX domain and a polymerization domain as described herein. In some embodiments, the kit is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses the corresponding mutation for a T. tengcongensis L144F H-NOX mutation and a domain of trimerization. In some embodiments, the kit is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses the mutation corresponding to a W9F/L144F mutation of the H-NOX T. tengcongensis and a trimerization domain. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses human H-NOX domains. In some embodiments, the trimeric H-NOX protein is characterized by the fact that it encompasses canine H-NOX domains. In some embodiments, the kit is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis L144F H-NOX domain and a foldon domain. In some embodiments, the kit is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some embodiments, the kit is characterized by the fact that it encompasses a trimeric H-NOX protein characterized by the fact that it encompasses three monomers, each monomer characterized by the fact that it encompasses a T. tengcongensis L144F H-NOX domain and a foldon domain.

[000274] Suitable packaging for compositions described herein are known in the art, and include, for example, vials (e.g., sealed vials), vessels, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may additionally be sterilized and/or sealed. Unit dosage forms characterized by the fact that they encompass the compositions described herein are also provided. These unit dosage forms can be stored in suitable single or multiple dosage unit packaging and can also be further sterilized and sealed. The instructions provided in kits of the invention are typically written instructions on a label or leaflet (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions included on a magnetic or optical disk of storage) are also acceptable. Instructions regarding the use of H-NOX proteins generally include information such as dosage, dosage schedule, and administration script for a treatment or industrial use. The kit may, in addition, cover the description of selection of appropriate individuals or treatment.

[000275] The container may be unit doses, bulk packages (e.g., multidose packages) or subunit doses. For example, kits may also be provided that contain sufficient doses of the H-NOX proteins mentioned herein to provide effective treatment for an individual for an extended period, such as approximately anywhere between a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks. weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or more. Kits may also include multiple dose units of H-NOX proteins and instructions for use and packaging with sufficient quantities for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies. In some embodiments, the kit is characterized by the fact that it includes a dry (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to generally form a stable aqueous suspension of H-NOX protein. Example of Methods for Production of H-NOX Proteins

[000276] The present invention also provides methods for producing any of the polymeric H-NOX proteins as described herein. In some embodiments, the method involves culturing a cell that has a nucleic acid encoding the polymeric H-NOX protein under conditions suitable for producing the polymeric H-NOX protein. In various embodiments, the polymeric H-NOX is also purified (such as purifying the H-NOX protein from cells or the culture medium). In some embodiments, the method involves culturing a cell that has a nucleic acid encoding an H-NOX protein monomer characterized by the fact that it encompasses an H-NOX domain and a polymerization domain. The monomers then associate in vivo or in vitro to form a polymeric H-NOX protein. The polymeric H-NOX protein characterized by the fact that it encompasses heterologous H-NOX domains can be generated by the co-introduction of two or more of two nucleic acids encoding monomeric H-NOX proteins with the desired H-NOX domains and where the two or more of two monomeric H-NOX proteins span the same polymerization domain.

[000277] In some embodiments, the polymeric H-NOX protein characterized by the fact that it encompasses heterologous H-NOX domains is prepared by separately preparing polymeric H-NOX proteins characterized by the fact that it encompasses homologous monomeric H-NOX subunits characterized by the fact that it encompasses the desired H-NOX domains and a common polymerization domain. The different homologous H-NOX proteins are mixed in the desired proportion of heterologous H-NOX subunits, the homologous polymeric H-NOX proteins are dissociated (e.g., by heat, denatured, high salt content, etc.), then allowed to associate to form heterologous polymeric H-NOX proteins. The mixture of heterologous polymeric H-NOX proteins can be further purified by screening for the presence of the desired subunits in the desired proportion. For example, each different H-NOX monomer may have a different tag to aid in the purification of heterologous polymeric H-NOX proteins and identification and quantification of heterologous subunits.

[000278] As noted above, the sequences of several H-NOX proteins and wild-type nucleic acids are known and can be used to generate H-NOX domains and mutant nucleic acids of the present invention. Techniques for 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. (August 10, 2004). Biochemistry 43(31):10203- 10211, which is hereby incorporated by reference in its entirety, particularly with respect to 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.

[000279] In particular, the mutant H-NOX proteins described here can be generated by numerous methods that are known in the art. Mutation can occur at the amino acid level by chemical modification of an amino acid or at the codon level by changing the nucleotide sequence that codes for a particular amino acid. Substitution of an amino acid at any given position in the protein can be achieved by changing the codon that codes for the amino acid. This can be accomplished by site-specific 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. (December 20, 1985). Nucleic Acids Res. 13(24):8749-8764; Taylor, J. W. et al. (December 20, 1985). Nucleic Acids Res. 13(24):8765-8785;Nakamaye, K. L. et al. (December 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. academic Sci USA 82(2):488-492; Kunkel, T. A. (1987). Methods Enzymol. 154:367-382; Kunkel, U.S. Pat. No. 4,873,192, all of which are incorporated herein by reference in their entirety, particularly with respect to protein mutagenesis. Mutagenesis can also be accomplished by other available or non-commercial means, such as those using site-specific mutagenesis with mutant oligonucleotides.

[000280] Site-specific mutagenesis can also be achieved 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, all of which are incorporated herein by reference in their entirety, particularly with respect to protein mutagenesis. Site-specific mutagenesis can also be achieved using cassette mutagenesis with techniques that are known to those skilled in the art.

[000281] 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 H-NOX protein. encoded.

[000282] In particular, heterologous proteins have been expressed in numerous biological expression systems, such as insect cells, plant cells, yeast cells, and bacterial cells. Thus, any suitable biological protein expression system can be used 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.

[000283] If desired, H-NOX proteins can be purified using standard techniques. In some embodiments, the protein is at least approximately 60%, by weight, free of 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. Purified protein can be obtained, for example, by purification (e.g., extraction) from a natural source, a recombinant expression system, or reaction mixtures for chemical synthesis. Example of purification methods include immunoprecipitation, column chromatography such as immunoaffinity chromatography, magnetic bead immunoaffinity purification, and panning with the bound antibody plate, as well as other techniques known to those skilled in the art. Purity may be tested 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.

[000284] In some embodiments, the polymeric H-NOX protein is characterized by the fact that it encompasses one or more than one His6 tag. An H-NOX protein characterized by the fact that it encompasses at least one His6 tag can be purified using chromatography; for example, using Ni2+-affinity chromatography. Following purification, the His6 tag can be removed; for example, when using an exopeptidase. In some embodiments, the invention provides purified polymeric H-NOX protein, in which the polymeric H-NOX protein has been purified through the use of a His6 tag. In some embodiments, the purified H-NOX protein is treated with an exopeptidase to remove the His6 tags.

EXAMPLES

[000285] The examples, which are intended to be purely examples of the invention and, for this reason, should not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention analyzed above. The examples are not intended to represent that the experiments below are all and/or only experiments performed. Unless otherwise stated, temperature is in degrees Celsius and pressure is at or near atmospheric.

Example 1. Creation and Expression of the Trimerized H-NOX Protein

[000286] To increase the circulation half-life of the H-NOX protein, fusion protein chimerics were designed to combine the H-NOX sequence from Thermoanaerobacter tengcongensis with the trimerization domain of the bacteriophage T4 fibritin protein. This fusion strategy produces a more than 3-fold increase in molecular weight and is completely modular (the trimerization domain could be added to any H-NOX sequence and even used to combine multiple H-NOX sequences into a single trimer molecule).

Fusion of the foldon domain to H-NOX

[000287] The foldon domain was genetically fused to the C-terminus of the H-NOX sequence from Thermoanaerobacter tengcongensis using three amino acid linkers (Gly-Ser-Gly) between the Xho I restriction site in the C-terminus of the H-NOX sequence and the initial glycine of the foldon domain. Purification of the His6 tag protein was added to the C-terminus of the foldon domain using three linker amino acids (Arg-Gly-Ser) between the foldon tag and His6. The DNA and amino acid sequence of the full-length fusion protein are shown in Figure 2. The full-length fusion protein encodes a 229 amino acid protein with a molecular weight of 26,677 AMU (as a monomer).

Construction of the H-NOX-foldon fusion protein sequence

[000288] The construction of the H-NOX-foldon fusion sequence was designed to use restriction endonucleases to cut the segment of DNA encoding His6 and stop codon from the parent H-NOX plasmid and replace it with the cassette encoding the foldon sequence, His6 marker, and stop codon. DNA encoding the foldon domain, His6 tag, and stop codon flanked by enzyme restriction sites (Xho I at the 5' terminus and Hind III at the 3' terminus after a His6 tag and stop codon) was purchased from GenScript (Piscataway, NJ). The sequence was carried into the GenScript pUC57 cloning vector.

[000289] Xho I and Hind III restriction enzymes were used to digest the pUC57 plasmid to release the His6 foldon fragment (147 base pairs in length). The pCW a vector encoding the L144F variant of Thermoanaerobacter tengcongensis H-NOX was also digested with Xho I and Hind III to remove the 48 base pairs fragment encoding the His6 tag and stop codon of an H-NOX sequence. The desired restriction fragment digestion reactions were isolated by preparative agarose gel electrophoresis and the DNA fragments were purified from the agarose. The fragments encoding the H-NOX sequence and His6 foldon tag were ligated using T4 ligase and the ligation reaction was used to transform competent E. coli cells. Sequencing with the pCW specific sequencing primer confirmed that one of the E. coli clones (clone 3I-A) combined the desired fusion sequence and encoded the full H-NOX-foldon His6 chimeric protein (Figure 3A shows the sequencing data aligned with the desired sequence). The clone 3I-A sequence was renamed v01-f002 to designate the pCW vector (v01) encoding the L144F H-NOX sequence (002) with the fused foldon domain (f). The wild-type T. thermoanaerobacter H-NOX-foldon monomer sequences with and without His6 tags and the C. lupus H-NOX-foldon monomer sequence are shown in Figure 3.

Expression and purification of the H-NOX-foldon fusion protein

[000290] The v01-f002 plasmid derived from the parent vector pCW (Gegner, JA & Dahlquist, FW (1991) Proc Natl Acad Sci USA 88, 75075; Muchmore, DC, et al. (1989) Methods Enzymol 177: 44-73 ) codes for expression of the f002 open reading frame by multiple tac (hybrid trp-lac) promoters. Plasmid v01-f002 was transformed into competent E. coli strain RP523 (Li, JM, et al., (1988) J Bacteriol 170:1021-1025) for efficient expression of H-NOX-linked heme protein. Expression was tested at a range of induction temperatures (37°C, 30°C, 18°C) and also in Rosetta2 cells (Merck Millipore, Darmstadt, Germany).

