WO2025222059A1

WO2025222059A1 - Method of treating cancer using pegylated bovine hemoglobin

Info

Publication number: WO2025222059A1
Application number: PCT/US2025/025254
Authority: WO
Inventors: Ronald Jubin
Original assignee: Prolong Pharmaceuticals LLC
Current assignee: Prolong Pharmaceuticals LLC
Priority date: 2024-04-17
Filing date: 2025-04-17
Publication date: 2025-10-23
Anticipated expiration: 2026-10-17

Abstract

The present invention provides a method of treating cancer by administering PEGylated HB-CO (PEG-HB-CO) in combination with radiation therapy. Further, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer by providing combination therapy to a subject that exhibits improvement by reducing tumor volume and improving the tumor free survival period. The present invention discloses a method of treating cancer by administering PEGylated HB-CO (PEG-HB-CO) simultaneously or sequentially or in any order before or after the radiation therapy.

Description

Attorney Docket No. PLG-021WO METHOD OF TREATING CANCER USING PEGYLATED BOVINE HEMOGLOBIN CROSS-REFERENCE TO RELATED APPLICATIONS [001] This application claims the benefit of and priority to U.S. Provisional Application No. 63/635,102, filed April 17, 2024, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION [002] The present invention provides a method of treating cancer by administering PEGylated HB-CO (PEG-HB-CO) in combination with radiation therapy. Further, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer by providing combination therapy to a subject that exhibits improvement by reducing tumor volume and improving the tumor free survival period. The present invention discloses a method of treating cancer by administering PEGylated HB-CO (PEG-HB-CO) simultaneously or sequentially or in any order before or after the radiation therapy. BACKGROUND OF THE INVENTION: [003] Cancer progression and response to treatment both play a significant role in how a cancer may impact and individual living with cancer. It was realized and appreciated as early as 1950’s that lack of oxygen (i.e. hypoxia) was associated with a diminished treatment response (Thomlison and Gray, 1955). Non-invasive methods including advanced imaging and radiolabeled tracers have increased the capability to identify tumor regions of hypoxia accurately locating and determining tumor-specific hypoxia. [004] Cancer cells grow rapidly as a result of genetic changes and often outpace the growth of supporting blood vessels. This sets up a proportional O2 concentration gradient wherein cells closest to the vessels have adequate supply and replicate rapidly. Conversely the cells the furthest away do not receive sufficient O2 and undergo necrosis. There is a middle region that with very limited O2 slow down considerably due in part to the increased activation of the hypoxia inducible transcription factor complex that include hypoxia inducible factors and von Hippel Landau protein. The HIF-1a protein binding is tightly regulated by oxygen. When O2 levels are low, the HIF-1a protein binds to the other factors that upregulate numerous genes that promotes cell survival (normal function). When O2 levels are restored, then HIF-1a is rapidly degraded and the transcription is turned off. This elaborate transcription system can be corrupted and usurped by tumor cells in a state of chronic hypoxia. These cancer cells in Attorney Docket No. PLG-021WO hypoxia become resistant to therapy due to the complex transcriptional changes and promote tumor advancement. These tumors may also metastasize with hypoxia being a key factor. [005] The control of tumor hypoxia has emerged as a therapeutic target in cancer treatment. The HIF-1a was identified as a key target based on its pivotal role in O2-specific gene expression and blocking the interaction with the larger complex would turn off the hypoxia expression and chemically restore normoxia. This would be expected to restore sensitivity to cancer drugs and reduce tumor growth. An alternative approach at the system level included hyperbaric oxygen therapy (HBOT) that would be expected to directly increase O2 levels and similarly, turn off HIF-1a mediated gene expression. Both approaches had treatment associated toxicity and limited clinical benefits so none of these treatments received FDA approval. [006] The primary challenge to targeting the hypoxic cells is access to the cells. Detailed imaging studies have shown the distance from a capillary to the zone of hypoxia is between 100 and 150 uM. Any closer cells are normoxic and any further cells are anoxic. The amount of drug needed is proportional to the ability to interact with its target and in the case of HIF-1a the drug levels were not achievable without dose-limiting toxicity. Similarly, HBOT at 90- 100% pure O2 can only be tolerated for a short period of time, and once the patient is out of the chamber, O2 blood levels drop quickly. [007] What is needed to selectively treat hypoxic tumor cells is a drug product with low toxicity, ability to specifically access the hypoxic cells and the ability to keep cells normoxic for an extended period of time. [008] (PEG-HB-CO) is a unique gas transfer agent comprised of PEGylated bovine hemoglobin and loaded with carbon monoxide (CO). The PEGylated Hb has a “left-shifted” p50 thereby preventing release of O2 until regions of severe hypoxia (e.g. p50 <10 mmHg) are encountered. [009] The CO and the PEG can function together to improve microvascular circulation. This along with the small molecular size of PEG-HB-CO ensures robust transit in the microcirculation that is often compromised in tumors (ref). Moreover, the drug can readily diffuse selectively to cells of low oxygen (p50<10) across a distance of up to 150uM. The ability to access and selectively deliver are needed to switch off the HIF-1a gene expression circuit. Additionally, with a half-life of ~24 h the drug can be administered in advance of any other cancer treatment (small molecule, biologic or radiation) restoring the tumor cell response to known therapies. Lastly, PEG-HB-CO has shown good tolerability and safety in both animal toxicology and human clinical trials. Attorney Docket No. PLG-021WO SUMMARY OF THE INVENTION [010] The invention discloses an improved method of treating a tumor and/or cancer by providing combination therapy of PEGylated hemoglobin and/or PEG-HB-CO with radiation therapy. [011] In some embodiments, the invention improves survival period or response to treatment of tumor and/or cancer by providing combination therapy of PEGylated hemoglobin and/or PEG-HB-CO with radiation therapy.In some embodiments, the improved method of treatment alleviates at least one symptom associated with tumor and/or cancer treatment selected from lung cancer, breast cancer. [012] In some embodiments, the present invention treats lung cancer, breast cancer with combination therapy of PEGylated hemoglobin and/or PEG-HB-CO with radiation therapy. [013] In some embodiments, the present invention discloses a method of treating a cancer in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated H_{B-CO (PEG-HB-} b. wherein the PEG-HB-CO is administered simultaneously or sequentially or in any wherein the radiation therapy does not wherein and wherein the method improves the cancer treatment. [014] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor in a subject in need thereof comprising: a_{. administering to the subject a combination of a dose of PEG-HB- and} _{b. treating the subject with radiation therapy} wherein the PEG-HB-CO is administered simultaneously or sequentially or in any wherein the radiation therapy does not wherein wherein the method improves the cancer treatment. [015] In some embodiments, the present invention discloses a method of treating cancer wherein the cancer treatment is improved by inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a) and/or improving one Attorney Docket No. PLG-021WO or more incidence of tumor-free survivors, increase in time to progression (ITP).In some embodiments, the radiation therapy is focal beam radiation therapy and/or stereotactic radiation surgery. [016] In some embodiments, the radiation therapy is focal beam radiation therapy. [017] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor in a subject in need thereof comprising: a_{. administering to the subject a combination of a dose of PEG-HB-} b. wherein the subject exhibits s a higher reduction in the size and/or volume of a tumor compared to the subject treated either with PEG-HB-CO or radiation therapy alone. [018] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEG-HB- and b. wherein the administration of PEG-HB-CO and radiation therapy to the subject exhibits s improvement in reduction of tumor volume compared to subject administered with PEG-HB-CO or radiation therapy alone. [019] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein subject administered with combination therapy of the PEG-HB-CO and the radiation therapy exhibits at least about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, and about 70%, improvement in tumor volume reduction compared to the subject treated either with PEG-HB-CO or radiation therapy alone. [020] In another embodiment, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein subject administered with combination therapy of the PEG-HB-CO and the radiation therapy exhibits about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, and about 65% improvement in tumor volume compared to the subject treated either with PEG- HB-CO or radiation therapy alone. [021] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor, wherein the reduction of volume comprising inhibiting tumor growth Attorney Docket No. PLG-021WO by reducing one or more incidence selected from partial tumor regression, complete tumor regression, tumor volume and hypoxia inducible factor 1 alpha (HIF-1a) [022] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a_{. administering a dose of PEG-HB-} b. wherein the administration of PEG-HB-CO and radiation therapy to the subject provides tumor free survivors. [023] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein tumor free survivors improves the increase in time to progression (ITP) and/or inhibiting tumor growth by reducing one or more incidence selected from partial tumor regression, complete tumor regression, tumor volume and hypoxia inducible factor 1 alpha (HIF-1a). [024] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. treating the subject and wherein the cancer treatment is improved by inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a) and/or improving one or more incidence of tumor free survivors, increase in time to progression (ITP). [025] In some embodiments, the method improves the incidence of partial tumor regression by about 30%, about 35%, about 40%, about 45%, and about 50%. [026] In some embodiments, the method improves