Modulation of tumor immunity by protein-mediated 02 delivery

Abstract

The invention provides methods to modulate hypoxia-mediated tumor immunity by administration of an O 2 carrier polypeptide (e.g., an H-NOX protein). The methods of the invention target both hypoxia inducible factor 1 alpha (HIF-1α) pathways and non-HIF-1α pathways of tumor immunity. Such methods are useful in the treatment of a wide variety of cancers and may be used alone or in combination with other anti-cancer therapies.

What is claimed is: 
     
         1 . A method for treating cancer in an individual comprising administering to the individual an effective amount of an O 2  carrier polypeptide. 
     
     
         2 . The method of  claim 1 , wherein the cancer is brain cancer, glioblastoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma, lung cancer, uterine cancer, ovarian cancer, colorectal cancer, anal cancer, liver cancer, hepatocellular carcinoma, stomach cancer, testicular cancer, endometrial cancer, cervical cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, esophageal cancer, intestinal cancer, thyroid cancer, adrenal cancer, bladder cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma, or squamous cell cancer. 
     
     
         3 . A method for modulating tumor immunity in an individual with a tumor comprising administering to the individual an effective amount of an O 2  carrier polypeptide. 
     
     
         4 . The method of  claim 3 , wherein the modulating tumor immunity comprises enhancing an immune response to the tumor. 
     
     
         5 . A method for increasing lymphocyte infiltration to a tumor in an individual comprising administering to the individual an effective amount of an O 2  carrier polypeptide. 
     
     
         6 . The method of  claim 5 , wherein the increase in lymphocyte infiltration to the tumor comprises an increase in infiltration of one or more of CD4 cells, CD8 cells, or NK cells. 
     
     
         7 . The method of  claim 5  or  6 , wherein the increase in lymphocyte infiltration to the tumor is accompanied by inhibition of one or more of Treg cells, tumor associated macrophages or myeloid derived suppressor cells in the tumor. 
     
     
         8 . The method of any one of  claims 5 - 7 , wherein the increase in lymphocyte infiltration to the tumor is accompanied by an increase in MHC1 expression on the tumor cells. 
     
     
         9 . A method for decreasing expression of hypoxia inducible factor 1α (HIF-1α) and/or 2α (HIF-2α) in a tumor in an individual comprising administering to the individual an effective amount of an O 2  carrier polypeptide. 
     
     
         10 . A method for decreasing expression of programmed death ligand-1 (PD-L1) in a tumor in an individual comprising administering to the individual an effective amount of an O 2  carrier polypeptide. 
     
     
         11 . A method for decreasing expression of A2A adenosine receptor (A2AR) in a tumor in an individual comprising administering to the individual an effective amount of an O 2  carrier polypeptide. 
     
     
         12 . The method of any one of  claims 3 - 11 , wherein the tumor is a brain tumor, a glioblastoma, a bone tumor, a pancreatic tumor, a skin tumor, a tumor of the head or neck, a melanoma, a lung tumor, a uterine tumor, an ovarian tumor, a colorectal tumor, an anal tumor, a liver tumor, a hepatocellular carcinoma, a stomach tumor, a testicular tumor, an endometrial tumor, a cervical tumor, a vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an esophageal tumor, an intestinal tumor, a thyroid tumor, an adrenal tumor, a bladder tumor, a kidney tumor, breast tumor, a multiple myeloma tumor, a sarcoma, or a squamous cell tumor. 
     
     
         13 . The method of any one of  claims 1 - 12 , wherein the individual is a mammal. 
     
     
         14 . The method of  claim 13 , wherein the mammal is a human. 
     
     
         15 . The method of  claim 14 , wherein the mammal is a pet, a laboratory research animal, or a farm animal. 
     
     
         16 . The method of  claim 15 , wherein the pet, research animal or farm animal is a dog, a cat, a horse, a monkey, a rabbit, a rat, a mouse, a guinea pig, a hamster, a pig, or a cow. 
     
     
         17 . The method of any one of  claims 1 - 16 , wherein the O 2  carrier polypeptide is administered by intravenous, intra-arterial, intratumoral, intravesicular, inhalation, intraperitoneal, intrapulmonary, intramuscular, subcutaneous, intra-tracheal, transmucosal, intraocular, intrathecal, or transdermal administration. 
     