[000291] Robust expression of the v01-f002 plasmid was achieved in RP523 cells using an initial overnight Luria Broth input culture supplemented with 100 mg/L Ampicillin and 30 mg/L hemin grown at 30 °C. The starter culture was then used to inoculate a 6 L Terrific Broth expression culture supplemented with 100 mg/L Ampicillin, 30 mg/L hemin, and 1.5% glucose. The expression culture was grown at 30 °C to an OD600 of ~0.5 and then protein expression was induced by the addition of IPTG to a final concentration of 0.1 mM. Expression was continued at 30°C overnight (24.5 hours of induction) before cells were collected by centrifugation and frozen at -80°C for purification.

[000292] The fusion protein was purified from the expressed granule cell using one-step heating treatment and two-step chromatography. First, the expressed granule cell was resuspended in 50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, and 5% glycerol buffer at pH 7.9. The solution was homogenized using an EmulsiFlex C-50 homogenizer (Avestin, Ottawa, Canada) to lyse the bacterial cells. The lysed cell was heated for 15 min at 75°C to precipitate non-thermostable proteins. The precipitate was removed by centrifugation at 27,000 g for 15 minutes to obtain clarified supernatant. The clarified supernatant was applied to HisTrap FastFlow column (GE, Piscataway, NJ) to bind the fusion protein His6 tag. Bound protein was eluted with a buffer of 50 mM sodium phosphate, 500 mM NaCl, 250 mM imidazole, and 5% glycerol buffer at pH 7.9. The eluted protein was buffer exchanged into a 30 mM Triethanolamine, 50 mM NaCl, pH 7.4 buffer on a DEAE Sepharose FastFlow column (GE, Piscataway, NJ). The H-NOX-foldon fusion protein circulated through the DEAE column, while contaminating host cell and endotoxin were retained on the column. The H-NOX-foldon fusion Protein could then be concentrated for storage at -80 °C. An SDS-PAGE gel showing the H-NOX-foldon fusion protein at each stage of the purification process from the initial purification is shown in Figure 4.

Characterization of purified H-NOX-foldon fusion proteins

[000293] Numerous techniques have been used to characterize the purified H-NOX-foldon protein. The purity of the final purified protein has been analyzed by SDS-PAGE (see Figures 4 and 5). Gel electrophoresis shows that H-NOX-foldon fusion Protein is >95% pure after heating treatment and two chromatography steps. On the denaturing SDS-PAGE gel, the H-NOX-foldon Fusion functions as a monomer with mobility consistent with the monomeric molecular weight of 26.7 kDa.

[000294] Further quantitative size calculations of the fusion protein have been obtained using both LC-MS to determine the exact mass of the monomeric unit and analytical size exclusion chromatography to calculate the size and dispersion of the trimer in the standard buffer solution. Figure 6 shows LC-MS analysis of Protein H-NOX-foldon fusion. The LC-MS derived mass of 26.678 AMU is consistent with the predicted mass of 26.677 AMU for the monomeric H-NOX-foldon fusion. Figure 7 shows analytical size exclusion chromatography using the Superdex 200 10/300 GL (GE, Piscataway, NJ) column and the 30 mM Triethanolamine, 50 mM NaCl, pH 7.4 buffer. Under SEC analytical conditions, an H-NOX-foldon fusion would remain trimerized and the resulting retention volume is consistent with the predicted molecular weight of 80.0 kDa for an H-NOX-foldon trimer.

[000295] Heme protein spectroscopy is used to characterize the nature of bound ligands and the oxidation state of the iron atom in heme. UV-vis spectroscopic analysis of the H-NOX-foldon protein shows that fusion of the foldon domain does not alter the characteristic H-NOX spectrum. The characteristic spectral peaks including the Soret peak (415 nm), and α/β peaks (550-600 nm) are all preserved between the H-NOX monomer and the H-NOX-foldon fusion protein and indicate that the H-NOX fusion -NOX-foldon binds the heme cofactor and diatomic oxygen gas in a similar way to the original H-NOX monomer (Figure 8).

[000296] A structural model of the H-NOX-foldon trimer was constructed using the crystal structures of the foldon domain (1V1H) and the H-NOX from Thermoanaerobacter tengcongensis monomer (1U4H) found in the RCSB Protein Database (Figure 9).

Example 2. Production of a panel of H-NOX trimers that demonstrate an extended half-life and a range of oxygen affinities.

[000297] Using computational structure-based design, H-NOX amino acids were systematically mutated to create a panel of H-NOX monomer variants that bind oxygen with varying affinities. It was determined that 23 kDa H-NOX small monomers were eliminated from the rat circulatory system with a half-life of 30 minutes. It was hypothesized that by raising the molecular weight above renal filtration limits the circulation half-life of the H-NOX protein could possibly increase and therefore allow sustainable oxygenation for longer durations. With the aim of determining whether higher molecular weight increases the circulation half-life of the H-NOX protein, H-NOX variants with higher molecular weights were generated by the trimerization of H-NOX monomers that had been successful in oxygenating hypoxic tissue. To generate a panel of trimerized variants, a small amino acid trimerization motif of the fibritin protein called the "foldon" domain was genetically linked to the C-terminus of several H-NOX monomers with different oxygen affinities ranging from 1 to 20 mmHg. The panel included H-NOX trimers grouped with wild-type H-NOX, H-NOX variant L144F, H-NOX variant L144F with no His6 tag at the C-terminus, H-NOX variant L144F/L189C, H-NOX variant L144F with in the linker between H-NOX and foldon, H-NOX variant L144F with three linker amino acids between H-NOX and foldon, H-NOX variant with nine linker amino acids between H-NOX and foldon, or H-NOX variant W9F/L144F. The foldon domain consisted of 27 amino acid residues, GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO:4), corresponding to amino acid residues 457 to 483 in fibritin and had a predicted mass of 3.08 kDa. As described above, the foldon domain has previously been shown to increase protein stability and has been widely used to trimerize proteins both in vitro and in vivo. Briefly, a plasmid containing the gene encoding the foldon domain with XhoI and HindIII restriction sites at the 5' and 3' termini, respectively, was digested with XhoI and HindIII restriction enzymes to clone the foldon domain gene into a plasmid that encodes the H-NOX monomer protein. After plasmid expression in bacterial cells (E. coli), H-NOX+foldon protein was purified and tested to verify trimerization of the H-NOX+foldon monomers into the H-NOX+ foldon trimers (e.g. e.g., H-NOX trimer). For purification, bacterial cells were lysed with lysozyme and homogenized. The supernatant was collected and heat treated at 75°C for 15 minutes before centrifugation at 14 krpm in the JA-17 rotor to remove insoluble protein. Soluble H-NOX+foldon was attached to a HisTrap column (IMAC) and eluted with imidazole, and the eluted samples were subjected to buffer change to provide a protein sample in low-salt buffer without imidazole. Additionally, contaminated proteins in the sample were removed by subjecting a protein sample to the DEAE ion exchange column and the eluted protein samples were concentrated prior to analysis. Mass Spectroscopy and Size Exclusion Chromatography was used to verify the monomeric molecular weight (approximately 26.7 kDa) and the trimeric molecular weight (approximately 80.1 kDa), respectively. Each trimerized variant was tested to determine if it satisfied biochemical parameters that include: homogeneous molecular weight, oxygen binding affinities between 1 and 20 mmHg as measured by koff and interpolating known kon values to arrive at the Kd's that correspond to mmHg. The Koff for homotrimers is essentially the same as for the corresponding monomers when measured by koff, minimal reactivity to nitric oxide (NO), low rates of autoxidation, and circulation time persistence of 3 hours or longer (Table 2). Spectroscopy was used to determine the nature of gas bound to the STP and stopped-flow spectroscopy was used to determine the rate constant of oxygen kinetic dissociation. From the analysis, H-NOX trimers were identified with oxygen affinities of 2 μM, an affinity that could possibly allow oxygen release in tissues with low oxygen levels. The H-NOX trimer also demonstrated reactivity to nitric oxide approximately less than 1 μM-1s-1 which was considered minimal as it is compared to hemoglobin which has a reactivity to nitric oxide of 58 μM-1s-1 which previously it had been proven to be vasoactive and toxic. Table 2. Biochemical and Release Properties of polymerized H-NOX trimers

[000298] Trimers were produced with high purity and low levels of endotoxin for release studies in rats. The H-NOX trimers were formulated for in vivo injection in the physiological buffer of 150 mM NaCl, 25 mM HEPES buffer (pH 7.4), and concentrated to 100 mg/mL for use in doses up to 100 mg/kg. To verify the persistence times of the extended circulation of the H-NOX trimers, the candidate trimers were each tested using a group of four male Wister rats. Anesthesia was induced in an induction chamber with 2-3% isoflurane in N2O:O2 (2:1), and maintained with 1-1.5% isoflurane via the nose cone. Adequate depth of anesthesia was assessed by the lack of pinch withdrawal to the lower limbs and loss of eye closing reflex. The animals received 40 mg/kg of cefazolin sodium via intraperitoneal injection and 0.1 mg/kg of buprenorphine subcutaneously. One day before the release test, catheters were surgically implanted into the femoral vein and artery in each animal to allow injection and removal of blood, respectively. Candidate H-NOX trimers were injected at time 0 at a dose of 100 mg/kg by intravenous bolus injection into the venous catheter. Approximately 250 μL of blood was collected from each mouse's arterial catheter at 5 min, 30 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, and 24 h after candidate injection. H-NOX trimer or buffer control. The collected blood was processed into serum and plasma and subsequently analyzed for the presence of H-NOX trimer using ELISA and SDS-PAGE assays with a polyclonal antibody compared to the H-NOX protein. Table 3: Pharmacokinetic parameters of HNOX monomer and trimer when administered as an intravenous bolus (100 mg/kg) to the rat

[000299] Furthermore, to measure the release time for each trimer, preliminary safety in animals was also monitored. A safety study such as Good Laboratory Practice (GLP) with H-NOX monomer showed no major adverse or immunological events within 48 hours, even at the maximum practicable dose of 1 g/kg. The candidate H-NOX trimers were similarly tested by monitoring gross toxicity, blood composition, and antitherapeutic antibodies up to 4 weeks after the initial injection. For preliminary testing safety, groups of six mice were administered candidate H-NOX trimers or buffer control at time 0 at a dose of 750 mg/kg by intravenous bolus injection into the tail vein. Blood samples were collected from the jugular vein of each rat prior to administration of H-NOX trimers and compared with blood collected each week for up to four weeks following administration of H-NOX trimers. The collected blood was processed for serum and plasma and subsequently subjected to clinical composition analysis and antitherapeutic antibody production as detected by ELISA. All animals were observed daily to monitor any evidence of gross toxicity.

[000300] The plasma profiles of H-NOX trimer after intravenous bolus (100 mg/kg) in two different rats are shown in Figure 10. The IgG and IgM antibody responses after administration of the H-NOX trimer are shown in Figure 11.

Example 3. Identification of candidate H-NOX trimers that demonstrate penetration and reduction of hypoxia into brain tissue in vivo in a mouse model of ischemia.