the incidence of partial tumor regression by about 30%, about 35%, and about 40%. [027] In some embodiments, the method improves the incidence of complete tumor regression by about 15%, about 20%, about 25%, about 30%, about 35%, and about 40%. [028] In some embodiments, the method improves the incidence of complete tumor regression by about 20%, about 25%, and about 30%. [029] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein the administration of PEG-HB-CO and Attorney Docket No. PLG-021WO radiation therapy increases the tumor-free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. [030] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. treating the wherein the treatment exhibits s about 40% incidence of partial regressions and about and wherein the administration of PEG-HB-CO and radiation therapy increases the tumor- free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. [031] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. treating the subject wherein the administration of PEG-HB-CO is performed simultaneously, sequentially, and wherein the reduction of tumor improves the increase in time to progression (ITP). [032] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer wherein the reduction of tumor improves the increase in time to progression (ITP) at least by 10% to 75 % or at least about 10% to about 50% or about 50% to about 75% compared to the increase in time to progression (ITP) when the subject treated either with PEG-HB-CO or radiation therapy alone. [033] In some embodiments, the combination therapy treatment produced anti-cancer activity with an ITP of about 160%, about 200% and about 216%. [034] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. wherein the combination of the PEG-HB-CO and the radiation therapy improves the cancer treatment and body weight management in the subject compared to the subject treated either with PEG-HB-CO or radiation therapy alone. Attorney Docket No. PLG-021WO [035] In some embodiments, the treatment maintains the body weight of the subject during the treatment. [036] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a_{. administering a dose of PEG-HB-} b. wherein the step (a) and (b) are performed sequentially, or simultaneously or in any and wherein the process improves anti-cancer activity compared to the process performed with either with PEG-HB-CO or radiation therapy alone. [037] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject comprising: a_{. administering to the subject a dose of PEG-HB- and} b. treating the subject with wherein the combination of the PEG-HB-CO and the radiation therapy thereof inhibits growth of the tumor in the subject compared to the subject treated either with PEG- HB-CO or radiation therapy alone. [038] In some embodiments, the subject is selected from animal model suitable for cancer research, and human suffering from cancer. [039] In some embodiments, the animal model suitable for cancer research is selected from guinea pig, mouse, and rat. [040] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein the type of cancer is selected from lung cancer, breast cancer, melanoma, renal cell carcinoma, squamous cell non-small cell lung cancer (NSCLC), non-squamous NSCLC, colon-rectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, head and neck. [041] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof by administering a combination therapy of PEG-HB-CO and radiation therapy, wherein the therapeutic effective amount PEG-HB-CO is administered in subject intravenously and/or subcutaneously at a dose selected in the range of about 300 mg/kg, to about 1500 mg/kg [042] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof by administering a combination therapy of PEG-HB-CO and radiation Attorney Docket No. PLG-021WO therapy, wherein the therapeutic effective amount PEG-HB-CO is administered in subject intravenously and/or subcutaneously at a dose selected in the range of about 500 mg/kg, to about 1200 mg/kg. [043] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof by administering a combination therapy of PEG-HB-CO and radiation therapy, wherein the therapeutic effective amount PEG-HB-CO is administered in subject intravenously and/or subcutaneously at a dose selected in the range of about 800 mg/kg, to about 1000 mg/kg. [044] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof by administering a combination therapy of PEG-HB-CO and radiation therapy, wherein the radiation therapy is focal beam radiation therapy. [045] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof by administering a combination therapy of PEG-HB-CO and radiation therapy, wherein the subject is treated with focal radiation of about 5 Gy to about 10 Gy.In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB- and} _{b. treating the subject with a radiation therapy} wherein the dose of PEG-HB-CO is about at 1000 mg/kg and focal radiation is about wherein the treatment produced anti-cancer activity. [046] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. wherein the subject exhibits s improvement in the treatment of cancer compared to the subject treated either with PEG-HB-Co or radiation therapy alone. [047] In some embodiments, the subject in need of treatment of cancer, exhibits s improvement in the %T/C value compared to the subject treated either with PEG-HB-CO or radiation therapy alone. [048] In some embodiments, the %T/C value is derived or measured by bioluminescence imaging (BLI). [049] In some embodiments, the present invention discloses a composition containing hemoglobin, wherein the hemoglobin composition delivers O2 to cells with severe hypoxia. In Attorney Docket No. PLG-021WO some embodiments, the hemoglobin is a PEGylated hemoglobin. In some embodiments, the PEGylated hemoglobin is PEG-Hb-CO. [050] In some embodiments, the present invention discloses a composition containing hemoglobin, particularly PEGylated hemoglobin, wherein the hemoglobin composition inhibits HIF-1a gene expression. [051] In some embodiments, the hemoglobin composition inhibits HIF-1a gene expression in hypoxia state. [052] In some embodiments, the present disclosure discloses a method for inhibiting tumor progression by reducing HIF-1a mediated gene expression. [053] In some embodiments, wherein the hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression provides an EC50 value in the range of about 0.5 mg/ml to about 2.0 mg/ml, or in the range of about 0.5 mg/ml to about 1.0 mg/ml. [054] In some embodiments, the PEGylated hemoglobin reduces or alleviate HIF-1a mediated gene expression in suitable cell line and provides desirable EC50 value. [055] In some embodiments, the PEGylated hemoglobin reduces or alleviate HIF-1a mediated gene expression in suitable cell line A549 cell line (human lung cancer). [056] In some embodiments, the PEGylated hemoglobin reduces or alleviate HIF-1a mediated gene expression in suitable cell line MCF7 cell line (human breast cancer). [057] In some embodiments, the suitable cell lines are selected from A549 cell line (human lung cancer), [058] MCF7 breast cancer cell line, human HNSCC cell lines. [059] In some embodiments, the present invention discloses a method of treating cancer wherein the PEG-HB-CO is administered in dose is dependent manner, wherein PEG-HB-CO dose is dependent on tumor hypoxia volume. [060] In some embodiments, the present invention discloses a method of treating cancer wherein the PEG-HB-CO dose in a dose dependent manner are selected from about 0.1 mg/mL, to about 11 mg/mL. [061] In some embodiments, the present invention discloses a composition of PEG-HB-CO comprises bovine hemoglobin and poly(ethylene glycol), wherein bovine hemoglobin is bound to carbon monoxide, wherein the composition comprises stabilizing agent. [062] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: Attorney Docket No. PLG-021WO a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB- and} b. treating the subject wherein the combination therapy of the PEG-HB-CO and the radiation therapy inhibits growth of the tumor in the subject compared to the subject treated either with PEG- _HB- wherein the method exhibits s at least 50% to about 70% improvement in reduction of tumor volume compared to the subject treated either with PEG-HB-CO or radiation therapy wherein the method reduces or alleviates hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, providing an EC50 value in the range of about 0.5 mg/ml to about wherein the method reduces the incidence of partial tumor regression in the range of about wherein the method reduces the incidence of complete tumor regression in the range of wherein and wherein the administration of PEG-HB-CO and radiation therapy increases the tumor- free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. [063] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject in need thereof, wherein the treatment is performed in at least one cycle. [064] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject in need thereof, wherein the treatment is performed in more than one cycle. [065] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, the method comprising: a_{. culturing the cells capable to express hypoxia-inducible factor 1-alpha (HIF-} b. c_{. treating with PEGylated HB-CO (PEG-HB-} d. incubating the assay plate e. determining the HIF- reduction in HIF-1a or Attorney Docket No. PLG-021WO f. collect the sample from step (d) in suitable apparatus and treated with fixation buffer g. h. separating the supernatant in suitable apparatus i. treating the supernatant with permeabilization buffer and incubate for suitable j. resuspending in staining buffer to prepare sample and transfer in suitable k. treating the sample of step (j) with Phycoerythrin conjugated mouse anti-human H_IF- _{l. incubating for suitable time} m. analyzing sample to read phycoerythrin fluorescent. [066] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression,, wherein the cell line is A549 or MCF7. [067] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the PEGylated HB-CO (PEG-HB-CO) is added in 1:10 dilution. [068] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the assay plate is incubated for suitable time for suitable period in normoxia or hypoxia. [069] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the assay plate is incubated for suitable time for suitable period in normoxia. [070] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the assay plate is incubated at temperature 30°C to 40°C. [071] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the hours. [072] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the determination of the HIF-1a expression is performed by the Luciferase assay. Attorney Docket No. PLG-021WO [073] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, the method comprising: a_{. culturing the cells capable to express hypoxia-inducible factor 1-alpha (HIF-} b. c_{. treating