     
         18 . The method of any one of  claims 1 - 17 , wherein administration of the O 2  carrier polypeptide is repeated. 
     
     
         19 . The method of  claim 18 , wherein administration of the O 2  carrier polypeptide is repeated daily, twice a day or about 1-4 times a week from about 4 weeks to about 8 weeks. 
     
     
         20 . The method of  claim 18  or  19  wherein the O 2  carrier polypeptide is administered every four, every 8, every 12 or every 24 hours for a period of about one to about 10 days. 
     
     
         21 . The method of any one of  claims 1 - 20 , wherein the O 2  carrier polypeptide is administered as a bolus. 
     
     
         22 . The method of any one of  claims 1 - 20 , wherein the O 2  carrier polypeptide is administered by infusion. 
     
     
         23 . The method of  claim 22 , wherein the 02 carrier polypeptide is infused in the individual for about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 6 hours, about 12 hours or about 24 hours. 
     
     
         24 . The method of any one of  claims 1 - 23 , wherein the O 2  carrier polypeptide is administered in combination with radiation therapy. 
     
     
         25 . The method of  claim 24 , wherein the radiation therapy is administered to the individual 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or 24 hours after the O 2  carrier polypeptide is administered. 
     
     
         26 . The method of  claim 24  or  25 , wherein the radiation is X-radiation. 
     
     
         27 . The method of  claim 26 , wherein the X-radiation is administered at about 0.5 gray to about 75 gray. 
     
     
         28 . The method of any one of  claims 24 - 27 , wherein the administration of the O 2  carrier polypeptide and/or the administration of the radiation is repeated. 
     
     
         29 . The method of  claim 28 , wherein the administration is repeated any number of times between about two times to about forty times or more. 
     
     
         30 . The method of  claim 28  or  29 , wherein the administration is repeated after one week, two weeks, three weeks, or four weeks or more. 
     
     
         31 . The method of any one of  claims 1 - 23 , wherein the O 2  carrier polypeptide is administered in combination with chemotherapy or immunotherapy. 
     
     
         32 . The method of  claim 31 , wherein the chemotherapy comprises a cytotoxin. 
     
     
         33 . The method of  claim 32 , wherein the administration of the O 2  carrier polypeptide and/or the administration of the chemotherapy is repeated. 
     
     
         34 . The method of  claim 31 , wherein the immunotherapy is one or more of an anticancer vaccine, an adoptive immune cell therapy or an agent that targets an immune checkpoint regulator. 
     
     
         35 . The method of  claim 31  or  34 , wherein the immunotherapy targets one or more of CTLA-4, PD1, PD-L1, or an immune checkpoint regulator. 
     
     
         36 . The method of  claim 31  or  34 , wherein the adoptive immude therapy is a chimeric antigen receptor expressing T cell or an engineered TCR-T cell. 
     
     
         37 . The method of any one of  claims 31  or  34 - 36 , wherein the administration of the O 2  carrier polypeptide and/or the administration of the immunotherapy is repeated. 
     
     
         38 . The method of any one of  claims 1 - 37 , wherein the O 2  carrier polypeptide is in a pharmaceutical composition. 
     
     
         39 . The method of  claim 38 , wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. 
     
     
         40 . The method of any one of  claims 1 - 39  wherein the O 2  carrier polypeptide is an H-NOX protein. 
     
     
         41 . A method for treating cancer in an individual comprising administering to the individual an effective amount of an H-NOX protein. 
     
     
         42 . The method of  claim 41 , wherein the cancer is brain cancer, glioblastoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma, lung cancer, uterine cancer, ovarian cancer, colorectal cancer, anal cancer, liver cancer, hepatocellular carcinoma, stomach cancer, testicular cancer, endometrial cancer, cervical cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, esophageal cancer, intestinal cancer, thyroid cancer, adrenal cancer, bladder cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma, or squamous cell cancer. 
     
     
         43 . A method for modulating tumor immunity in an individual with a tumor comprising administering to the individual an effective amount of an H-NOX protein. 
     
     
         44 . The method of  claim 43 , wherein the modulating tumor immunity comprises enhancing an immune response to the tumor. 
     
     
         45 . A method for increasing lymphocyte infiltration to a tumor in an individual comprising administering to the individual an effective amount of an H-NOX protein. 
     