[000301] Candidate H-NOX trimers with circulation half-lives of at least 3 hours were tested in a rodent model of temporary MCA occlusion (tMCAO) of ischemic stroke to identify H-NOX trimers that perfuse and persist in tissue cerebral. Examples of candidate H-NOX trimers were each tested using a group of 24 male Wister rats. Anesthesia was induced in an induction chamber with 2-3% isoflurane in N2O:O2 (2:1), and maintained with 1-1.5% isoflurane via the nose cone. Adequate depth of anesthesia was assessed by the lack of withdrawal due to pinching of the lower limbs and loss of the eye-closing reflex. The animals received 40 mg/kg of cefazolin sodium via intraperitoneal injection and 0.1 mg/kg of buprenorphine subcutaneously. Temporary occlusion of the middle cerebral artery was initiated on Day 0 using standard suture-based surgical technique. Briefly, a skin incision was made over the right common carotid artery (CCA) to allow temporary locking of the CCA and ligation of the distal segment of the external carotid artery (ECA). ). A nylon suture was inserted through the proximal ECA and advanced into the internal carotid artery (ICA) to occlude blood flow to the brain. The CCA clip was removed and the skin incision was closed with surgical staples, and the animal was allowed to awaken. A dose of 750 mg/kg of a candidate H-NOX trimer was infused by tail vein injection into the rats 30 minutes after middle cerebral artery occlusion. In order to analyze hypoxic tissue, the hypoxia marker pimonidazole was injected intraperitoneally at 60 mg/kg approximately 15 minutes after H-NOX trimer injection to identify irreversibly ischemic tissue. The occlusion was released 30 minutes after H-NOX trimer infusion by providing anesthesia to the rats, reopening the lesion and removing the ECA intravascular suture before closing the lesion again with the aim of providing a total of 1 hour of occlusion before reperfusion. . To analyze tissue distribution of H-NOX trimers and quantify the reduction in hypoxia, rats were sacrificed at 2, 4, 12, and 24 hours after reperfusion (N= 6 rats per H-NOX trimer per time point), and brain tissue and blood were collected. Brains from euthanized animals were sectioned and stained for distribution of H-NOX and hypoxic tissue using a polyclonal antibody to H-NOX and the Hydroxyprobe antibody in the hypoxia marker pimonidazole (Hydroxyprobe International), respectively, for subsequent immunohistochemical analysis. Images were taken using a microscope equipped with a camera and staining was quantified. Ischemia and reperfusion are known to trigger neurological cell death and initiate inflammatory responses that result in post-ischemic damage. As secondary endpoints, apoptosis was measured in brain tissue by TUNEL staining assay (Roche) and counting TUNEL-positive cells, and by staining for apoptotic markers that include Cleaved Caspase-1, -3, Bax, and Bcl-2. Blood tests were performed on samples taken at multiple time points to assess the reduction of inflammatory markers and any hematopoietic and systemic effects due to the use of the candidate H-NOX trimers. Upregulation of pro-inflammatory cytokines including TNFα, IL-1α, IL-1β IL-6, MCP-1, Rantes, and MIP were quantified from serum using ELISA (Signosis Rat Inflammation ELISA kit) and RT-PCR. Candidate H-NOX trimers were proven to penetrate ischemic tissue and significantly reduce hypoxia.

Example 4: H-NOX trimers reduced infarct volume and improved neurological outcomes in an in vivo mouse model of ischemia.

[000302] Candidate H-NOX trimers that demonstrated brain tissue penetration and reduced hypoxic tissue were further evaluated in the rat tMCAO stroke rodent model with a focus on clinically relevant assessments extended at time points after ischemia and reperfusion. Example candidate H-NOX trimers were all tested using a group of 12 male Wister rats. Anesthesia was induced in an induction chamber with 2-3% isoflurane in N2O:O2 (2:1), and maintained with 1-1.5% isoflurane via the nose cone. Adequate depth of anesthesia was assessed by the lack of withdrawal due to pinching of the lower limbs and loss of the eye-closing reflex. The animals received 40 mg/kg of cefazolin sodium via intraperitoneal injection and 0.1 mg/kg of buprenorphine subcutaneously. Temporary occlusion of the middle cerebral artery was initiated on Day 0 using standard suture-based surgical technique. Briefly, a skin incision was made over the right common carotid artery (CCA) to allow temporary locking of the CCA and ligation of the distal segment of the external carotid artery (ECA). A nylon suture was inserted through the proximal ECA and advanced into the internal carotid artery (ICA) to occlude blood flow to the brain. The CCA clips were removed and the skin incision was closed with surgical clips, and the animal was allowed to awaken. A dose of 750 mg/kg of a candidate H-NOX trimer or buffer control was infused by tail vein injection into the rats 30 minutes after onset of middle cerebral artery occlusion. The occlusion was released 30 minutes after H-NOX trimer infusion by providing anesthesia to the rats, reopening the lesion and removing the ECA intravascular suture before closing the lesion again with the aim of providing a total of 1 hour of occlusion before reperfusion. . For neurological testing, each animal was evaluated at 72 hours, 1 week, 2 weeks, 3 weeks, and 4 weeks after reperfusion. Animals were evaluated for neurological deficits by rating on a scale of 0-4 with 0 indicating no deficit, 1 indicating failure to extend the forelimbs when placed, 2 indicating circulating, 3 indicating unilateral weakness, and 4 indicating lack of spontaneous motor activity. as previously described. See Borlongan, C.V., et al., (1998) Exp Neurol., 149:310-321, which is incorporated herein in its entirety by reference. Four weeks after reperfusion, animals were sacrificed and brain tissue was harvested for H&E staining and subsequent histological analysis to assess infarct volume. To measure infarct volumes, seven sections of the brain of each animal (+4.7, +2.7, +0.7, -1.3, -3.3, -5.3, and -7, 3, compared to bregma, respectively) were photographed with a digital camera. The infarcted area of each slice was quantified by Image J using the "indirect method" (area of intact contralateral [left] hemisphere - area of intact regions of the ipsilateral [right] hemisphere) to correct for cerebral edema. Infarct areas were summed across slices and multiplied by the slice thickness to obtain the total infarct volume, which was expressed as a percentage of intact contralateral hemispheric volume. Infarct volume was measured by comparison with buffer control animals and qualifications for each group were compared by multiple aNOVA and Newman-Keuls range tests at the 0.05 level of significance. Long-term evaluations up to 4 weeks post-reperfusion ensured that any improvements in neurological function or reduction in infarct volume resulted from a neuroprotective effect of the H-NOX protein and not simply postponing the onset of neurological consequences due to slowing infarct maturation. .

Example 5. H-NOX proteins demonstrated tumor penetration and oxygenation in an in vivo mouse model of cancer.

[000303] Oxygen is a critical factor that ameliorates radiation-induced DNA damage and kills the tumor. Low oxygen levels or hypoxia within solid tumors can cut off 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 that contain 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 the wide variation of tumors arising from, among others, prostate, sarcoma, pancreatic, head and neck, cervical, and brain cancer. See Moeller, BJ 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; all of which are incorporated herein in their entirety by reference.

[000304] To determine the ability of H-NOX proteins to penetrate tumors, groups of 6 mice bearing colon-derived HCT116 subcutaneous tumors were injected via the tail vein with 750 mg/kg of H-NOX monomer, 750 mg/kg of T. tengcongensis L144F H-NOX trimer or saline control. Mice were subsequently sacrificed at 30 minutes or 60 minutes after injection. Tumors were resected, sectioned, stained with an anti-H-NOX antibody, and photographed for intensity of H-NOX staining (Figure 12A). Quantification of the color of HCT-116 tumor sections demonstrated that 23 kDa T. tengcongensis L144F H-NOX monomer accumulated in tumors by 30 minutes and exhibited partial release by 60 minutes (Figure 12B). In comparison, the 80 kDa L144F H-NOX trimer accumulated in tumors by 30 minutes and continued to persist in tumors 60 minutes after injection with accumulation peaking at 4 hours after injection (Figure 12B). Two hours after intravenous injection, H-NOX trimer diffuses from the vasculature into the tumor tissue (Figure 12C). Immunohistochemical staining of tumor sections with H-NOX Antibody and CD31 Antibody (vascularization marker, BD Bioscience). No fluorescent staining is detected in buffer-injected mice.

[000305] To determine whether H-NOX proteins reduced hypoxia in tumors, groups of 6 mice bearing colon-derived HCT116 subcutaneous tumors were injected via the tail vein with 750 mg/kg of L144F H-NOX monomer, 750 mg/kg kg of L144F H-NOX trimer or control saline solution. Before euthanasia, mice were determined with hypoxia marker pimonidazole via intraperitoneal injection and active vascularization marker DiOC73 via intravenous injection. Tumors were harvested at 30 minutes or 60 minutes after H-NOX protein injection, and tested by immunohistochemistry by imonidazole with Hydroxyprobe-1 monoclonal antibody and total vascularization with anti-CD31 antibody (Figure 13A). Quantification of the color of HCT-116 tumor sections demonstrated that in contrast to treated control mice, the 23 kDa H-NOX monomer decreased hypoxia 30 minutes after injection but that there was no recovery in hypoxia 60 minutes after injection ( Figure 13B). In comparison, the 80 kDa L144F H-NOX trimer did not appear to reduce hypoxia at 30 minutes after injection, but substantially reduced hypoxia at 60 minutes after injection (Figure 13B). Additional experiments confirmed that in mice bearing colon-derived HCT116 subcutaneous tumors the H-NOX monomer distributed throughout the tumor tissue (Figure 14A, bottom panel) and released tumor hypoxia at distances away from the vasculature as detected by antipimonidazole antibody ( Figure 14B, the panel at the bottom). The hypoxia-1 (antipimonidazole 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 mean hypoxia-1 test staining was reduced from approximately 13 μM in mice treated with saline to 5 μM in H-NOX monomer treated mice at a distance of approximately 150 μm from the nearest blood vessel (Figure 14C). . These results were further confirmed in mice bearing RIF-1 murine sarcoma xenotransplants.

[000306] Mice bearing RIF-1 sarcoma tumors were injected via the tail vein with 750 mg/kg of T. tengcongensis L144F H-NOX trimer or control saline. Before euthanasia, mice were determined by hypoxia marker pimonidazole via intraperitoneal injection. Tumors were collected at 120 minutes after L144F H-NOX trimer injection, and tested by immunofluorescence imaging for H-NOX trimer distribution (Figure 15), or by western blot for pimonidazole with Hydroxyprobe-1 monoclonal antibody, hypoxia inducible factor 1 (HIF-1α) with anti-HIF-1α antibody, H-NOX Protein with an anti-H-NOX antibody, and total protein with anti-actin antibody (Figure 16). Immunofluorescence staining demonstrated distribution of L144F H-NOX trimer in tumor sections prepared from large isolated tumors approximately 400 mm3 and 800 mm3 in size (Figure 15). Western blot analysis of cell lysates of tumors collected from 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 (Figure 16A). Quantification of additional western blots confirmed the low levels of pimonidazole protein adducts as well as the low levels of HIF-1α protein in tumors from treated mice as compared to saline-treated mice (Figures 16B and 16C).