with PEGylated HB-CO (PEG-HB-} d. incubating the assay plate e. collect the sample from step (d) in suitable apparatus and treated with fixation buffer f. g. h. treating the supernatant with permeabilization buffer and incubate for suitable i. j. treating the sample of step (i) with Phycoerythrin conjugated mouse anti-human H_IF- k. l. analyzing the sample to read phycoerythrin fluorescent. [074] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the PEGylated HB-CO (PEG-HB-CO) is added in 4.0, 0.4 mg/ml PEG-HB-CO or media diluent [075] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the suitable apparatus is microfuge tube. [076] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the incubation time in step (f) is 25⁰C to 40⁰C for 5 minutes to 30 minutes. [077] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the incubation time in step (f) is 37°C for 10 minutes. [078] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the incubation time in step (h) and (k) on ice for 5 minutes to 30 minutes. Attorney Docket No. PLG-021WO [079] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the incubation time in step (h) on ice for 10 minutes. [080] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the cell line is U266 human multiple myeloma cells. DETAILED DESCRIPTION OF THE INVENTION Definitions [081] The term “about”, as used herein, is intended to refer to ranges of approximately 10- 20% greater than or less than the referenced value. In certain circumstances, one skill in the art will recognize that, due to the nature of the referenced value, the term “about” can mean more or less than a 10-20% deviation from that value. [082] The term “Sanguinate™” or “Sg” or “PP-007” or “PEGylated bovine hemoglobin” or “PEGylated carboxyhemoglobin” as used herein are interchangeable and refers to a PEG-Hb- CO composition of the invention. [083] The term “Hemoglobin,” as used herein refers to an oxygen-binding (or CO-binding), active polypeptide that is not chemically cross-linked through treatment with chemical cross- linking agents, e.g., dialdehydes, etc. An exemplary hemoglobin is the native protein with no modifications other than the conjugation of one or more PEG (e.g., m-PEG) moieties. As used herein, “substantially free of chemical cross-linking agents,” refers to hemoglobin molecules that are not purposely cross-linked with chemical cross-linking agents. These hemoglobin preparations include less than 5%, less than 3% or less than 1% cross-linked hemoglobin. [084] The term “therapy” refers to “treating” or “treatment” of a disease or condition including providing relief from the symptoms or side-effects of the disease (including palliative treatment) and relieving the disease (causing regression of the disease). These terms also refer to the treatment of injury, including hemorrhagic shock, stroke, ischemia/reperfusion injury, trauma and the like. In various embodiments, these terms also refer to preventing the disease or condition from occurring in a subject that may be predisposed to the disease but does not yet experience or exhibits symptoms of the disease (prophylactic treatment), inhibiting the disease (slowing or arresting its development). [085] The term “anticancer activity” herein refers to ability of the present combination therapy to inhibit the tumor growth by reducing one or more incidence selected from partial Attorney Docket No. PLG-021WO tumor regression, complete tumor regression, tumor volume and hypoxia inducible factor 1 alpha (HIF-1a). The term “effective amount” or “an amount effective to” or a “therapeutically effective amount” or “therapeutic effective amount” or “therapeutic effective dose” any grammatically equivalent term means the amount that, when administered to a subject for treating a disease, condition or injury, is sufficient to effect treatment for that disease. In exemplary embodiments, this term refers to any amount of a conjugate of the invention (or a formulation including a conjugate of the invention) sufficient to repay at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or up to about 100% of tissue or organ oxygen debt attributable to disease, insult or injury. When used in the context of delivery of CO to a tissue, this term refers to an amount administered sufficient to derive a detectable therapeutic effect from the delivery of CO to a tissue. [086] In some embodiments, the therapeutic effective amount of PEG-HB-CO is selected from about 300 mg/kg to about 1500 mg/kg. In some embodiments, the therapeutic effective amount of PEG-HB-CO is selected from about 500 mg/kg to about 1200 mg/kg. In some embodiments, the therapeutic effective amount of PEG-HB-CO is selected from about 800 mg/kg to about 1000 mg/kg. [087] Exemplary hemoglobin composition of the invention is referred to as “capable of transferring a member selected from oxygen and carbon monoxide bound thereto to a tissue.” This phrase refers to a hemoglobin composition having the capability to transfer oxygen or carbon monoxide bound to the iron atom of the hemoglobin to a tissue in. In exemplary compositions, the transfer is measurable by alteration in a tissue parameter (e.g., vasodilation, tissue oxygenation) or by a detectable alteration in a clinically relevant endpoint (e.g., termination of necrotic process, decreased ischemia/reperfusion injury). In an exemplary embodiment, the transfer of carbon monoxide or oxygen to a tissue is measured in terms of the amount of an oxygen debt “repaid” by administration of a selected volume of a composition of the invention to a subject (or tissue) with an oxygen debt. In another exemplary embodiment, the amount of oxygen or carbon monoxide delivered to a tissue is measured in terms of the mass of oxygen or CO transferred to a pre-selected mass of tissue (e.g., one gram) by administration of a pre-selected dosage of a composition of the invention. The ability to transfer oxygen or CO to a tissue can also be measured functionally in vivo, and by comparison with known hemoglobin-based blood substitutes. In an exemplary embodiment, the hemoglobin is in “contact” or “operative contact” with the tissue to which it is delivering oxygen or carbon monoxide. By operative contact is meant that the hemoglobin is sufficiently proximate to the Attorney Docket No. PLG-021WO tissue that the oxygen or carbon monoxide is transferred directly, through an intermediate carrier or through diffusion to the tissue. [088] Hemoglobin of use in the present invention is derived from substantially any mammalian source. Exemplary sources of hemoglobin include common livestock animals, e.g., cows, pigs, sheep and the like. The invention is not limited by the source of hemoglobin. In various embodiments, the hemoglobin is bovine hemoglobin. [089] The “subject” or “individual” refers to animal, mammals, or human subject. In some embodiments, mammal is preferably a human. Mammals also include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. [090] In some embodiments, the animal model is selected from rats, mouse, and guinea pig.The term “normoxia” used herein refers to the level of oxygen realized as normal based on the system upper limits: for example, when referring to an in vitro cell based assay system atmospheric oxygen (21%) would be the upper limit. When referring to an in vivo systems, red blood cell p50 of 26mmHg this would be the upper limit. It is further realized that oxygen gradients may exist and vary based on spaciotemporal O2 levels as well as the metabolic requirements of a specific cells, tissues, organ systems with the body. Moreover, this may be further defined as stochastic and complex in the tumor microenvironment as a consequence of compromised intratumoral vasculature. [091] The term “Tumor Doubling Time”or “Td” used herein refers as follows – Td is an individual and group endpoint, typically expressed as the median Td of the group. It is measured in days. Td can be calculated from any type of volumetric data (caliper measurements, BLI signals, etc.,). For QC purposes it is calculated for the exponential portion of the tumor growth curve. Data points during any lag phase and in the Gompertzian advanced stage are not included. Typical tumor burden limits are between 100 and 1000 mm³, but actual selection is data driven. Td is calculated from a least square best fit of a log/linear plot of tumor burden vs day as: =_{log 2 /} [092] The term “Percent T/C” or “% T/C” as used herein refers or defined as the median tumor burden of the treated group divided by the median tumor burden of the control group. (Group endpoint). [093] The term “Time to Progression” or “TP” as used herein refers to the time to progression is an individual endpoint and can be used as a surrogate for lifespan or time on study. The selected tumor evaluation size is tumor model and study dependent. TP data is analyzed by Attorney Docket No. PLG-021WO Kaplan Meier methods just as traditional lifespan data. The Time to Progression for an individual animal is the number of days between initiation of treatment and death or the day that the animal reaches a selected evaluation size and may be “>” if animals survive to study termination if the evaluation size is not reached. The initiation of treatment is the day of first treatment in the study as a whole and is not specific to the group in question. Time to progression is a log-linear interpolation between the adjacent data points on either side of the selected tumor evaluation size. This normalizes the evaluation criteria for all animals. [094] If animals do not reach the selected evaluation size and are euthanized or found dead due to disease progression or lack of treatment tolerance, lifespan is reported instead of Time to Progression. Animals euthanized or found dead for causes unrelated to disease progression (technical errors, etc.) are excluded from this calculation and reported as “NA”. The median Time to Progression for a group is used to calculate the % Increase in Time to Progression (% ITP). [095] In BLI studies, TP is only evaluated within the duration that imaging is completed. Animals that do not reach the selected evaluation size by the final day of imaging will be awarded a > value in endpoint calculations. [096] The term “% Increase in Time to Progression” or “% ITP” used herein refers as follows – % ITP is a group endpoint. %ITP is “>” when the median animal is It is calculated as: %_{= [( ) − ( )]/ 100} [097] The term “Complete Regression” or “CR” used herein described as follows – Complete tumor regression refers to the disappearance of all signs of cancer in response to treatment or spontaneously, meaning the tumor and its associated cancer cells are no longer detectable. All tumors will be measured, even if they fall below the standard limit of measurement. Tumors _{below 63 mm} ³ _{may not be accurate in measurement or may not be established (viable).