     
         46 . The method of  claim 45 , wherein the increase in lymphocyte infiltration to the tumor comprises an increase in infiltration of one or more of CD4 cells, CD8 cells, or NK cells. 
     
     
         47 . The method of  claim 45  or  46 , wherein the increase in lymphocyte infiltration to the tumor is accompanied by inhibition of one or more of Treg cells, tumor associated macrophages or myeloid derived suppressor cells in the tumor. 
     
     
         48 . The method of any one of  claims 45 - 47 , wherein the increase in lymphocyte infiltration to the tumor is accompanied by an increase in MHC1 expression on the tumor cells. 
     
     
         49 . A method for decreasing expression of HIF-1α and/or HIF-2α in a tumor in an individual comprising administering to the individual an effective amount of an H-NOX protein. 
     
     
         50 . A method for decreasing expression of PD-L1 in a tumor in an individual comprising administering to the individual an effective amount of an H-NOX protein. 
     
     
         51 . A method for decreasing expression of A2AR in a tumor in an individual comprising administering to the individual an effective amount of an H-NOX protein. 
     
     
         52 . The method of any one of  claims 41 - 51 , wherein the tumor is a brain tumor, a glioblastoma, a bone tumor, a pancreatic tumor, a skin tumor, a tumor of the head or neck, a melanoma, a lung tumor, a uterine tumor, an ovarian tumor, a colorectal tumor, an anal tumor, a liver tumor, a hepatocellular carcinoma, a stomach tumor, a testicular tumor, an endometrial tumor, a cervical tumor, a vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an esophageal tumor, an intestinal tumor, a thyroid tumor, an adrenal tumor, a bladder tumor, a kidney tumor, a breast tumor, a multiple myeloma tumor, a sarcoma, or a squamous cell tumor. 
     
     
         53 . The method of any one of  claims 41 - 52 , wherein the individual is a mammal. 
     
     
         54 . The method of  claim 53 , wherein the mammal is a human. 
     
     
         55 . The method of  claim 52 , wherein the mammal is a pet, a laboratory research animal, or a farm animal. 
     
     
         56 . The method of  claim 55 , wherein the pet, research animal or farm animal is a dog, a cat, a horse, a monkey, a rabbit, a rat, a mouse, a guinea pig, a hamster, a pig, or a cow. 
     
     
         57 . The method of any one of  claims 41 - 56 , wherein the H-NOX protein is administered by intravenous, intra-arterial, intratumoral, intravesicular, inhalation, intraperitoneal, intrapulmonary, intramuscular, subcutaneous, intra-tracheal, transmucosal, intraocular, intrathecal, or transdermal administration. 
     
     
         58 . The method of any one of  claims 41 - 57 , wherein administration of the H-NOX protein is repeated. 
     
     
         59 . The method of  claim 58 , wherein administration of the H-NOX protein is repeated daily or twice a day from about 4 weeks to about 8 weeks. 
     
     
         60 . The method of  claim 58  or  59  wherein the H-NOX protein is administered every four, every 8, every 12 or every 24 hours for a period of about one to about 10 days. 
     
     
         61 . The method of any one of  claims 41 - 60 , wherein the H-NOX protein is administered as a bolus. 
     
     
         62 . The method of any one of  claims 41 - 60 , wherein the H-NOX protein is administered by infusion. 
     
     
         63 . The method of  claim 62 , wherein the H-NOX protein is infused in the individual for about 15 minutes, about 30 minutes, about 1 hour, about 1 hour, about 2 hours, about 3 hours, about 6 hours, about 12 hours or about 24 hours. 
     
     
         64 . The method of any one of  claims 41 - 63 , wherein the H-NOX protein is administered in combination with radiation therapy. 
     
     
         65 . The method of  claim 64 , wherein the radiation therapy is administered to the individual 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours after the H-NOX protein is administered. 
     
     
         66 . The method of  claim 64  or  65 , wherein the radiation is X-radiation. 
     
     
         67 . The method of  claim 66 , wherein the X-radiation is administered at about 0.5 gray to about 75 gray. 
     
     
         68 . The method of any one of  claims 64 - 67 , wherein the administration of the H-NOX protein and/or the administration of the radiation is repeated. 
     