Example 6. H-NOX proteins demonstrated tumor penetration and oxygenation in an in vivo mouse model of glioblastoma.

[000307] To further characterize the ability of H-NOX proteins to penetrate tumor tissue, three mouse models of glioblastoma were used to evaluate the distribution of T. tengcongensis L144F H-NOX monomer and H-NOX T. tengcongensis L144F trimer in brain tumors. BT-12 cells, an atypical teradoid/rhabdoid infantile brain tumor line that is highly invasive in the spine, was used to generate a mouse model of infantile glioblastoma, GBM-43 cells were used to generate a glioblastoma model adult radioresistant, and U251 cells were used to generate a hypoxic model of adult glioblastoma. Glioblastoma mouse models were generated as previously described. See Ozawa, T, et al., (2010) J Vis Exp, Jul 13;(41) which is incorporated in its entirety here by reference. Briefly, BT-12 cells, U251 cells, or GBM-42 cells were collected by intracranial injection and resuspended in Dulbecco's Modified Eagle Medium (DMEM) at a concentration of approximately 1 x 108 cells per mL. Mice were anesthetized by intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Anesthetic depth was monitored prior to the first incision as well as at regular intervals throughout the procedure, using the foot withdrawal reflex by planting the foot 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 25g needle, 3 mm lateral and 0.5 mm anterior to bregma. Using a sterile Hamilton syringe (Stoelting), 3 x 105 cells in 3μl were injected to a depth of 3 mm over a period of 60 seconds. 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 that received a subcutaneous injection of 0.1 mg/kg buprenorphine were placed on a heated pad and monitored until they regained mobility for use in these studies.

[000308] To determine whether L144F H-NOX trimer could penetrate brain tissue, mice bearing orthotopic U251 brain tumors were injected via tail vein with 750 mg/kg T. tengcongensis L144F H-NOX monomer or 750 mg/kg T. tengcongensis L144F H-NOX trimer. Before euthanasia, the hypoxia marker pimonidazole was determined in the mice by intraperitoneal injection. For immunohistochemical analysis, brains were isolated, sectioned, and stained with pimonidazole with Hydroxyprobe-1 monoclonal antibody, hypoxia-inducible factor 1 (HIF-1α) with anti-HIF-1α antibody, H-NOX protein with an anti-HIF-1α antibody. H-NOX, and HLA-ABC protein with an anti-HLA-ABC antibody (NvusBiological rat monoclonal antibody clone #YTH862.2) approximately two hours after administration of the H-NOX protein. A set of brain tissue samples was additionally stained with secondary antibodies combined with FITC-conjugated anti-rabbit antibody (green channel) manufactured by Jackson ImmunoResearch and DAPI for immunofluorescence imaging. Mice treated with H-NOX trimer demonstrated greater staining for H-NOX as compared to control treated mice indicating that H-NOX trimer penetrated brain tissue (Figure 17A). Furthermore, decreased staining for pimonidazole with Hydroxyprobe-1 monoclonal antibody showed that administration of the H-NOX trimer substantially reduced hypoxia at 60 minutes after injection (Figure 17B). Decreased staining for pimonidazole and HIF-1α protein was further observed in immunofluorescence images (Figures 18A and 18C). Quantification of the immunofluorescence images demonstrated the low levels of pimonidazole staining as well as the low levels of HIF-1α protein staining in the tumors of L144F H-NOX trimer-treated mice as compared to saline-treated mice (Figures 18B and 18D ).

[000309] Figure 19 shows the biodistribution of H-NOX trimer in orthotopic U251 brain tumor and healthy brain. Fluorescent image of H-NOX trimer at high magnification shows weak diffusion outside vessels in healthy brain.

[000310] To compare the 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 the tail vein with 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 fluorescent labeling of H-NOX proteins as follows.

[000311] Purified proteins, H-NOX protein monomer, H-NOX trimer, or BSA (Sigma, used as a control), were thawed on ice and buffer exchanged into endoxin-free Labeling Buffer (50 mM HEPES, 50 mM NaCl, pH 8.0) using endoxin-free dialysis cassettes (Pierce Slide-A-Lyzer, 7 kDa MWCO). The protein concentration after dialysis in Labeling Buffer was determined by UV-vis spectroscopy. Alexa 647 dye (Alexa Fluor® 647 carboxylic acid, succinimidyl ester, Invitrogen #A-20006) was prepared immediately before adding 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 the dye was added to each labeling reaction. Labeling reactions used varying protein:dye ratios with control over the extent of Alexa tagging. Reactions consisted of protein (in Labeling Buffer) and dye to a final DMSO concentration of 5-10%. The reactions were incubated for 1 hour at room temperature (protected from light) with moderate agitation. After the reaction, free Alexa dye was removed by extensive dialysis in endoxin-free buffer formulation (30mM Triethanolamine, 50mM NaCl, pH 7.4) using endoxin-free dialysis cassettes (Pierce Slide-A-Lyzer, 7kDa MWCO cutoff).

[000312] After dialysis in the buffer formulation, the protein concentration and extent of labeling were 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 proportion molar of dye for protein after labeling. The fluorescence of the tagged protein was analyzed by excitation at 647 nm to obtain an emission spectrum. The emission spectrum of the tagged protein was consistent with published data and data from Invitrogen. The tagged protein was further analyzed by size exclusion chromatography to ensure that tagging did not affect the oligomerization state of the protein. Final endotoxin contamination in the tagged protein was determined using the Charles River LAL Gel Clot assay (0.03 EU/mL sensitivity).

[000313] For bioluminescence imaging, mice were anesthetized by IP Injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and then IP injected with 33.3 mg of D-luciferin (salt potassium, 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, CA, USA) and LivingImage software, as the sum of photons counted per second in regions of interest defined by the lowest values threshold of 25% peak pixel intensity. Image acquisition was non-invasive, and the animal's body temperature was maintained using the 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 hours after 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 hours after 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 hours after injection. Tumor bioluminescence had previously been shown to be directly proportional to tumor volume in mice bearing orthotopic GB xenotransplants. See Moeller, BJ 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 BT-12 in a mouse model of glioblastoma, both the L144F H-NOX monomer (Figure 20A) and the L144F H-NOX trimers (Figure 20B) penetrated brain tumors. H-NOX trimer had a significantly longer retention time in tumors as compared to H-NOX monomers. Where HNOX monomer was largely eliminated from tumors by 2 hours (Figure 20A), H-NOX trimer continued to accumulate in tumors for several hours (Figure 20B). Intracranial H-NOX localization was confirmed by imaging ex vivo isolated mouse brain tissue 30 and 60 minutes after injection with the H-NOX monomer (Figures 21A and 18B) and 60 minutes after injection with an H-NOX trimer (Figure 21C ). Additional bioluminescence visualization at 30, 60, 120, and 240 minutes after injection demonstrated that the H-NOX monomer (Figure 22) and H-NOX trimer (Figure 23) also localized to metastatic colonies in the spine. Where H-NOX monomers substantially accumulated in the spine within 30 minutes (Figure 22A) as compared to H-NOX trimer (Figure 23A), it was largely eliminated by 2 hours (Figure 22B-D) while H-NOX trimer -NOX continued to accumulate in the spine for several hours (Figure 23B-D). By using smaller amounts of labeled protein and increasing signal intensity, it was revealed that H-NOX monomer accumulated in the kidneys during this time suggests an elimination route (Figure 24).

[000314] The accumulation of L144F H-NOX trimers in the brain and spine was confirmed in GBM-43 (Figure 25) and U251 in a mouse model (Figure 26). 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 after injection demonstrated that L144F H-NOX trimer accumulated in mouse brain tumors with both administration of high and low concentrations of H-NOX trimer. (Figures 27 and 28, respectively). In comparison, localization of a clustered H-NOX trimer of a variant H-NOX L144F monomer did not accumulate in small brain tumors as evidenced by bioluminescence images 0, 0.5, 1, 2, 4, and 6 hr after injection. with 30 mg/kg (Figure 29). Ex vivo bioluminescence imaging of isolated mouse brain treated with 30 mg/kg L144F H-NOX trimer (Figure 30A) or 750 mg/kg L144F trimer (Figure 30B) showed that the amount of H-NOX protein in a single dose had little effect on localization of H-NOX in intracranial tumors. Furthermore, real-time bioluminescence imaging of mice bearing large (Figure 30C) or small tumors (Figure 30D) showed that after administration of 295 mg/kg of L144F H-NOX trimer, the trimer distributed into intracranial tumors regardless of the tumor size (Figure 30B). Real-time ex vivo bioluminescence imaging of three mouse models of glioblastoma, GBM, U251, and BT-12, demonstrated that L144F H-NOX trimer distributed in intracranial tumors and spinal tumors in all three models (Figures 31 and 32 ). Immunofluorescent imaging of a tumor section stained with antibodies to the H-NOX protein and the vasculature showed that L144F H-NOX trimer left the vasculature and fused throughout the brain tumor (Figure 33). Overall, these data identified H-NOX proteins with clinically relevant tumor biodistribution profiles.

[000315] To verify the partition of H-NOX trimers between the brain and plasma, L144F trimer was tested using a group of three female FVB mice (Figure 34). The 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 after injection of the candidate H-NOX trimer or buffer control, mice were sacrificed. Approximately one ml of blood was collected by intracardiac puncture and the brain was collected. The collected blood was processed for brain samples and the plasma was lysed to extract proteins. Brain and plasma were subsequently analyzed for the presence of H-NOX trimer using an ELISA Assay with a polyclonal antibody compared to H-NOX protein.

Example 7. H-NOX trimers improved the effects of radiation in mouse models of glioblastoma in vivo.

[000316] To determine whether oxygenation of hypoxic tumors due to H-NOX penetration could improve radiation performance by inducing tumor death, studies were performed on groups of 10 U251 mice bearing athymic intracranial glioblastoma tumors to evaluate the effects of therapy. radiation (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 after tumor implantation with or without administration of 750 mg/kg Alexa-647 labeled T. tengcongensis L144F H-NOX trimer. transported by intravenous injection. Mice were monitored until day 29 and subjected to bioluminescence imaging on days 15, 17, 20, 22, 24, and 29. Mean bioluminescence imaging (BLI) qualifications determined for each treatment group demonstrated that multiple doses of trimer L144F H-NOX resulted in statistically significant postponement of tumor growth (Figures 35A and 35B) and despite the aggressive and mildly hypoxic nature of orthotopic U251 treated tumors, animal survival was also significantly improved in L144F H-NOX treated groups (Figure 35C). Tumors were also collected by immunohistochemical staining and analysis.