} Therefore, an animal is credited a complete regression if its tumor burden is reduced to a volume below the standard limit of measurement (63 mm³) at any point after the first treatment. For caliper driven studies, the CR must be maintained for at least 2 consecutive measurements, unless the credited value occurs on the last measurement day of the study. In BLI studies, an animal is credited a complete regression if its tumor burden decreases to less than a declared background BLI signal level. Only a single measurement below background is required to meet CR criteria for a BLI study due the dynamic range of the measurements and typically longer intervals between imaging. (Individual and Group efficacy endpoint). Attorney Docket No. PLG-021WO [098] The term “Partial Regression” or “PR” are interchangeable herein and means that the tumor has shrunk or decreased in size or number, but not completely disappeared, in response to treatment or, rarely, spontaneously. An animal is credited with a partial regression if its tumor burden decreases to less than half of the tumor burden at first treatment. For caliper driven studies, the PR must be maintained for at least 2 consecutive measurements unless the credited value occurs on the last measurement day of the study. For BLI driven studies, only a single measurement is required because of the dynamic range of the measurements and typically longer intervals between imaging. PRs are tabulated exclusive of CRs, thus an animal that achieves a CR is not also counted as a PR. (Individual and Group efficacy endpoint). [099] The term “Tumor-Free Survivor” or “TFS” or “progression free survivors” or “PFS” are interchangeable and herein refers as follows – A TFS is any subject that (1) survives until termination of the study, and (2) has no reliably measurable evidence of disease (defined as subcutaneous tumor volumes below the limit of measurement, 63 mm³) at study termination. For BLI studies, this means that tumor burden has decreased to less than a declared background BLI signal level at the final imaging timepoint and there is no evidence of disease upon final necropsy (no observed mass >63 mm³). Subjects hat are tumor–free at some point during the study but are then euthanized for sampling or other purposes prior to the end of the study are not considered TFS. They are excluded from calculation of the %TFS. (Individual and Group efficacy endpoint). [0100] “BLI” or “bioluminescence imaging” are interchangeable is a very sensitive, noninvasive optical technique, widely used for monitoring tumors in small animals. Bioluminescence imaging (BLI) relies on the light emitted by luciferase, an enzyme that catalyzes the oxidation of a substrate (luciferin) to produce light, enabling non-invasive visualization of biological processes in living organisms. [0101] In clinical studies, "good responders" refer to patients who show a clinically meaningful improvement or response to a treatment or intervention, often assessed using responder analysis, which dichotomizes continuous endpoints into "responders" and "non-responders" based on a pre-specified threshold. Herein the murine cancer models when detected through BLI on day 75 are showing reduction in the tumor volume between 10^6 - 10^5 are termed as good responders (figure 10). [0102] In clinical studies, “high responders” refer to participants who exhibits a significantly greater or more favorable response to treatment or intervention compared to the average or other participants. Herein the murine cancer models when detected through BLI on day 75 are Attorney Docket No. PLG-021WO showing reduction in the tumor volume between 10^5 - 10^4 are termed as High responders (figure 10). [0103] “A549” herein refers to human lung cancer cell line. [0104] “MCF7” herein refers to human breast cancer cell line. _{[0105] “HIF-1 ” or “HIF-1a” or “Hypoxia-inducible factor 1-alpha" is a subunit of the} heterodimeric transcription factor, HIF-1, that is crucial for cellular and systemic responses to hypoxia (low oxygen levels) by regulating genes involved in energy metabolism, angiogenesis, and other processes. [0106] The term “EC50” used herein refers to half maximal effective concentration (EC50) that is a measure of the concentration of a drug, antibody or toxicant which induces a biological response halfway between the baseline and maximum after a specified exposure time. More simply, EC50 can be defined as the concentration required to obtain a 50% effect and may be also written as [A]50. [0107] The term “RLU” refers to Relative Light Units. [0108] The “Heme oxygenase –1" or “HO-1” is a protein upregulated in stress including hypoxia. HO-1 is regulated by Keap1-Nrf2 transcription complex that activated a pathway is a cellular defense mechanism against oxidative and electrophilic stress, with Nrf2 (nuclear factor erythroid 2-related factor 2) being a transcription factor and Keap1 (Kelch-like ECH-associated protein 1 [0109] The “Keap1-Nrf2" pathway is a crucial cellular defense mechanism against oxidative and electrophilic stress, with Nrf2 being a transcription factor that, when activated, promotes the expression of genes involved in detoxification and antioxidant defense, and Keap1 acting as a repressor of Nrf2 under normal conditions. Methods of Use and Treatment [0110] Disclosed herein is a method of treating and/or alleviating one or more symptoms of cancer, wherein the method exhibits s surprising improvements in reduction of tumor volume, exhibits s improvement in the incidence of partial regression, and complete regression, and improves survival period. It was surprisingly found that when PEG-HB-CO is administered in combination with Focal beam radiation therapy, the method improves incidence of partial tumor regression, complete tumor regression and tumor free survival and reduction in tumor volume and hypoxia inducible factor 1 alpha (HIF-1a). Attorney Docket No. PLG-021WO [0111] The present disclosure provides a method of treatment of tumor and/or cancer by providing combination therapy of PEGylated hemoglobin and/or PEG-HB-CO with radiation therapy. [0112] The present disclosure provides a method of treating cancer by providing combination therapy of PEGylated carboxyhemoglobin with radiation therapy. In some embodiments, the invention improves survival period or response to treatment of tumor and/or cancer by providing combination therapy of PEGylated hemoglobin and/or PEG-HB-CO with radiation therapy. [0113] In some embodiments, the improved method of treatment alleviates at least one symptom associated with tumor and/or cancer treatment selected from lung cancer, breast cancer, melanoma, renal cell carcinoma, squamous cell non-small cell lung cancer (NSCLC), non-squamous NSCLC, colon-rectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, head and neck. [0114] In some embodiments, the present invention treats lung cancer, breast cancer with combination therapy of PEGylated hemoglobin and/or PEG-HB-CO with radiation therapy. [0115] In some embodiments, the present invention method of treating a cancer in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the PEG-HB-CO is administered simultaneously or sequentially or in any wherein the radiation therapy does not substantially affect wherein wherein the method improves the cancer treatment. [0116] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the PEG-HB-CO is administered simultaneously or sequentially or in any wherein the radiation therapy does not wherein Attorney Docket No. PLG-021WO wherein the method improves the cancer treatment. [0117] In some embodiments, the present invention a method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the PEG-HB-CO is administered simultaneously or sequentially or in any wherein the radiation therapy does not substantially affect wherein wherein the method improves the cancer treatment. [0118] In some embodiments, the present invention discloses a method of treating cancer, wherein the cancer treatment is improved by inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a) and/or improving one or more incidence of tumor-free survivors, increase in time to progression (ITP). [0119] In some embodiments, the present invention discloses a method of treating a cancer in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the PEG-HB-CO is administered simultaneously or sequentially or in any order before or after the radiation therapy. [0120] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the PEG-HB-CO is administered simultaneously or sequentially or in any order before or after the radiation therapy. [0121] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: Attorney Docket No. PLG-021WO a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the PEG-HB-CO is administered simultaneously or sequentially or in any order before or after the radiation therapy. [0122] In some embodiments, the present invention discloses a method, wherein the subject is administered with PEG-HB-CO and simultaneously treated with radiation therapy. [0123] In another embodiment, the present invention discloses a method, wherein the subject is administered with PEG-HB-CO and sequentially treated with radiation therapy. [0124] In some embodiments, the PEG-HB-CO is administered in any order before or after the radiation therapy. [0125] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor in a subject in need thereof, the method comprising: a. administering to the subject a combination of a dose of PEGylated HB-CO (PEG- H_B- b. wherein the combination of the PEG-HB-CO is administered in any order before or after treating the subject with radiation therapy. [0126] In some embodiments, the present invention discloses a method of treating cancer in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the subject is administered with PEG-HB-CO and sequentially treated with radiation therapy. [0127] In another embodiment, the present invention discloses a method of reducing the size and/or volume of a tumor in a subject in need thereof comprising: a_{. administering to the subject a combination of a dose of PEG-HB-} b. wherein the combination of the PEG-HB-CO is administered simultaneously to the subject with radiation therapy. [0128] In some embodiments, the invention provides an improved method of treating and/or alleviating one or more symptoms of cancer comprising the steps: Attorney Docket No. PLG-021WO a_{. administering a dose of PEG-HB-} b. where in the step (a) and (b) is performed sequentially, or simultaneously or any _{order and} wherein the process improves anti-cancer activity compared to the process performed with either with PEG-HB-CO or radiation therapy alone. [0129] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. treating the subject wherein the administration of PEG-HB-CO and radiation therapy to the subject exhibits s improvement in tumor volume reduction compared to the