     
         69 . The method of  claim 68 , wherein the administration is repeated any number of times between about two times to about forty times or more. 
     
     
         70 . The method of  claim 68  or  69 , wherein the administration is repeated after one week, two weeks, three weeks, or four weeks or more. 
     
     
         71 . The method of any one of  claims 41 - 63 , wherein the H-NOX protein is administered in combination with chemotherapy or immunotherapy. 
     
     
         72 . The method of  claim 71 , wherein the chemotherapy comprises a cytotoxin. 
     
     
         73 . The method of  claim 72 , wherein the administration of the H-NOX protein and/or the administration of the chemotherapy is repeated. 
     
     
         74 . The method of  claim 71 , wherein the immunotherapy is one or more of an anticancer vaccine, an adoptive immune cell therapy or an agent that targets an immune checkpoint regulator. 
     
     
         75 . The method of  claim 71  or  74 , wherein the immunotherapy targets one or more of CTLA-4, PD1, PD-L1, or an immune checkpoint regulator. 
     
     
         76 . The method of  claim 71 ,  74  or  75 , wherein the adoptive immude therapy is a chimeric antigen receptor expressing T cell or an engineered TCR-T cell. 
     
     
         77 . The method of any one of  claims 71 , or  74 - 76 , wherein the administration of the H-NOX protein and/or the administration of the immunotherapy is repeated. 
     
     
         78 . The method of any one of  claims 41 - 77 , wherein the H-NOX protein is a  T. tengcongensis  H-NOX, a  L. pneumophilia  2 H-NOX, a  H. sapiens  β1, a  R. norvegicus  β1, a  C. lupus  H-NOX, a  D. melangaster  β1, a  D. melangaster  CG14885-PA, a  C. elegans  GCY-35, a  N. punctiforme  H-NOX,  C. crescentus  H-NOX, a  S. oneidensis  H-NOX, or  C. acetobutylicum  H-NOX. 
     
     
         79 . The method of any one of  claims 41 - 77 , wherein the H-NOX protein comprises a H-NOX domain corresponding to the H-NOX domain of  T. tengcongensis  set forth in SEQ ID NO:2. 
     
     
         80 . The method of any one of  claims 41 - 78 , wherein the H-NOX comprises one or more distal pocket mutations. 
     
     
         81 . The method of  claim 80 , wherein the distal pocket mutation is an amino acid substitution at a site corresponding to L144 of  T. tengcongensis  H-NOX. 
     
     
         82 . The method of  claim 80  or  81 , wherein the H-NOX is a  T. tengcongensis  H-NOX comprising an amino acid substitution at position 144. 
     
     
         83 . The method of  claim 82 , wherein the amino acid substitution at position 144 is an L144F substitution. 
     
     
         84 . The method of any one of  claims 41 - 83 , wherein the H-NOX protein is a polymeric H-NOX protein. 
     
     
         85 . The method of  claim 84 , wherein the polymeric H-NOX protein comprises monomers, wherein the monomers comprise an H-NOX domain and a polymerization domain. 
     
     
         86 . The method of  claim 85 , wherein the H-NOX domain is covalently linked to the polymerization domain. 
     
     
         87 . The method of any one of  claims 84 - 86 , wherein the polymeric H-NOX protein is a trimeric H-NOX protein. 
     
     
         88 . The method of  claim 87 , wherein the trimeric H-NOX protein comprises one or more trimerization domains. 
     
     
         89 . The method of  claim 88 , wherein the trimeric H-NOX protein comprises three monomers, wherein the monomers comprise an H-NOX domain and a trimerization domain, wherein the trimerization domain is a bacteriophage T4 trimerization domain. 
     
     
         90 . The method of  claim 88  or  89 , wherein the trimerization domain is a foldon domain. 
     
     
         91 . The method of  claim 90 , wherein the foldon domain comprises the amino acid sequence of SEQ ID NO:4. 
     
     
         92 . The method of any one of  claims 41 - 91 , wherein the H-NOX protein is fused to an Fc domain of an immunoglobulin. 
     
     
         93 . The method of any one of  claims 41 - 92 , wherein the H-NOX protein is covalently bound to polyethylene glycol. 
     