[000317] 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 1) buffer treatment alone, 2) buffer treatment in combination with a single dose of 2 Gy radiation (irradiator setting = 0.81; a Cesium dose rate from the 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 configuration = 0.81; the Cesium dose rate from the irradiator was 247 CGy/min). Mice receiving the combination treatment were irradiated for 2 hours after L144F H-NOX transporter trimer in the suprasensory portion of the brain. Treatment for all mice began 14 days after intracranial injection of mice with 3.0 x 105 U251 cells. Animal survival was found to be increased in cohorts that received the combined treatment of L144F H-NOX trimer and 2 Gy radiation (Figure 36A). In another study, groups of 10 GBM43 mice bearing intracranial glioblastoma tumors were treated with 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) buffer treatment. Mice receiving the combined treatment were irradiated 1 to 1.5 hours after trimer H-NOX carrier and mice receiving multiple doses of RT had RT administration separated by 4 days. Radiation treatment was administered to the suprasensory portion of the brain to all RT groups. Treatment of all mice began 7 days after tumor implantation. It was concluded that animal survival in cohorts that received combined L144F H-NOX trimer treatment and 4 Gy radiation was similar to animal survival in cohorts that received 4 Gy treatment alone (Figure 36B).

Example 8. Toxicology test of H-NOX proteins.

[000318] To evaluate the safety profile of H-NOX monomers in anticipation of IND-qualifying toxicology studies, a preliminary toxicology study on GLP in Sprague-Dawley rats was performed at an independent FDA-accredited Contract Research Organization (MPI Research Laboratories). No adverse events or control differences were detected at doses of 100 and 300 mg/kg of H-NOX T. tengcongensis L144F monomer 48 hours after injection (IV). At the maximum practicable dose of 1000 mg/kg, some mild signs of toxicity were observed (Table 4). The high number of white blood cells recorded in the 48 hours were probably due to trace amounts of endotoxin present in the protein formulation and these levels were reduced 100-fold in production verified by additional studies. In the separate study, mice were injected with 50mg/kg L144F H-NOX monomer or L144F H-NOX trimer and followed on Day 32. Both antitherapeutic IgM and IgG antibodies were generated, however, there were no cases of anaphylactic shock regardless of the H-NOX variant or number of doses (up to 4 doses tested). Table 4. H-NOX monomer was well tolerated in rats.

Example 9. Characterization of minimal H-NOX trimer dosing program in in vivo animal models of glioblastoma.

[000319] To better inform the design of INDs that enable toxicity studies and clinical studies, the schedules and dosage levels of the example H-NOX trimer are characterized using a mouse model of U251 glioblastoma. The outcome of these studies is further validated in animal models of glioblastoma to better inform patient selection.

Identify the minimum effective dose of the H-NOX trimer example that improves RT

[000320] To minimize adverse events in patients who received the H-NOX trimer example and quantify the pharmacodynamics (PD) of the H-NOX trimer example in oxygenation and radiosensitization of tumors, the minimum effective dose (MED, according to its acronym in English) of the H-NOX trimer is identified in the U251 glioblastoma mouse model. Preliminary studies demonstrated that a dose of 750 mg/kg H-NOX trimer resulted in substantial reduction of tumor hypoxia two hours after administration (Figures 17A and 17B) and that this effect was sufficient to significantly improve tumor responses to RT ( Figure 35). Other studies of xenotransplant tumors have demonstrated that H-NOX trimer accumulates in tumors at doses as low as 10 mg/kg, establishing a wide range of potential for effective dosing. To identify H-NOX trimer MED, dosages ranging from 7.5 mg/kg to 750 mg/kg were used to improve tumor responses to RT.

[000321] To identify the H-NOX trimer MED, the radiosensitization effects of doses of 75 mg/kg and 7.5 mg/kg compared to the previously established efficient dose of 750 mg/kg are compared (Table 5) . Efficacy is tested in comparison to orthotopics in a mouse model of GB, using the luciferase modified U251 human GB linear cell. A cohort of 10 mice is used to follow tumor growth by bioluminescence imaging and to follow survival. Furthermore, three cohorts of 3 mice each were used to examine molecular mechanisms behind H-NOX trimer action, which include H-NOX trimer localization by immunohistochemistry and quantitative ELISA, tumor oxygenation by immunohistochemistry using EF5 as a marker. by hypoxia, and assessment of DNA damage by immunohistochemistry using YH2AX staining (Table 5).

[000322] For these studies, U251 cells are resuspended in Dulbecco's Modified Eagle Medium (DMEM) at a concentration of approximately 1 x 108 cells per mL. Athymic mice are anesthetized by intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). A 1 cm sagittal incision is made along the scalp and a small hole is created by puncture with a 25 g needle, 3 mm lateral and 0.5 mm anterior to bregma. Using the sterile Hamilton syringe (Stoelting), 3 x 10 cells in 3 μ| volume is injected to a depth of 3 mm over a period of 60 seconds. After injection, the syringe is removed and the skull is sealed with bone wax before closing the scalp using 7mm surgical staples (Stoelting). See Ozawa, T et al., (2010) J Vis Exp, Jul 13;(41). Mice receive a subcutaneous injection of 0.1 mg/kg buprenorphine and are monitored until they regain mobility. Between 21 and 25 days after tumor implantation, H-NOX trimer at the dosage indicated for each cohort is administered by tail vein injection two hours before RT, which is administered at 2 Gy/dose to the entire suprasensory brain (Table 6). For all administration of RT to the brain, a 137Cs source carrying a dose rate of approximately 280 cGy/min is used and mice are irradiated for the duration of time that results in 2 Gy. Table 5. Cohort dose escalation studies for H-NOX trimer

[000323] All RT programs consist of 3 weekly doses of 2 Gy/dose

[000324] Non-invasive tumor growth is monitored by bioluminescence imaging during the course of the study. For bioluminescence imaging, mice are anesthetized by IP injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and then injected IP with 33.3 mg of D-luciferin (potassium salt, Gold Biotechnology, St. Louis, MO, USA) dissolved in sterile saline. Tumor bioluminescence is determined 10 minutes after luciferin injection, using the IVIS Lumina System (Caliper Life Sciences, Alameda, CA, USA) and LivingImage software, as the sum of photons counted per second in regions of interest defined by the lowest values threshold of 25% of peak pixel intensity. For survival analysis, mice are euthanized when body weight decreases by more than 15% or when neurological deficits are observed (Efficacy, Table 5).

[000325] To research tumor oxygenation, or reduction of hypoxia, and localization of the H-NOX trimer in the tumor, three mice from each dosage group are injected with 10 mM EF5, a clinical biomarker for hypoxia, to IV and sacrificed immediately after RT for IHC analysis on all brain sections (Nhypoxia and H-NOX IHC trimer, Table 5). Additionally, a cohort of three mice are sacrificed immediately after RT and tumors are resected for analysis of H-NOX trimer content by quantitative ELISA (H-NOX Trimer ELISA, Table 5). Two hours after completion of RT, additionally, cohort of three mice are sacrificed for DNA damage analysis by YH2AX IHC in all brain sections (NDDNA damage IHC, Table 5). For analysis of RT efficacy and analysis of molecular hypoxia, tumor location, and DNA damage, statistical analyzes are performed by aNOVA with subsequent paired t-tests and the change at P < 0.05 is considered statistically significant. For survival analysis, the Log-Rank test is used with two-tailed alpha equal to 0.05 for 88% to be able to detect an effect size of 1.5. Effect size is defined as the difference in mean survival divided by the within-treatment standard deviation.

Correlate residence time of H-NOX trimer example and oxygenation in tumors with radiosensitization

[000326] To better inform the target clinical profile of H-NOX trimers, the longevity of the oxygenation effects of the H-NOX trimer on tumors resulting in radiosensitization was researched. RT is administered at a range of time points based on the peak intracranial tumor location of the H-NOX trimer schedule (Figure 26), and using the H-NOX trimer MED, the length of time between H-trimer administration -NOX and RT is varied (Table 6). Similar to studies to determine the MED of H-NOX trimer, several pharmacodynamic parameters are evaluated such as distribution of H-NOX trimer in tumor tissue, reduction of hypoxia, DNA damage, postponement of tumor growth, and improved overall survival. Table 6. Cutoffs by duration of radiosensitization studies

[000327] All RT programs consist of 3 weekly doses of 2 Gy/dose

Reproducibility of H-NOX trimer-mediated radiosensitization furthermore across GB classes

[000328] Three molecularly defined clinical GB subclasses are studied by H-NOX trimer mediated radiosensitization. Xenotransplant models derived from cell lines (GBM43, GBM6, and GBM14) of these three subclasses are established as previously described. See Verhaak et al., (2010) Cancer Cell, 17:98110 and Phillips et al., (2006) Cancer Cell, 9:157-173. Briefly, by producing these three mouse models, subcutaneous tumors from these three cancer types are ground with a scalpel and subjected to three rounds of passage through a 40 μm pore filter, with centrifugation after each round of filtration with increasing speed. 158 xg, 355 xg, 631 xga 10 minutes each. After the final round of centrifugation, cells are resuspended in 1 mL of sterile DMEM medium, counted, and diluted to 1 x 108 cells/mL for intracranial injection. Furthermore, H-NOX trimer-mediated radiosensitization is studied in an immunocompetent model of GB (GL261), which replicates the intact immune system in GB patients. For production of GL261 in a mouse model, tumor cell harvest and injection is performed similarly to U251 models using a mouse GB cell line that is syngeneic with C57BL/6 mice. See Newcomb et al., (2006) Cell Cycle, (5):93- 99. For efficacy testing four experimental groups for each tumor model are used (Table 7). As with the U251 models, treatment initiation occurs when the tumor model is 75% complete, with average survival days that reflect 100% completion. Similar to studies to determine the MED of H-NOX trimer, pharmacodynamic parameters are evaluated such as distribution of H-NOX trimer in tumor tissue, reduction of hypoxia, DNA damage, postponement of tumor growth, and improved overall survival. Table 7. Cohorts for studies in addition in GB models

[000329] All RT programs consist of 3 weekly doses of 2 Gy/dose Example 10. Pharmacodynamic Characterization of Single Dose H-NOX Trimer Toxicity in In Vivo Animal Models of glioblastoma.

[000330] To justify the selection of species for IND-qua enable GLP toxicity studies and to inform Phase 1 clinical, exploratory testing of non-GLP toxicity and GB PD studies are performed in rats (rodents) and dogs (non-rodents). To the extent that the H-NOX trimer does not bind to or react with a specific human target, non-primate species are acceptable to the FDA.

Study of the variation in toxicity of a single dose of non-GLP in rats

[000331] To identify the maximum tolerated dose (MTD, according to its acronym in English) and characterize the toxicity and toxic kinetic profile (TK, according to its acronym in English) of H-NOX trimer, a single dose study of non-GLP in Sprague-Dawley (SD) rats was performed.