subject administered with PEG-HB-CO or radiation therapy alone. [0130] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor in a subject in need thereof comprising: a_{. administering to the subject a combination of a dose of PEG-HB-} b. wherein the subject exhibits s a higher reduction in the size and/or volume of a tumor compared to the subjected treated either with PEG-HB-CO or radiation therapy. [0131] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein subject administered with combination therapy of the PEG-HB-CO and the radiation therapy exhibits at least about 50%, about 51%, about 52%, about 53%, about 54%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, and about 70%, improvement in tumor volume reduction compared to the subject treated either with PEG-HB-CO or radiation therapy alone. [0132] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein subject administered with combination therapy of the PEG-HB-CO and the radiation therapy exhibits at least about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, , improvement in tumor volume reduction compared to the subject treated either with PEG-HB-CO or radiation therapy alone. Attorney Docket No. PLG-021WO [0133] In another embodiment, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein subject administered with combination therapy of the PEG-HB-CO and the radiation therapy exhibits at least about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, and about 64% improvement in tumor volume compared to the subject treated either with PEG-HB-CO or radiation therapy alone. [0134] In some embodiments, the present invention discloses a method of reducing the size and/or volume of a tumor, wherein the reduction of volume comprising inhibiting tumor growth by reducing one or more incidence selected from partial tumor regression, complete tumor regression, tumor volume and hypoxia inducible factor 1 alpha (HIF-1a). [0135] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: [0136] In some embodiments, the present invention discloses method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a_{. administering a dose of PEG-HB-} b. wherein the administration of PEG-HB-CO and radiation therapy to the subject provides tumor-free survivors. [0137] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein tumor free survivors improves increase in time to progression (ITP) and/or inhibiting tumor growth by reducing one or more incidence selected from partial tumor regression, complete tumor regression, tumor volume and hypoxia inducible factor 1 alpha (HIF-1a). [0138] In some embodiments, the present invention discloses method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB- and} b. treating the subject wherein the treatment improves the incidence of partial tumor regression in the range of wherein the treatment reduces the incidence of complete tumor regression in the range of and Attorney Docket No. PLG-021WO wherein the administration of PEG-HB-CO and radiation therapy increases the tumor- free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. [0139] In some embodiments, the present invention discloses a method of treating cancer in a subject, wherein the treatment exhibits s incidence of partial progression about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, and about 50%. [0140] In some embodiments, the present invention discloses a method of treating cancer in a subject, wherein the treatment exhibits s incidence of partial progression about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, and about 40%,. [0141] In some embodiments, the present invention discloses a method of treating cancers in a subject, wherein the treatment exhibits s about 40% incidence of partial regressions. [0142] In some embodiments, the present invention discloses a method of treating cancer in a subject, wherein the treatment exhibits s incidence of complete progression about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, and about 40%. [0143] In some embodiments, the present invention discloses a method of treating cancer in a subject, wherein the treatment exhibits s incidence of complete progression about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, and about 30%, [0144] In some embodiments, the present invention discloses a method of treating cancers in a subject, wherein the treatment exhibits s about 20% incidence of complete regression. [0145] In some embodiments, the present invention discloses a method of treating cancers in a subject, wherein the treatment exhibits s about 40% incidence of partial regressions and about 20% incidence of complete regression. [0146] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, the administration of PEG-HB-CO and radiation therapy increases the tumor-free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. Attorney Docket No. PLG-021WO [0147] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. wherein the treatment exhibits s improvement in the incidence of partial regressions _{and incidence of complete regressio and} wherein the combination therapy provides tumor free survivors. [0148] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. wherein the treatment exhibits s about 40% incidence of partial regressions and about and wherein the administration of PEG-HB-CO and radiation therapy increases the tumor- free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. [0149] In some embodiments, the present invention discloses a method of treating a cancer in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the administration of PEG-HB-CO and radiation therapy to the subject exhibits s at least 50% to about 70% improvement in tumor volume reduction compared to _{the subject treated either with PEG-HB- and} wherein the administration of PEG-HB-CO and radiation therapy increases the tumor- free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. [0150] In some embodiments, the present invention discloses method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. treating the subject Attorney Docket No. PLG-021WO wherein the administration of PEG-HB-CO and radiation therapy to the subject exhibits s at least 50% to about 70% improvement in tumor volume reduction compared to _{the subject treated either with PEG-HB-CO or radiation therapy alone and} wherein the administration of PEG-HB-CO and radiation therapy increases the tumor- free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. [0151] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. treating the subject wherein the administration of PEG-HB-CO and radiation therapy to the subject exhibits s exhibits at least about 50% to about 70% improvement in tumor volume reduction _{compared to the subject treated either with PEG-HB-CO or radiation therapy alone and} wherein the administration of PEG-HB-CO and radiation therapy increases the tumor- free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. [0152] In some embodiments, the present invention provides a method of treating cancer in the subject, wherein the subject is administered with at least one cycle of the combination therapy. [0153] In some embodiments, the present invention provides a method of treating cancer in the subject, wherein the subject is administered with more than one cycle of the combination therapy. [0154] In some embodiments, the invention provides an improved method of treating and/or alleviating one or more symptoms of cancer comprising the steps: a_{. administering a dose of PEG-HB-} b. process improves anti-cancer activity compared to the process performed with either with _PEG-HB- wherein the administration of PEG-HB-CO and radiation therapy to the subject exhibits s improvement in tumor volume reduction compared to subject administered with _PEG-HB- wherein the administration of PEG-HB-CO and radiation therapy to the subject provides tumor free survivors. Attorney Docket No. PLG-021WO [0155] In some embodiments, the present invention discloses a method of treating cancer, wherein the subject is selected from animal model suitable for cancer research, and human suffering from cancer. [0156] In some embodiments, the animal model suitable for cancer research is selected from guinea pig, mouse and rat. In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein the type of cancer is selected from lung cancer, breast cancer, melanoma, renal cell carcinoma, squamous cell non-small cell lung cancer (NSCLC), non-squamous NSCLC, colon-rectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, head and neck. [0157] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the administration of PEG-HB-CO is performed simultaneously, sequentially, wherein the reduction of tumor improves increase in time to progression (ITP). [0158] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer wherein the reduction of tumor improves the increase in time to progression (ITP) at least by 10% to 75 % or at least about 10% to about 50% or about 50% to about 75% compared to the increase in time to progression (ITP) when the subject treated either with PEG-HB-CO or radiation therapy alone. [0159] In another embodiment, the present method exhibits s improvement in the increase in time to progression (ITP) atleast about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, and about 75% compared to the subject treated either with PEG-HB-CO or radiation therapy alone. In another embodiment, the present method exhibits s improvement in the increase in time to progression (ITP) atleast about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, and about 75% compared to the subject treated either with PEG-HB-CO or radiation therapy alone. [0160] In another embodiment, the present method exhibits s improvement in the increase in time to progression (ITP) atleast about 30%, about 35%, about 40%, about 45%, about 50%, Attorney Docket No. PLG-021WO about 55%, about 60%, about 65%, and about 75% compared to the subject treated either with PEG-HB-CO or radiation therapy alone. [0161] In another embodiment, the present method exhibits s improvement in the increase in time to progression (ITP) atleast about 50%, about 55%, about 60%, about 65%, about 70%, and about 75% compared to the subject treated either with PEG-HB-CO or radiation therapy alone. [0162] In another embodiment, the present method exhibits s improvement in the increase in time to progression (ITP) at least about 73% compared to the subject treated either with PEG- HB-CO or radiation therapy alone. [0163] In some embodiments, the combination therapy treatment produced anti-cancer activity with an ITP of about 160%, about 161%, about 162%, about 163%, about 164%, about 165%, about 167%, about 168%, about 169%, about 170%, about 171%, about 172%, about 173%, about 174%, about 175%, about 176%, about 177%, about 178%, about 179%, about 180%, about 181%, about 182%, about 183%, about 184%, about 185%, about 186%, about 187%, about 188%, about 