     
         94 . The method of any one of  claims 41 - 93 , wherein the O 2  dissociation constant of the H-NOX protein is within 2 orders of magnitude of that of hemoglobin, and wherein the NO reactivity of the H-NOX protein is at least 10-fold lower than that of hemoglobin. 
     
     
         95 . The method of any one of  claims 41 - 94 , wherein the O 2  dissociation constant of the polymeric H-NOX protein is between about 1 nM and about 1000 nM at 20° C. 
     
     
         96 . The method of any one of  claims 41 - 95 , wherein the O 2  dissociation constant of the H-NOX protein is between about 1 μM and about 10 μM at 20° C. 
     
     
         97 . The method of any one of  claims 41 - 96 , wherein the Oz dissociation constant of the H-NOX protein is between about 10 μM and about 50 μM at 20° C. 
     
     
         98 . The method of any one of  claims 41 - 97 , wherein the NO reactivity of the H-NOX protein is less than about 700 s −1  at 20° C. 
     
     
         99 . The method of any one of  claims 41 - 98 , wherein the NO reactivity of the H-NOX protein is at least 100-fold lower than that of hemoglobin. 
     
     
         100 . The method of  claim 99 , wherein the NO reactivity of the H-NOX protein is at least 1,000-fold lower than that of hemoglobin. 
     
     
         101 . The method of any one of  claims 41 - 100 , wherein the k off  for oxygen of the H-NOX protein is less than or equal to about 0.65 s −1  at 20° C. 
     
     
         102 . The method of any one of  claims 41 - 101 , wherein the k off  for oxygen of the H-NOX protein is between about 0.21 s −1  and about 0.65 s −1  at 20° C. 
     
     
         103 . The method of any one of  claims 41 - 102 , wherein the k off  for oxygen of the H-NOX protein is between about 1.35 s −1  and about 2.9 s −1  at 20° C. 
     
     
         104 . The method of any one of  claims 41 - 103 , wherein the rate of heme autoxidation of the H-NOX protein is less than about 1 h −1  at 37° C. 
     
     
         105 . The method of any one of  claims 41 - 104 , wherein the H-NOX protein is in a pharmaceutical composition. 
     
     
         106 . The method of  claim 105 , wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. 
     
     
         107 . Use of an O 2  carrier protein for treating cancer in an individual. 
     
     
         108 . The use of  claim 107 , wherein the cancer is brain cancer, glioblastoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, melanoma, lung cancer, uterine cancer, ovarian cancer, colorectal cancer, anal cancer, liver cancer, hepatocellular carcinoma, stomach cancer, testicular cancer, endometrial cancer, cervical cancer, Hodgkin's Disease, non-Hodgkin's lymphoma, esophageal cancer, intestinal cancer, thyroid cancer, adrenal cancer, bladder cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma, or squamous cell cancer. 
     
     
         109 . Use of an O 2  carrier protein for modulating tumor immunity in an individual. 
     
     
         110 . The use of  claim 109 , wherein the modulating tumor immunity comprises enhancing an immune response to the tumor. 
     
     
         111 . Use of an O 2  carrier polypeptide for increasing lymphocyte infiltration to a tumor in an individual. 
     
     
         112 . The use of  claim 111 , wherein the increase in lymphocyte infiltration to the tumor comprises an increase in infiltration of one or more of CD4 cells, CD8 cells, or NK cells. 
     
     
         113 . The use of  claim 111  or  112 , wherein the increase in lymphocyte infiltration to the tumor is accompanied by inhibition of one or more of Treg cells, tumor associated macrophages or myeloid derived suppressor cells in the tumor. 
     
     
         114 . The use of any one of  claims 111 - 113 , wherein the increase in lymphocyte infiltration to the tumor is accompanied by an increase in MHC1 expression on the tumor cells. 
     
     
         115 . Use of an O 2  carrier polypeptide for decreasing expression of HIF-1α and/or HIF-2α in a tumor in an individual. 
     
     
         116 . Use of an O 2  carrier polypeptide for decreasing expression of PD-L1 in a tumor in an individual. 
     
     
         117 . Use of an O 2  carrier polypeptide for decreasing expression of A2AR in a tumor in an individual. 
     