[000332] Male and female SD rats at least 6 to 8 weeks old are assigned to 5 study groups (Table 8). Each animal receives vehicle or H-NOX trimer to slow IV bolus administration (4-5 minutes) into the tail vein in volumes up to 10 mL/kg. The animals are dosed per group, with 3 days between each dose. The range of MED H-NOX trimer dose levels approximates the maximum practicable dose. Plasma samples are taken at regular intervals after dosing to evaluate plasma pharmacokinetics (PK) of H-NOX trimer. Time points for toxicokinetic analysis are based on PK results. Doses are administered volumetrically, based on the animal's most recent body weight. Clinical observations, body weights, food consumption, and clinical pathology are reviewed before selecting the next dose (Table 9). The recovery period of up to 14 days makes it impossible to evaluate the persistence, occurrence of postponement and recovery of any toxicity events. Table 8. Cohorts for the single dose toxicity study in rats Table 9. Measurements and observations for the single dose toxicity study in rats

Assess tumor distribution of H-NOX trimer and PD in a GB mouse model

[000333] To verify that tumor H-NOX distribution and hypoxia reduction is similar in rats and mice, tumor distribution and PD of H-NOX trimer in the rat 9L glioma model are evaluated. To produce the rat 9L glioma model cells are implanted intracranially into Wistar rats as previously described. See Stojiljkovic et al., (2003) J. Neurooncol, (63):1-7. Briefly, 9L cells, a linear rat glioma cell, are collected by intracranial injection by monolayer trypsinization and resuspended in DMEM at a concentration of 4 x 104 cells in 5 μL. Anesthesia is induced by IP injection of 10 mM ketamine and 7.5 mg/kg xylazine. A 2 cm sagittal incision is made along the scalp and a hole is created using the small dental splint at 3 mm lateral and 0.5 mm anterior to the bregma. Using the sterile Hamilton syringe (Stoelting), 5 x 105 cells in 10 μl are injected to a depth of 4.5 mm over a period of 60 seconds. After injection, the syringe is removed and the skull is sealed with bone wax before closing the scalp using 7 mm surgical staples (Stoelting). H-NOX trimer is administered to the rats in tail vein injection using a 27g needle at a dosage volume not exceeding 10 mL/kg. One hour after administration of the H-NOX trimer, mice receive a 10 mM dose of the hypoxia marker EF5 by IV. Two hours after H-NOX administration, mice are euthanized and tumor-bearing brains are collected for H-NOX trimer localization and hypoxia quantification by immunohistochemical analysis or for H-NOX trimer quantification by ELISA (Table 10). Cohorts for H-NOX trimer biodistribution and oxygenation studies in rats.

Non-GLP Single Dose Toxicity Ranging Study in Canines

[000334] The toxicity and toxicokinetics of H-NOX trimer were researched in Beagle dogs. Briefly, male and female Beagle dogs at least 5 months of age are assigned to 4 dosing groups (Table 11). Range H-NOX trimer dose levels from the approximate MED, as calculated equivalent in this species, to the maximum practicable dose. Each animal receives H-NOX trimer by slow IV bolus through the cephalic vein. Plasma samples are taken at regular intervals after dosing for plasma pharmacokinetic (PK) analysis of H-NOX trimer. Time points for toxicokinetic analysis are based on PK results. Doses are administered volumetrically, based on the animal's most recent body weight. Clinical observations, body weights, food consumption, and clinical pathology are reviewed before selecting the next dose (Table 12). A recovery period of up to 14 days allows assessment of persistence, postponement of occurrence, and recovery from any toxicity event. Statistical analysis is performed using Graph Pad Prism (Version 4.03). Comparisons are made between the vehicle and H-NOX trimer study groups at each corresponding analysis data time point using parametric statistical procedures (e.g., repeated measures analysis of variance followed by multiple Dunnett's t-test comparisons). or non-parametric (e.g., Friedman Test and Dunn's post-hoc Test). The choice of parametric or non-parametric statistics is based on whether the groups compared meet the homogeneity of variance criteria. Differences between treatment with vehicle and H-NOX trimer are observed as p < 0.05. Table 11. Cohorts for single dose toxicity study in canines Table 12. Measurements and observations for single dose toxicity study in canines

Evaluate H-NOX trimer distribution in tumor and PD in a canine model of GB

[000335] To confirm that tumor distribution and oxygenation seen in GB rodent models is representative of tumors similar to human tumors, Phase 1b testing in canines with GB is performed to measure spontaneous H-NOX trimer accumulation in tumors. brain cells, and to quantify changes in hypoxia using a clinically relevant real-time PET test for hypoxia (and PET Test can be used; e.g., 18F-EF5 of 18F-MISO). Ten dogs belonging to clients (hereinafter referred to as "canine patients") with spontaneous GB are included in the study. In general, canine patients receive a single dose of H-NOX trimer accompanied by pre- and post-18F-EF5 PET scanning to assess any change in tumor hypoxia resulting from H-NOX trimer treatment. Dogs that do not have hypoxic tumors (defined as a brain tumor:normal ratio 2.0) are removed from the study prior to administration of the H-NOX trimer. Canine patients with sufficiently hypoxic tumors are scheduled for treatment with H-NOX trimer no less than 72 hours after the initial PET scan. H-NOX trimer is administered as an IV bolus injection at a dosage volume not exceeding 10 mL/kg. H-NOX trimer dosage levels start at MED and escalate according to a 3 + 3 dose escalation design. Dose escalation is stopped after 3 dose levels or when dose toxicity limit is reached in 1 or more dogs. Two hours after H-NOX trimer administration, canine patients undergo a second PET scan and tumor hypoxia is quantified. For pet scans, food is withheld for 12 hours prior to anesthesia. An intravenous catheter is placed in each canine patient and anesthesia is induced with 10-25 mg/kg thiopental or 6 mg/kg propofol and maintained with 1-5% isoflurane in oxygen. 18F-EF5 is administered briefly after anesthesia is induced, and pet scanning/CT is initiated one hour after 18F-EF5 administration. For canine patients undergoing surgery as part of standard care, the resected portion of the tumor is reserved for analysis of H-NOX trimer content. To assess tumor H-NOX trimer localization, resected tumors are evaluated by quantitative IHC or ELISA using antibodies to H-NOX protein and pimonidazole. Small amounts of whole blood are obtained pre-dose and at 24 hours, 48 hours, and 14 days after administration of the H-NOX trimer to analyze safety parameters such as maximum tolerated dose and to test for the presence of antitherapeutic antibodies. Safety data are available pre-dosing and 14 days post-dosing. Furthermore, hypoxia biomarkers are used in this study such as copper(II) (diacetyl-bis(N4-methylthiosemicarbazone)) (64Cu-ATSM).

H-NOX trimer-mediated radiosensitization in canines

[000336] Using information from single dose toxicity studies in canines, H-NOX trimer-mediated radiosensitization is further studied in these animals. Male and female Beagle dogs of at least 5 months of age are assigned to dosage groups consisting of H-NOX trimer or vehicle treatment with or without high dose radiation therapy in a single 8 Gy fraction. Tumors are resected in these canine patients for analysis of H-NOX trimer content. For localization of the tumor H-NOX trimer, resected sections of the tumors are evaluated by IHC or Quantitative ELISA using antibodies to H-NOX Protein and pimonidazole. Furthermore, hypoxia biomarkers are used in this study such as 64Cu-ATSM.

Example 11. GMP production of H-NOX trimers.

[000337] The development of H-NOX trimer, which includes cGMP production, GLP safety testing, and regulatory preparation necessary to obtain FDA approval to begin clinical trials was researched.

GMP fabrication of H-NOX trimer candidate

[000338] Large quantities (e.g., kilo quantities) of GMP protein to support toxicological testing, rat and canine PD studies, and clinical trials are produced. Cell lines, plasmids, and growth culture conditions required to cultivate cells and create the GMP cell banks are provided here. Methods for producing H-NOX trimer from 4L fermentation with ODs reaching as high as 115 are also provided here. Methods for purification of H-NOX trimer protein which can generate up to 1g of purified H-NOX trimer protein from 12L of cell culture are further provided here. Also provided here are quality control assays for H-NOX trimer, which include, but are not limited to, SDS-PAGE, UV/Vis spectral analysis, LC-MS, SEC analytical, SEC-HPLC, endoxin testing, filtration testing for particulates, viral contamination testing, and oxygen release rates for effectiveness. Additional systems are provided here for the production of H-NOX trimer which include but are not limited to microreactor systems, scale process equipment, pumps and filters. H-NOX trimer undergo QC/QA testing and validation before being released for pre-clinical and clinical studies.

H-NOX trimer GLP toxicity tests in rats and dogs

[000339] GLP toxicity studies are carried out in accordance with ICH guidelines S6(R1) and S9. According to ICH guidelines, toxicological tests are performed on rodent (Sprague-Dawley rat) and non-rodent (Beagle dog) species. For a 7-day repeat dose rat study, male and female Sprague-Dawley rats are assigned to four treatment groups (Table 14). Varying MED H-NOX trimer dose levels approximate the maximum practicable dose or maximum tolerated dose. Each animal receives vehicle or H-NOX trimer to slow IV bolus administration into the tail vein in volumes up to 10 mL/kg. Doses are administered volumetrically based on the animal's most recent body weight. Animals are dosed once daily for 7 consecutive days. Animals assigned to terminal necropsy (up to the first 10 mice/sex/group) undergo necropsy on Day 8. Animals assigned to recovery necropsy (up to the last 5 mice/sex/group) undergo 7 consecutive dosing days for 7 days of recovery and undergo necropsy on Day 15 with the aim of evaluating the toxicity and toxicokinetics of H-NOX trimer. Standard toxicity parameters, which include assessment of vital organs (respiratory, cardiovascular, and central nervous systems) are monitored (Table 15). Table 14. Cohorts for LPG toxicity study in rats Table 15. Measurements and observations for GLP toxicity study in rats

[000340] For a 7-day repeat dose study in dog, male and female purebred beagle dogs of at least 5 months of age are assigned to 4 groups (Table 16). The range of MED H-NOX trimer dose levels approximates the maximum practicable dose or the maximum tolerated dose. Each animal receives vehicle or H-NOX trimer to slow IV bolus administration into the cephalic vein in volumes up to 10 mL/kg. Animals are administered vehicle or H-NOX trimer once daily for 7 consecutive days. Animals assigned to terminal necropsy (up to the first 3 canines/sex/group) undergo necropsy on Day 8. Animals assigned to recovery necropsy (up to the last 2 canines/sex/group) undergo 7 days of dosing followed by 7 days of recovery and undergo necropsy on Day 15 with the aim of evaluating the toxicity and toxicokinetics of H-NOX trimer. Standard toxicity parameters, which include assessment of vital organs (respiratory, cardiovascular, and central nervous systems) are monitored (Table 17). Cardiac safety is assessed as part of this study by monitoring electrocardiograms (ECGs) during the dosing phase. Table 16. Cohorts for Canine GLP Toxicity Study Table 17. Measurements and observations for LPG toxicity study in canines