189%, about 190%, about 191%, about 192%, about 193%, about 194%, about 195%, about 196%, about 197%, about 198%, about 199%, about 200%, about 201%, about 202%, about 203%, about 204%, about 205%, about 206%, about 207%, about 208%, about 209%, about 210%, about 211%, about 212%, about 213%, about 214%, about 215%, about 216%, about 217%, about 218%, about 219%, and about 220%. [0164] In some embodiments, the combination therapy treatment produced anti-cancer activity with an ITP of about 160%, about 200% and about 216%. [0165] In one aspect of the embodiment, anti-cancer activity is determined by ITP. In some embodiments, the ITP is achieved about 216.6%. [0166] In some embodiments, the present invention discloses a method of treating cancer in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the administration of PEG-HB-CO is performed simultaneously, sequentially, or in any wherein the increase in time to progression (ITP) wherein the reduction of tumor improves the increase in time to progression (ITP) at least by 10% to 75 % or at least about 10% to about 50% or about 50% to about 75% Attorney Docket No. PLG-021WO compared to the increase in time to progression (ITP) when the subject is treated either with PEG-HB-CO or radiation therapy alone. [0167] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. wherein the combination of the PEG-HB-CO and the radiation therapy improves the cancer treatment and body weight management in the subject compared to the subject treated either with PEG-HB-CO or radiation therapy alone. [0168] In some embodiments, the treatment maintains body weight of the subject during the treatment. [0169] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a_{. administering a dose of PEG-HB-} b. wherein the step (a) and (b) are performed sequentially, or simultaneously or in any wherein the process improves anti-cancer activity compared to the process performed with either with PEG-HB-CO or radiation therapy alone. [0170] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject comprising: a_{. administering to the subject a dose of PEG-HB-} b. wherein the combination of the PEG-HB-CO and the radiation therapy thereof inhibits growth of the tumor in the subject compared to the subject treated either with PEG- HB-CO or radiation therapy alone. [0171] In some embodiments, the present invention discloses a method of treating cancer, wherein the subject is selected from animal model suitable for cancer research, and human suffering from cancer. [0172] In some embodiments, the animal model suitable for cancer research is selected from guinea pig, mouse and rat. [0173] In some embodiments, the present invention discloses a method of treating and/or alleviating one or more symptoms of cancer, wherein the type of cancer is selected from lung Attorney Docket No. PLG-021WO cancer, breast cancer, melanoma, renal cell carcinoma, squamous cell non-small cell lung cancer (NSCLC), non-squamous NSCLC, colon-rectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, head and neck. [0174] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof by administering a combination therapy of PEG-HB-CO and radiation therapy, wherein the therapeutic effective amount PEG-HB-CO is administered in subject intravenously and/or subcutaneously at a dose selected in the range of about 300 mg/kg, to about 1500 mg/kg [0175] In some embodiments, the dose of PEG-HB-CO is about at 300 mg/kg, about at 400 mg/kg, about at 500 mg/kg, about at 600 mg/kg, 700 mg/kg, about at 800 mg/kg, about at 900 mg/kg, about at 1000 mg/kg, about at 1100 mg/kg, about at 1200 mg/kg, about at 1300 mg/kg, about at 1400 mg/kg, and about at 1500 mg/kg. [0176] In some embodiments, the dose of PEG-HB-CO is about at 500 mg/kg, about at 600 mg/kg, 700 mg/kg, about at 800 mg/kg, about at 900 mg/kg, about at 1000 mg/kg, about at 1100 mg/kg, about at 1200 mg/kg. [0177] In some embodiments, the dose of PEG-HB-CO is about at 800 mg/kg, about at 900 mg/kg, and about at 1000 mg/kg. [0178] In some embodiments, the dose of PEG-HB-CO is about at 1000 mg/kg. [0179] In some embodiments, the radiation therapy is focal beam radiation therapy and/or stereotactic radiation surgery. [0180] In some embodiments, the radiation therapy is focal beam radiation therapy. [0181] In some embodiments, the dose of PEG-HB-CO is about at 1000 mg/kg and focal beam radiation is about 5.0 Gy, about 5.5 Gy, about 6.0 Gy, about 6.5 Gy, about 7.0 Gy, about 7.5 Gy, about 8.0 Gy, about 8.5 Gy, about 9.0 Gy, about 9.5 Gy, about 10 Gy. [0182] In some embodiments, the dose of PEG-HB-CO is about at 1000 mg/kg and focal radiation is about 7.5 Gy. [0183] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. wherein the dose of PEG-HB-CO is about at 320 mg/kg to about at 1500 mg/kg and -cancer activity. Attorney Docket No. PLG-021WO [0184] In some embodiments, the present invention discloses a method for of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. wherein the dose of PEG-HB-CO is about at 1000 mg/kg and focal radiation is about and wherein the treatment produced anti-cancer activity. [0185] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. wherein the subject exhibits s improvement in the treatment of cancer compared to the subject treated either with PEG-HB-Co or radiation therapy alone. [0186] In some embodiments, the present invention discloses a method of treating cancers in a subject in need thereof comprising: a_{. administering a dose of PEG-HB-} b. treating the patient with wherein the subject exhibits s improvement in the treatment of cancer compared to the subject treated either with PEG-HB-Co or radiation therapy alone. [0187] In some embodiments, the subject in need of treatment of cancer, exhibits s improvement in the %T/C value compared to the subject treated with PEG-HB-CO alone. [0188] In some embodiments the %T/C value is derived or measure by bioluminescence imaging (BLI). [0189] In some embodiments, the present invention discloses a method of treating cancers in a subject, wherein the subject is administered with single dose of PEG-HB-CO in combination with radiation therapy. [0190] In some embodiments, the present invention discloses a method of treating cancers in a subject, wherein the subject is administered with more than one dose of PEG-HB-CO in combination with radiation therapy. [0191] In some embodiments, the present invention discloses a composition containing hemoglobin, wherein the hemoglobin composition delivers O2 to cells with severe hypoxia. In some embodiments, the hemoglobin is a PEGylated hemoglobin. In some embodiments, the PEGylated hemoglobin is PEG-Hb-CO. Attorney Docket No. PLG-021WO [0192] In some embodiments, the present invention discloses a composition containing hemoglobin, particularly PEGylated hemoglobin, wherein the hemoglobin composition inhibits HIF-1a gene expression. [0193] In some embodiments, the hemoglobin composition inhibits HIF-1a gene expression in hypoxia state. [0194] In some embodiments, the present disclosure discloses a method of inhibiting tumor progression by reducing HIF-1a mediated gene expression. [0195] In some embodiments, the PEGylated hemoglobin reduces or alleviate HIF-1a mediated gene expression in suitable cell line and provides desirable EC50 value. [0196] In some embodiments, the PEGylated hemoglobin reduces or alleviate HIF-1a mediated gene expression in suitable cell line A549 cell line (human lung cancer). [0197] In some embodiments, the PEGylated hemoglobin reduces or alleviate HIF-1a mediated gene expression in suitable cell line MCF7 cell line (human breast cancer). [0198] In some embodiments, the EC50 value is about 0.52 mg/mL. [0199] In some embodiments, the suitable cell lines are selected from A549 cell line (human lung cancer), [0200] MCF7 breast cancer cell line, human HNSCC cell lines. [0201] In some embodiments, the EC50 value for reducing HIF-1a mediated gene expression is about 0.52 mg/mL. [0202] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB- and} b. treating the subject wherein the combination therapy of the PEG-HB-CO and the radiation therapy inhibits growth of the tumor in the subject compared to the subject treated either with PEG- _{HB- and} wherein the combination therapy reduces or alleviates hypoxia-inducible factor 1- alpha (HIF-1a) mediated gene expression, by providing an EC50 value in the range of about 0.5 mg/ml to about 2.0 mg/ml. [0203] In some embodiments, the present invention discloses a method of treating cancer by providing combination therapy to the subject, wherein the combination therapy reduces or alleviates hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, providing an Attorney Docket No. PLG-021WO EC50 value of about 0.50 mg/ml, about 0.60 mg/ml, about 0.70 mg/ml, about 0.80 mg/ml, about 0.90 mg/ml, about 1.0 mg/ml, about 1.1 mg/ml, about 1.2 mg/ml, about 1.3 mg/ml, about 1.4 mg/ml, about 1.5 mg/ml, about 1.6 mg/ml, about 1.7 mg/ml, about 1.8 mg/ml, and about 1.9 mg/ml. [0204] In some embodiments, the present invention discloses a method of treating cancer by providing combination therapy to the subject, wherein the combination therapy reduces or alleviates hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, providing an EC50 value of about 0.50 mg/ml, about 0.60 mg/ml, about 0.70 mg/ml, about 0.80 mg/ml, about 0.90 mg/ml, and about 1.0 mg/ml, [0205] In some embodiments, the present invention discloses a method of treating cancer by providing combination therapy to the subject, wherein the combination therapy reduces or alleviates hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, providing an EC50 value of about 0.52 mg/ml. [0206] In some embodiments, the present invention discloses a composition of PEG-HB-CO comprises bovine hemoglobin and poly (ethylene glycol), wherein bovine hemoglobin is bound to carbon monoxide, wherein the composition comprises stabilizing agent. [0207] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the combination therapy of the PEG-HB-CO and the radiation therapy inhibits growth of the tumor in the subject compared to the subject treated either with PEG- _HB- wherein the method exhibits s at least 50% to about 70% improvement in reduction of tumor volume compared to the subject treated either with PEG-HB-CO or radiation therapy wherein the method reduces or alleviates hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, providing an EC50 value in the range of about 0.5 mg/ml to about wherein the method reduces the incidence of partial tumor regression in the range of Attorney Docket No. PLG-021WO wherein the method reduces the incidence of complete tumor regression in the range wherein wherein the administration of PEG-HB-CO and radiation therapy increases the tumor- free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. [0208] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject in need thereof, wherein the treatment is performed