     
         118 . The use of any one of  claims 109 - 117 , wherein the tumor is a brain tumor, a glioblastoma, a bone tumor, a pancreatic tumor, a skin tumor, a tumor of the head or neck, a melanoma, a lung tumor, a uterine tumor, an ovarian tumor, a colorectal tumor, an anal tumor, a liver tumor, a hepatocellular carcinoma, a stomach tumor, a testicular tumor, an endometrial tumor, a cervical tumor, a vaginal tumor, a Hodgkin's lymphoma, a non-Hodgkin's lymphoma, an esophageal tumor, an intestinal tumor, a thyroid tumor, an adrenal tumor, a bladder tumor, a kidney tumor, a breast tumor, a multiple myeloma tumor, a sarcoma, or a squamous cell tumor. 
     
     
         119 . The use of any one of  claims 107 - 118 , wherein the individual is a mammal. 
     
     
         120 . The use of  claim 119 , wherein the mammal is a human. 
     
     
         121 . The use of any one of  claims 107 - 120 , wherein the O 2  carrier polypeptide is an H-NOX protein. 
     
     
         122 . The use of  claim 121 , wherein the H-NOX protein is a  T. tengcongensis  H-NOX, a  L. pneumophilia  2 H-NOX, a  H. sapiens  β1, a  R. norvegicus  β1, a  C. lupus  H-NOX domain, a  D. melangaster  β1, a  D. melangaster  CG14885-PA, a  C. elegans  GCY-35, a  N. punctiforme  H-NOX,  C. crescentus  H-NOX, a  S. oneidensis  H-NOX, or  C. acetobutylicum  H-NOX. 
     
     
         123 . The use of any one of  claims 121 - 122 , wherein the H-NOX protein comprises a H-NOX domain corresponding to the H-NOX domain of  T. tengcongensis  set forth in SEQ ID NO:2. 
     
     
         124 . The use of any one of  claims 121 - 123 , wherein the H-NOX comprises one or more distal pocket mutations. 
     
     
         125 . The use of  claim 124 , wherein the distal pocket mutation is an amino acid substitution at a site corresponding to L144 of  T. tengcongensis  H-NOX. 
     
     
         126 . The use of  claim 124  or  125 , wherein the H-NOX is a  T. tengcongensis  H-NOX comprising an amino acid substitution at position 144. 
     
     
         127 . The use of  claim 126 , wherein the amino acid substitution at position 144 is an L144F substitution. 
     
     
         128 . The use of any one of  claims 121 - 127 , wherein the H-NOX protein is a polymeric H-NOX protein. 
     
     
         129 . The use of  claim 128 , wherein the polymeric H-NOX protein comprises monomers, wherein the monomers comprise an H-NOX domain and a polymerization domain. 
     
     
         130 . The use of  claim 129 , wherein the H-NOX domain is covalently linked to the polymerization domain. 
     
     
         131 . The use of any one of  claims 128 - 130 , wherein the polymeric H-NOX protein is a trimeric H-NOX protein. 
     
     
         132 . The use of  claim 131 , wherein the trimeric H-NOX protein comprises one or more trimerization domains. 
     
     
         133 . The use of  claim 132 , wherein the trimeric H-NOX protein comprises three monomers, wherein the monomers comprise an H-NOX domain and a trimerization domain, wherein the trimerization domain is a bacteriophage T4 trimerization domain. 
     
     
         134 . The use of  claim 132  or  133 , wherein the trimerization domain is a foldon domain. 
     
     
         135 . The use of  claim 134 , wherein the foldon domain comprises the amino acid sequence of SEQ ID NO:4. 
     
     
         136 . The use of any one of  claims 131 - 135 , wherein the H-NOX protein is fused to an Fc domain of an immnunoglobulin. 
     
     
         137 . The use of any one of  claims 121 - 136 , wherein the H-NOX protein is covalently bound to polyethylene glycol. 
     
     
         138 . A kit for modulating tumor immunity in an individual comprising an O 2  carrier protein for use in the method of any one of  claims 1 - 106 . 
     
     
         139 . The kit of  claim 138 , wherein the kit further comprises one or more of a vial, a vessel, an ampule, a bottle, a jars, or flexible packaging. 
     
     
         140 . The kit of  claim 138  or  139 , wherein the kit further comprises one or more buffers. 
     
     
         141 . The kit of any one of  claims 138 - 140 , wherein the kit further comprises instructions for use.