[000341] Furthermore, since the H-NOX trimer is a new therapeutic protein, a standard cardiac safety study is performed only in canines to monitor effects on heart function and vasoactivity. Four non-authentic, male Beagle dogs (9 to 18 kg), previously implanted with radiotelemetric devices (DSI: TL11M2-D70-PCT) are dosed by IV bolus injection. Canines are administered vehicle and 3 dose levels of H-NOX trimer using an ascending or Latin Square dosing regimen (Table 18). Each animal receives 1 of 4 doses (in predetermined order) once per week (Days 1, 8, 15 and 22 of the dosing phase). Doses are chosen based on studies previously carried out in canines. All animals are observed frequently for clinically relevant signs of reactions to the H-NOX trimer or vehicle from the day of dosing, and then observed from the side of the cage at least once daily during the in-life phase of the study. Hemodynamic parameters (arterial blood pressure, heart rate, and electrocardiogram [ECG] example) are recorded. Recordings begin at least 1 hour before dosing and continue for at least 24 hours after dosing. Parameters assessed include; systolic, diastolic, and mean blood pressure, heart rate, PR, QRS, QT, and RR intervals, QRS duration, and QTcVW and QTcI corrected QT intervals (Table 19). Data is visually inspected for accuracy and values tabulated at 10 predetermined time points based on the article's pharmacokinetic testing. A visual inspection of all ECG waveforms for disturbances in rhythm morphology and waveforms is added. Statistical analysis of hemodynamics and ECG data is performed using Graph Pad Prism (Version 4.03). Comparisons are made between the vehicle and H-NOX trimer treatment groups at each corresponding data analysis time point using parametric statistical procedures (e.g., repeated measures analysis of variance followed by Dunnett's multiple t-test comparison). or non-parametric (e.g., Friedman Test and Dunn's post-hoc Test). The choice of parametric or nonparametric statistics is based on whether the compared groups satisfy a criterion of homogeneity of variance. Differences between vehicle and H-NOX trimer treatment are observed as p < 0.05. Table 18. Cohorts for telemetric cardiovascular study in canines Table 19. Measurements and observations for telemetric cardiovascular study in canines

Example 11. Phase 1 clinical trials for the use of H-NOX trimer in patients with glioblastoma.

[000342] The Phase 1b study is carried out to evaluate the safety of H-NOX trimer, biodistribution in tumors, and reduction of hypoxia in patients with recurrent GB. A second Phase 1b study is conducted to evaluate the safety of the H-NOX trimer in patients recently diagnosed with GB.

Phase 1b Trial in Recurrent GB Patients

[000343] The first Phase 1b Trial is a single-dose escalation study aimed at terminally ill patients with recurrent GB who are candidates for a second resection. This study provides direction in terms of dose level selection and route of administration in a clinically relevant population. Twenty patients with recurrent GB who meet the inclusion and exclusion criteria are included in the study.

Inclusion criteria:

[000344] 1. Patients with imaging evidence of recurrent GB who plan to have a repeat resection as part of standard care are eligible.

[000345] 2. Patients are eligible if the original histological diagnosis was low-grade glioma and the subsequent histological diagnosis is GB.

[000346] 3. All patients must sign an informed consent indicating that they are aware of the investigational nature of this study. Patients must sign an authorization for the release of their protected health information. Patients must register in the database prior to study drug treatment.

[000347] 4. Patients must be over 18 years old, and with a life expectancy of > 8 weeks.

[000348] 5. Patients must have a Karnofsky performance status > 60.

[000349] 6. At the time of registration: patients must be recovered from the toxic effects of prior therapy: >28 days of any investigational agent, >28 days of prior cytotoxic therapy, >14 days of vincristine, >42 days of nitrosoureas, >21 days of procarbazine administration, and >7 days for non-cytotoxic agents, e.g., interferon, tamoxifen, thalidomide, cis-retinpoic acid, etc. Any questions regarding the definition of non-cytotoxic agents should be addressed directly to the Study Chair.

[000350] 7. Patients must have adequate bone marrow function (WBC > 3,000/μl, ANC > 1,500/mm3, platelet count > 100,000/mm3, and hemoglobin > 10 gm/dl), adequate liver function (SGOT and bilirubin < 2 times ULN), and adequate renal function (creatinine < 1.5 mg/dL) before starting therapy. These tests must be performed within 14 days prior to registration. The hemoglobin eligibility level may not be met by transfusion.

[000351] 8. Patients must have shown unequivocal radiographic evidence for tumor progression by MRI or CT. A scan must be performed within 14 days prior to registration and a steroid dose that has been stable for at least 5 days. If the steroid dose is increased between the imaging date and registration of a new MR/CT reference line is required. The same type of scan, i.e. MRI or CT should be used throughout the treatment protocol period for tumor measurement.

[000352] 9. Patients may have been treated for any number of previous relapses.

[000353] 10. Women and girls of carrier potential must have a negative B-HCG pregnancy test documented within 14 days prior to registration.

Exclusion Criteria:

[000354] 1. Patients must not have previously received therapy with agents bevacizumab (Avastin), other VEGF or VEGFR, or other agents considered anti-angiogenic agents.

[000355] 2. Any patient with PET evidence of low hypoxic fraction (SUVtumor/SUVcerebellum ratio 2.0) is excluded.

[000356] 3. Any patient on antihypertensive medication therapy is excluded.

[000357] 4. Patients must not have any significant medical condition that in the opinion of the researcher cannot be adequately controlled with appropriate therapy or that would compromise the patient's ability to tolerate this therapy.

[000358] 5. Patients with a history of any other cancer (except non-melanoma skin cancer or carcinoma in situ of the cervix), unless in complete remission and off all therapy for the disease for a minimum of 3 years , are ineligible.

[000359] 6. Patients must not have an active infection or serious intercurrent medical illness.

[000360] 7. Patients must not have any illness that will obscure the toxicity or dangerously alter the metabolism of the medication.

[000361] Patients in this study receive only H-NOX trimer, with no other competing therapy. There are four dose levels with exact dosage levels determined from levels identified as well tolerated in preclinical toxicology studies, and are escalated according to a 3+3 design. Breakpoints and dose escalation are primarily based on reducing hypoxia, due to the expected low toxicity of a single dose of H-NOX trimer. Based on preliminary toxicity testing of the H-NOX protein, little or no toxicity is expected to be associated with the H-NOX trimer. However, if the dose toxicity limit (DLT) is met, the escalation criteria are immediately modified and based on the DLT and the escalation rules based on the standard 3+3 design. DLTs for the study are defined as any of the following events that may be attributable to the H-NOX trimer:

[000362] Grade 3 thrombocytopenia

[000363] Grade 4 anemia and/or grade 4 neutropenia

[000364] Any grade 3 non-hematological toxicity, excluding alopecia.

[000365] To evaluate biological activity of H-NOX trimer, patients receive an 18F-EF5 PET Scan 72 hours before dosing with H-NOX trimer. The pet scan allows the quantification of the hypoxic fraction, characterized as a proportion of the standard assimilation value (SUV) of the tumor to the SUV of the cerebellum (normal brain) and establishes the baseline of tumor hypoxia levels before surgery. Patients with low levels of hypoxia (defined as a tumor:normal brain ratio below 2.0) are excluded. Patients with sufficiently hypoxic tumors receive a single dose of H-NOX intravenously and a second 18F-EF5 PET scan two hours after H-NOX trimer dosing to assess changes in the hypoxic fraction. The second PET Scan is quantified and any change from the baseline scan is recorded. A change in hypoxic fraction of at least 15% is considered as clinically promising and constitutes a 'positive' change where a change of less than 15% is declared a 'negative' change. Proceed with planned surgical resections within 24 hours of H-NOX trimer dosing. Resected tumors are examined via quantitative immunohistochemistry (IHC) for H-NOX trimer penetration, as well as Quantitative IHC for EF5 staining to confirm PET results. H-NOX trimer distribution data is confirmed by Quantitative ELISA, using homogenized tissue samples when possible. All clinical laboratory tests for safety (e.g., liver function, kidney function, CBC) are performed prior to dosing and 24 hours, 48 hours, and 14 days after dosing. Blood samples from each of these time points are used to determine plasma pharmacokinetics (PK), as well as to test for the presence of antitherapeutic antibodies (ATAs). The primary endpoint is characterized by the fact that it includes a single agent safety and secondary endpoints include tumor PK, reduction in hypoxia, and time to ATA production.

Phase 1b Trial in Newly Diagnosed GB Patients

[000366] The second Phase 1b Trial is a classic 3+3 dose escalation study combining H-NOX trimer with current standard of care therapy for newly diagnosed GB. After diagnosis and an initial resection, GB patients currently receive approximately 60 Gy in fractionated RT in conjunction with temozolomide (TMZ), an oral DNA alkylating chemotherapy. Since the mechanism of action of the H-NOX trimer involves reducing hypoxia in solid tumors, only patients with partial (i.e., subtotal) resections are included in the study. Twenty patients with newly diagnosed GB who meet the inclusion and exclusion criteria are included in the study.

Inclusion criteria:

[000367] 1. Patients with histologically proven newly diagnosed intracranial GB will be eligible for this protocol.

[000368] 2. Patients must have significant hypoxic fraction on the 18F-EF5 PET image to be taken before starting therapy.

[000369] 3. Residual and evaluable disease after resection of newly diagnosed GB is considered for study eligibility. To best assess the extent of residual disease after surgery, CT/MRI should be done no later than 96 hours in the immediate period after surgery or at least 4 weeks after surgery, within 14 days before registration. If the scan after 96 hours is more than 14 days before registration, the scan needs to be repeated if the steroid dose is increased between the date of imaging and registration, a new baseline MRI/CT is required at a stable dosage of steroids for at least 5 days.

[000370] 4. Biopsy or resection must have been performed no more than 5 weeks before treatment.

[000371] 5. All patients must sign an informed consent indicating that they are aware of the investigational nature of this study. Patients must have signed an authorization for the release of their protected health information. Patients must be registered in the database prior to study drug treatment.

[000372] 6. Patients must be over 18 years old, and with a life expectancy > 8 weeks.

[000373] 7. Patients must have a Karnofsky performance status > 60.

[000374] 8. Patients must have adequate bone marrow function (WBC > 3,000/μl, ANC > 1,500/mm3, platelet count > 100,000/mm3, and hemoglobin > 10 gm/dl), adequate liver function (SGOT and bilirubin < 2 times ULN), and adequate renal function (creatinine < 1.5 mg/dL) before starting therapy. These tests must be performed within 14 days prior to registration. Hemoglobin eligibility level may NOT be met by transfusion.

[000375] 9. Female carriers of potential must have a negative B-HCG pregnancy test documented within 14 days prior to registration.