in at least one cycle. [0209] In some embodiments, the present invention discloses a method of inhibiting tumor growth in a subject in need thereof, wherein the treatment is performed in more than one cycle. [0210] In some embodiments, the present disclosure discloses a method of inhibiting tumor progression by reducing the HO-1/Nrf2-KEAP expression pathway. [0211] In some embodiments, the EC50 value for reducing HIF-1a mediated gene expression is about 1.61 mg/ml. [0212] In some embodiments, the EC50 value for reducing HIF-1a mediated gene expression and HO-1/Nrf2-KEAP expression pathway is less than 2.0 mg/mL. [0213] In some embodiments, the present disclosure discloses a method of inhibiting tumor progression by reducing the HO-1/Nrf2-KEAP and HIF-1a mediated gene expression rapidly by utilizing PEG-HB-CO in a dose-dependent manner. [0214] In some embodiments, the present invention discloses a method of treating cancer, wherein PEG-HB-CO dose is dependent on tumor hypoxia volume. [0215] In some embodiments, the present invention discloses a method of treating cancer, wherein PEG-HB-CO dose is directly proportional to the tumor hypoxia volume. [0216] In some embodiments, the present invention discloses a method of treating cancer, wherein PEG-HB-CO dose is high if the tumor hypoxia volume is high. In another embodiment, the present invention discloses a method of treating cancer, wherein PEG-HB- CO dose is low if the tumor hypoxia volume is low.In some embodiments, the use of PEG-HB- CO in a dose dependent manner are selected from about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, and about 11 mg/mL. [0217] In some embodiments, the present invention discloses a composition containing hemoglobin, particularly PEGylated hemoglobin, wherein use of an effective amount of the hemoglobin composition in A549 HIF-1a-LUC transfected cells shows a dose-dependent Attorney Docket No. PLG-021WO decrease in RLUs with a calculated drug EC50 of 0.52 mg/mL. In some embodiments, the A549 HIF-1a-LUC transfected cells were preconditioned to hypoxia (0.5% O2). [0218] In some embodiments, the present invention discloses a composition containing hemoglobin, particularly PEGylated hemoglobin, wherein the hemoglobin composition improves HO-1/Nrf2-KEAP signaling. [0219] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, the method comprising: a_{. culturing the cells capable to express hypoxia-inducible factor 1-alpha (HIF-} b. c_{. treating with PEGylated HB-CO (PEG-HB-} d. incubating the assay plate e. determining the HIF- exhibits reduction in HIF-1a or f. collect the sample from step (d) in suitable apparatus and treated with fixation buffer g. h. separating the supernatant in suitable apparatus i. treating the supernatant with permeabilization buffer and incubate for suitable j. Resuspend in staining buffer to prepare sample and transfer in suitable k. Treating the sample of step (j) with Phycoerythrin conjugated mouse anti- h_{uman HIF-} l. Incubating for suitable time m. Analyze sample to read phycoerythrin fluorescent. [0220] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression,, wherein the cell line is A549 or MCF7. [0221] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the PEGylated HB-CO (PEG-HB-CO) is added in 1:10 dilution. Attorney Docket No. PLG-021WO [0222] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the assay plate is incubated for suitable time for suitable period in normoxia or hypoxia. [0223] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the assay plate is incubated for suitable time for suitable period in normoxia. [0224] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the assay plate is incubated at temperature 30°C to 40°C. [0225] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the hours. [0226] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the determination of the HIF-1a expression is performed by the Luciferase assay. [0227] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, the method comprising: a_{. culturing the cells capable to express hypoxia-inducible factor 1-alpha (HIF-} b. c_{. treating with PEGylated HB-CO (PEG-HB-} d. incubating the assay plate e. collect the sample from step (d) in suitable apparatus and treated with fixation buffer f. g. h. treating the supernatant with permeabilization buffer and incubate for suitable i. resuspend in staining buffer to prepare sample and transfer in suitable j. treating the sample of step (i) with Phycoerythrin conjugated mouse anti- h_{uman HIF-} k. Attorney Docket No. PLG-021WO l. analyzing the sample to read phycoerythrin fluorescent. [0228] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the PEGylated HB-CO (PEG-HB-CO) is added in 4.0, 0.4 mg/ml PEG-HB-CO or media diluent [0229] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the suitable apparatus is microfuge tube. [0230] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the incubation time in step (f) is 25⁰C to 40⁰C for 5 minutes to 30 minutes. [0231] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the incubation time in step (f) is 37°C for 10 minutes. [0232] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the incubation time in step (h) and (k) on ice for 5 minutes to 30 minutes. [0233] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the incubation time in step (h) on ice for 10 minutes. [0234] In some embodiments, the present invention discloses a method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, wherein the cell line is U266 human multiple myeloma cells. [0235] In some embodiments, the present invention provides a method of treating and/or alleviating one or more symptoms of cancer, wherein the method exhibits s improvement in reduction of tumor volume, exhibits s improvement in the incidence of partial regression, and complete regression, and improves survival period. EXAMPLES Example 1: Detection of Hypoxia inducible factor 1 alpha transcription activity in A549 human lung cancer cells in an oxygen-controlled cell culture assay system: [0236] The effects of PEGylated bovine hemoglobin treatment was evaluated in cell culture experiments. The A549 cell line (human lung cancer) was used to determine treatment effects Attorney Docket No. PLG-021WO on HIF-1a. An experimental system was developed in house that used the HIF-1a promoter element controlling expression of the Firefly luciferase gene. [0237] A549 cell line (ATCC CCL-185) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and supplements in 20% O2, 5% _{CO2 humidified cell incubator at 37 . Cells collected, centrifuged, washed and resuspended} into Phosphate buffered saline (PBS) without calcium or magnesium and kept on ice for preparation of plasmid DNA transfection. The Signal HIF Reporter (luc) Kit was used in combination with Attractene transfection agent and Opti-mem transfection media. [0238] Transfection complexes including the HIF-FLUC gene cassette and the control plasmid CMV-RLUC were combined and incubated RT for 5 minutes (Set 1) while the Attractene and Opti-mem were combined separately (Set 2). Sets were combined and incubated for 20 minutes while cells were prepared for transfections. [0239] A549 cells were collected from flask (T75) by first washing with 5 mL of PBS w/o Ca or Mg and then followed by washing with 0.5% trypsin/EDTA. Following trypsin aspiration, flask was placed in 37° incubator for 5 minutes. 10 mL of DMEM (10%FBS) was added to flask and cell suspension was transferred to a 12 mL conical tube and cells were pelleted by centrifugation at 300xg for 5 minutes and supernatant was removed by aspiration. [0240] Cell Pellet was suspended in 1 mL of Optimem using P1000 pipet to minimize cell clumping before bringing final volume of cell suspension to 10ml in Optimem and re-counted (6.6x105 cells per mL). [0241] An A549 cell suspension (16ml at 1.0x105 cells per mL) was then prepared and combined with the transfection complexes. Then the OptiMem was supplemented with 5% FBS. Plating & Transfection [0242] Transfection complexes in the cell suspension were (150 μL /well) were distributed to replicate wells in a 96 well plate. Plate was placed at 37° for 24 hours at either normoxia or hypoxia _{[0243] PEGylated bovine hemoglobin (PEG-HB-CO) (Sanguinate was added to} A549 cells transfected with HIF-1a responsive reporter plasmid at a 1:10 dilution (2mg/ml final) and incubated at 3 h or 6 h and then immediately assayed for LUC activity. In the same way saline (black bars) was added to A549 cells as shown in figure 1. [0244] In normoxia, PEGylated bovine hemoglobin (PEG-HB-CO) has no effect on luc activity. When exposed to hypoxic cells (0.5% O2) the Relative Luc activity is reduced at 3h Attorney Docket No. PLG-021WO and further reduced in a time-dependent manner. These results demonstrate the specific capability of PEGylated bovine hemoglobin (PEG-HB-CO ) to inhibit HIF-1a gene expression that increased over time as hypoxia increased, its effects did in proportion. Example 2: Dose-Dependent effects of PEGylated bovine hemoglobin in A549 HIF-1a- LUC reporter assay: [0245] PEGylated bovine hemoglobin (PEG-HB-CO) was added to the A549 HIF-1a-LUC transfected cells that are preconditioned to hypoxia (0.5% O2). The drug is serially diluted from 20 mg/mL to 0.02 mg/mL. Cells are incubated for 6h in hypoxia and luciferase assay is performed. The luciferase assay has following steps: • Cells were washed with PBS and 20 μL of 1x passive lysis buffer (E1910, Promega) was added to each well and plate stored at -80° until luciferase assay. • Dual Luciferase Assay (E1910, Promega) was used to quantify luciferase activity as per manufacturer’s protocol. • Lysates were thawed and 15μL of each lysate was transferred to a white walled 96 well assay plate (3917, Costar) containing 100 μL of LARII. • Firefly luciferase dependent luminescence was measured in micro-plate reader (Biotek) during 10 second integration period for each well. • Plate was then removed and 100μL of Stop & Glo Reagent supplemented with Renilla luciferase reagent to each well and returned to plate reader. • Firefly luciferase dependent luminescence was measured in micro-plate reader (Biotek) during 1 second integration period for each well. • Firefly luciferase relative light units (RLU) was normalized to Renilla RLU to determine relative expression levels. • Firefly luciferase dependent luminescence was measured in micro-plate reader (Biotek) during 1 second integration period for each well. • Firefly luciferase RLU was normalized to Renilla RLU to determine relative expression levels. [0246] The results showed a dose-dependent decrease in RLUs with a calculated drug EC50 of 0.52 mg/mL (Refer figure 2). Example 3: PEGylated bovine hemoglobin (PEG-HB-CO) improves HO-1/Nrf2-KEAP signaling: Attorney Docket No. PLG-021WO [0247] The PEGylated bovine hemoglobin (PEG-HB-CO) drug product was saturated with CO, and it will