[000376] 10. Patients must not have previously received cytotoxic drug therapy, non-cytotoxic drug therapy, or experimental drug therapy for brain tumors. Patients who received polyfespan 20 with carmustine (Gliadel) pellet implantation at the time of the original resection will be excluded.

[000377] 11. Patients should plan to begin partial brain radiotherapy the next day after starting H-NOX trimer and temozolomide. Radiation therapy must be a site affiliated such that the radiation oncologist can provide assurance that radiation can be performed as specified in this protocol. Radiotherapy should be given by external beam to a partial field of the brain in daily fractions of 1.8 to 2.0 Gy, up to the total dose planned for the tumors of 59.4 to 61.0 Gy. Stereotactic radiosurgery (e.g. Gamma-Knife treatment) and brachytherapy will not be permitted.

[000378] 12. Patients must be willing to undergo other cytotoxic and non-cytotoxic drug therapy compared to the tumor while being treated with H-NOX trimer and temozolomide.

[000379] 13. Male and female patients of reproductive potential should use an approved method of contraception if appropriate (e.g., intrauterine device [IUD], birth control pills, or barrier device) during and for 3 months after discontinuation of treatment of the study. Female carriers of potential must have a negative beta-HCG pregnancy test documented within 14 days prior to treatment. If a condom is used as a contraceptive barrier, a spermicidal agent must be added to ensure that pregnancy does not occur. If a woman becomes pregnant or suspects that she is pregnant while participating in this study, she must inform her treating physician immediately.

Exclusion criteria:

[000380] 1. Patients must not have previously received concurrent therapy with bevacizumab (Avastin), other VEGF or VEGFR agents, or other agents considered anti-angiogenic agents.

[000381] 2. 18F-EF5 PET evidence of low hypoxic fraction will result in exclusion.

[000382] 3. Any patient on antihypertensive medication therapy is excluded.

[000383] 4. Patients must not have any significant medical condition that in the researcher's opinion may not be adequately controlled with appropriate therapy or that would compromise the patient's ability to tolerate this therapy.

[000384] 5. Patients with a history of any other cancer (except non-melanoma skin cancer or carcinoma in situ of the cervix), unless in complete remission and off all therapy for this disease for a minimum of 3 years , are ineligible.

[000385] 6. Patients must not have an active infection or series of intercurrent illnesses.

[000386] 7. Patients must not have any illness that will obscure the toxicity or dangerously alter the metabolism of the medication.

[000387] 8. Those patients with a severe total resection are excluded.

[000388] 9. The patient must not have had cranial radiation therapy before.

[000389] A baseline 18F-EF5- PET Scan is performed, and patients with normoxic tumors (defined as a tumor:normal brain ratio < 2.0) are removed from the study. When starting on the first day of RT therapy, patients receive the H-NOX trimer IV dose two hours before RT. To minimize the chances of an allergic reaction to an H-NOX protein trimer, dosing is limited to the first 5 days of RT when residual tumor is largest. This regimen continues daily for 5 days, with dose levels escalating according to a 3+3 design. Toxic sites are graded as described in the first Phase 1b study with modifications to the DLT definition. DLT is defined as any of the following events occurring at Week 10 of the study and attributable solely to the H-NOX trimer or to the H-NOX trimer dosed in combination with TMZ and RT:

[000390] 1. Any grade 3 or 4 thrombocytopenia, grade 4 anemia, or grade 4 neutropenia that lasts more than 7 days.

[000391] 2. Any febrile neutropenia.

[000392] 3. Any grade 3 or greater non-hematological toxicity, excluding alopecia, despite maximum medicinal therapy. Non-hematological toxicity such as rash, nausea, vomiting and diarrhea will only be considered a DLT if it remains grade 3 or greater despite maximal medicinal therapy.

[000393] 4. Any grade 4 radiation-induced skin change.

[000394] 5. Current standard of care therapy for GB consisting of concurrent RT and temozolomide, manages to limit the dose to toxic sites in approximately 1 of every 6 patients. Dose escalation of H-NOX trimer by continuous dose cohort produces a DLT in <1 of every 3 patients or <2 of every 6 patients if the cohort size is increased. The cohort size is slightly higher than the more conventional size of 3 patients due to the relatively high frequency of known and expected toxic sites during treatment with RT and temozolomide. If the dosing cohort does not have 3 evaluable patients included due to dropout or if 3 out of 6 patients experience a DLT, then up to 3 additional patients are included in the cohort at one time (Table 20). Table 20. The regulated dose escalation for the second Phase 1b of the study

[000395] After completion of H-NOX trimer dosing, patients are evaluated for adverse events weekly for the duration of the concurrent radiation/TMZ phase (generally a six-week period). Patients will be followed for 12 months, with survival progress determined at 6 months and 12 months after treatment. The MTD of H-NOX trimer is a dose that is less than or equal to one-third of the DLTs that patients experience. The MTD is based on DLTs observed during the course of concurrent radiation and TMZ, and is defined for use in subsequent Phase 2 study. The primary end point is characterized by the fact that it includes safety in combination with standard treatment and the secondary end point is characterized by the fact that it includes time to production of ATA and PFS-12.

[000396] Due to the expectation of low toxicity of H-NOX trimer, the first Phase 1b of the study was able to find a biologically adequate dose, that is, the dose that produces 80% of the response rate. The [4/6] ratio design will be implemented with a binary response of positive/negative hypoxic fraction change. See Brown et al., (2010) Int J Radiat Oncol Biol Phys 78:323-327. This design ensures that if the true response rate associated with the dose is low (defined here as 0.3) there is a high probability of escalation to the next dose; as a high true response rate (defined as 0.8) results in a low probability of further escalation. Cohorts of 3 are used per escalation while < 1/3 of responses are observed. When > 2/3 responses are observed the cohort will be expanded to 6. Escalation is continued if < 3/6 responses are observed. The recommended dose for the second Phase 1b of the Test is the dose that achieves > 4/6 responses or the maximum dose level, i.e. 100 mg/kg. If starting the dose of 5 mg/kg achieves > 4/6 responses lower doses are investigated for sufficient activity. In contrast to the standard toxicity of the designs, if it is clear that the response criteria for stopping at a given dose are not met (i.e., 0/2 or 2/5 responses were observed) prior escalation to a higher dose will to occur. The probability of scaling when the true rate = 0.3 is 0.94 while the probability of scaling when the true rate is 0.8 is 0.15. A minimum of 9 and a maximum of 24 patients are used to determine the four dose levels.

[000397] If toxic sites are observed, testing is transferred from an escalation target endpoint study to one based on DLTs. As such, the MTD is based on assessment of DLT during the two weeks following H-NOX trimer treatment and is defined as a dose at which less than a third of patients experience a DLT; That is, the MTD is the dose level at which 0/3 or 1/6 of patients experience DLT with the next higher dose having at least 2/3 or 2/6 patients experiencing DLT. If DLT, as defined above, is not achieved in any cohort up to the 100 mg/kg dose level there is no further escalation.

Claims (20)

1. Polymeric H-NOX protein, characterized by the fact that it is a trimeric H-NOX protein comprising three H-NOX monomers, wherein each H-NOX monomer comprises an H-NOX domain and a trimerization domain; each of the H-NOX domains comprising a sequence of amino acids selected from the group consisting of SEQ ID NOs: 2, 14, 16, 18, 20, 22 and 24, optionally with the H-NOX domain of SEQ ID NO :2 comprises a L144F substitution; wherein the trimerization domain is a foldon domain of bacteriophage T4 fibritin comprising the amino acid sequence set forth in SEQ ID NO:4; each H-NOX monomer comprises an H-NOX domain covalently linked to the trimerization domain through an amino acid linker to the fibritin foldon domain of bacteriophage T4, with the amino acid linker having three, four, five, six, seven, eight, nine, or ten amino acids long. 2. Polymeric H-NOX protein according to claim 1, characterized by the fact that in each H-NOX monomer the C-terminus of the H-NOX domain is covalently linked to the trimerization domain. 3. Polymeric H-NOX protein according to claim 1, characterized by the fact that in each H-NOX monomer the N-terminus of the H-NOX domain is covalently linked to the trimerization domain. 4. Polymeric H-NOX protein according to any one of claims 1 to 3, characterized by the fact that the H-NOX domain in each H-NOX monomer comprises SEQ ID NO: 2, optionally comprising a L144F substitution. 5. Polymeric H-NOX protein according to claim 1, characterized in that the amino acid linker is three amino acids in length. 6. Polymeric H-NOX protein according to claim 5, characterized in that the amino acid linker is a Gly-Ser-Gly linker. 7. Polymeric H-NOX protein according to claim 1, characterized in that each H-NOX monomer comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, 12, 26 and 28. 8. Polymeric H-NOX protein according to claim 1, characterized in that each H-NOX monomer comprises the amino acid sequence of SEQ ID NO: 8. 9. Polymeric H-NOX protein according to any one of claims 1 to 18, characterized in that each H-NOX monomer is PEGylated. 10. Polymeric H-NOX protein according to any one of claims 1 to 9, characterized in that the O2 dissociation constant is within 2 orders of magnitude of that of hemoglobin, and the reactivity with NO is at least 10 times lower than that of hemoglobin. 11. Polymeric H-NOX protein according to any one of claims 1 to 10, characterized in that the O2 dissociation constant is between 1 mM and 10 mM at 20 ° C. 12. Polymeric H-NOX protein according to any one of claims 1 to 11, characterized by the fact that the reactivity with NO is less than 700 s-1 at 20 °C. 13. Polymeric H-NOX protein according to any one of claims 1 to 12, characterized in that the reactivity with NO is at least 100 times less than that of wild-type human hemoglobin. 14. Polymeric H-NOX protein according to any one of claims 1 to 13, characterized in that the heme autoxidation rate of the polymeric H-NOX protein is less than 1 h-1 at 37 ° C. 15. Pharmaceutical composition, characterized by the fact that it comprises the polymeric H-NOX protein, as defined in any one of claims 1 to 16, and a pharmaceutically acceptable carrier or excipient. 16. Recombinant nucleic acid, characterized by the fact that it comprises a nucleotide sequence selected from the group consisting of SEQ SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO: 25, SEQ ID NO:27, and degenerate sequences thereof that encode the same amino acid sequences. 17. Vector, characterized by the fact that it comprises the nucleic acid, as defined in claim 16. 18. Transgenic microorganism, characterized by the fact that it comprises the nucleic acid, as defined in claim 16, or the vector, as defined in claim 17. 19. Method for producing a polymeric H-NOX protein, characterized in that it comprises culturing a cell comprising the nucleic acid as defined in claim 16, or the vector, as defined in claim 17, under conditions suitable for production of the polymeric H-NOX protein. 20. Kit, characterized by the fact that it comprises: (a) the polymeric H-NOX protein, as defined in any one of claims 1 to 14; and (b) instructions for use.