initially release CO before it acquires and distributes O2 to severely hypoxic tumor cells. Test samples were analyzed using a 96-well plate format cell-based assay. The CO is known to signal through the HO-1/Nrf2-KEAP gene expression pathway that has been shown to improve tumor responses. PEGylated bovine hemoglobin (PEG-HB-CO) is evaluated in the MCF7 breast cancer cell line stably transfected with an NrF2-RLuc reporter plasmid. [0248] Herein the Nrf2 reporter-MCF7 cells were plated at 5 x 10^4 cells/well (100 ul volume) _{in 96-well white plates and incubated at 37 , 05% CO2 for 16 h. Cell media was replaced} with 50 ul of fresh medium. Serial dilutions of PP-007 were prepared and 50 ul added to cells in duplicate across the assay plate (1:2 final assay dilution) at increasing concentrations [0.156mg/ml, 0.31mg/ml, 0.625mg/ml, 1.25mg/ml, 2.5mg/ml, 5mg/ml, 10mg/ml and 20 mg/ml]. Positive controls and assay control were included in dilution ranges to determine and were prepared at concentrations to determine EC50. These included CORM-3 (CO releasing Increasing concentrations (Refer figure 3) of PEGylated bovine hemoglobin ( PEG-HB-CO) are added to MCF7/NrF2-RLuc cells in culture for a period of 6hrs. Following, a Renila Luc commercial assay, the increase of RLUC was shown to occur with PEGylated bovine hemoglobin ( PEG- HB-CO) exposure. Three repeated runs were conducted that showed and average EC50 value of 1.61 mg/mL. Example 4: Detection of Hypoxia inducible factor 1 alpha protein in U266 human multiple myeloma cells in an oxygen-controlled cell culture assay system [0249] U266 Multiple myeloma cell line (ATCC TIB-196) were cultured in Roswell Park Memorial Institute (RPMI 1640) with 15% FBS and supplements in 20% O2, 5% CO2 humidified cell incubator at 37C. Cells collected, centrifuged and seeded into 12-well culture plates. One plate was kept at normoxia and a second plate was transferred to hypoxia (0.5% O2, 5% CO2). Cells were incubated overnight and the two wells per plate were treated with a final concentration of 4.0, 0.4 mg/ml PEG-HB-CO or media diluent. [0250] After 3 hr samples were collected from each plate and condition transferred into 2.0ml microfuge tubes, centrifuged for 5 min, media mixtures aspirated and 100ul fixation buffer was added to each tube, and incubated back at 37°C respective incubator for 10 minutes. Add 800 ul of staining buffer to wash, spin again 5 minutes. Supernatant again removed and 200ul of permeabilization buffer was added per tube and incubate on ice for 30 minutes. Repeat the Attorney Docket No. PLG-021WO 800ul staining buffer wash step and resuspend in 300 ul staining buffer. Transfer each 300ul sample to 3 x 100ul new tubes. Add 1 ul of Phycoerythrin conjugated mouse anti-human HIF- 1 alpha (PE-HIF-1 alpha antibody), Isotype Control (PE: Mouse IgG2b Isotype antibody) and no antibody control to the 3 tubes/sample set. Incubate 30 min on ice. Wash again with 200 ul stain buffer per tube centrifuge and aspirate Ab, stain buffer. Resuspend the cell pellets into 100ul stain buffer and run samples on GUAVA Muse analyzer (Millipore) set to read phycoerythrin fluorescent read-out. Normoxia and hypoxia no drug samples served as negative (HIF-) and positive (HIF+) controls, respectively. Figure 4 represents PEG-Hb-CO at the increasing concentrations were analyzed in an identical manner as controls normalizing the final mean fluorescence intensity (MFI) to isotype and no Ab readings obtained per sample. MFI decrease was observed with increasing concentrations of drug. [0251] Reduction of HIF-1 alpha levels in U266 myeloma cells treated with SG (PEG-Hb- CO). The U266 experimental cell group were cultured for 3 hours in hypoxia (0.5% O2). One another 4.0 mg/mL (hashed bars). A separate cell group was a positive control cultured at normoxia (light gray bar). HIF-1a intracellular protein levels were measured by immunostaining procedures with a fluorescent labeled anti-HIF-1 alpha antibody and quantified by flow cytometry. The values are reported as mean fluorescent units (MFI). Example 5: Determination of radiation sensitivity [0252] Determination of radiation sensitivity of PEG-HB-CO in at least 4 HNSCC cell lines using radiation clonogenic assays at 21% oxygen vs 1% oxygen conditions. Example 6: Animal studies to test in vivo radiosensitization using heterotopic models: [0253] Eight to ten-week-old athymic mice are caged in groups of five or less. Cancer cell lines are injected subcutaneously into the left flank of each mouse with 1-2 × 106 cells suspended in 100 ul Matrigel/PBS mixture. For each xenograft model, mice are randomly divided into four groups treated with different regimens: Vehicle, Radiation + Vehicle, , and Radiation +PEG- HB-CO. Treatment regimens are started once tumors reach ~150-200mm3 in size (to ensure some level of hypoxia), usually about 2-4 weeks post-injection. PEG-HB-CO are administered to mice by IV (tail vein) twice per week (e.g. Monday and Thursday). Using a custom shielding apparatus to block non-targeted areas, 4 Gy of radiation are administered directly to tumors once daily for up to 5 consecutive days. For combination treatment, mice are treated with Attorney Docket No. PLG-021WO radiation 2-3 hours after PEG-HB-CO delivery. Mice are observed for a minimum of 30 days after treatment is complete. Example 7: An efficacy evaluation of PP-007 as monotherapy and in combination with focal beam radiation in the established orthotopic murine glioma GL261-luc2 tumor model in albino female C57BL/6 mice. Materials and Methods Animals and Husbandry Attorney Docket No. PLG-021WO Cell Preparation/Implantation Orthotopic Brain Implantation [0254] Prior to implant, the hair over the incision site is shaved using electric clippers. Approximately 30-minutes prior to surgery, the mice are dosed subcutaneously with 5 mg/kg carprofen. For surgical implantation preparation, mice are anesthetized using 2% isoflurane in air. The mice are then secured in a stereotaxic frame and ocular ointment is applied to the eyes of the mice to prevent drying during surgery. A re-circulating 37°C water heated pad is used to maintain the animal’s body temperature during the implant procedure. [0255] Once in the stereotaxic frame, the cranium is disinfected by alternating chlorhexidine solution and 70% ethanol-saturated swabs in preparation for incision. A median incision of ~1 Attorney Docket No. PLG-021WO cm was made through the skin and the skull is exposed. A burr hole was drilled into the skull over the right striatum. The needle was then lowered 3 mm into the brain and retracted 1 mm to form a “reservoir” for the deposition of the cell suspension. The cell suspension was then injected slowly into the brain tissue with any slight leakage being absorbed with a dry cotton swab. Following injection, the burr hole was sealed with bone wax and the incision closed. Following implantation, the mice were allowed to fully recover from anesthesia and placed in clean housing cages. Approximately 24-hours post-surgery, a second dose of carprofen was administered. Treatment [0256] All mice were sorted into study groups based on BLI estimation of tumor burden. The mice were distributed to ensure that the mean tumor burden for all groups is within 10% of the overall mean tumor burden for the study population. In vivo SARRP irradiation [0257] The Small Animal Radiation Research Platform (SARRP) has been designed to allow for highly targeted irradiation which mimics that applied in human patients. All treatment plans are optimized prior to study treatment and designed to minimize normal tissue toxicity and Attorney Docket No. PLG-021WO produce a homogeneous dose distribution in the target. Dose distribution maps, dose volume histograms and normal tissue interactions were all considered during the treatment planning process. Mechanical and X-ray systems were precisely calibrated daily, and the dose rate calculated for each individual treatment plan. Imaging In vivo Bioluminescence Imaging (BLI) [0258] Bioluminescence imaging (BLI) of luciferase-expressing tumor cell lines enables a noninvasive determination of site-localized tumor burden. The quantity of emitted light from the tumor after systemic injection of D-luciferin is expected to correlate with viable tumor burden. D-luciferin Attorney Docket No. PLG-021WO Reporting Endpoints Attorney Docket No. PLG-021WO Results Study validation: [0259] The mean estimated tumor burden for all groups in the experiment on the first day of treatment is 2.38E+07 p/s. All animals weighed at least 16.2 g at the initiation of therapy with an overall mean body weight of 18.8 g. Tumor burden and body weights for all groups in the experiment were well-matched (within 10% of overall mean). Tumor growth kinetics and parameters (clinical signs, body weight change, and time on study) in the Control Group (Group 1) are within historical norms. Treatment with vehicle control: [0260] Treatment with vehicle control resulted in a 10.5% mean body weight loss in the treatment window and a 60% incidence of deaths in the treatment window. Treatments within Groups 2-4 were tolerated with maximum body weight loss of 2.2% and a range of deaths in the treatment window of 0-10%. The high incidence of weight loss and death experienced by Group 1 was likely due to disease progression. Treatment with radiation therapy alone: [0261] Single treatment with focal radiation at 7.5 Gy (Group 2) resulted in an increase in time to progression (ITP) of 158.3% and a Day 20 median %T/C value of 1.5%. There was a 50% incidence of partial regression and a 10% incidence of complete regression. There were no tumor free survivors. Treatment with PEG-HB-CO alone [0262] Treatment with PP-007 alone at 1000 mg/kg (Group 3) did not produce anti-cancer activity with an ITP of -16.6% and a Day 20 median %T/C value of 263.9%. There was no incidence of regression in the group. Attorney Docket No. PLG-021WO Combination of focal radiation and PEG-HB-CO [0263] Combination of focal radiation at 7.5 Gy and PEG-HB-CO at 1000 mg/kg (Group 4) produced anti-cancer activity with an ITP of 216.6% and a Day 20 median %T/C value of 2.3%. There was a 40% incidence of partial regressions, and a 20% incidence of complete regression and increases tumor free survivors by at least 50% (Figure 6) when compared to the subject administered with PEG-HB-CO or radiation therapy alone. _{[0264] It is observed through figure 9 when the subject is administered with vehicle control} there is a sharp decrease in the body weight of the subjects. When the subjects in group 2 are treated with radiation therapy alone, the administration does not result in sharp change in the body weight of the subject, representing there is not much % Mean Body Weight change in the group 2 hence the weight is maintained. When subjects in group 3 administered with PEG-HB- CO alone, there is significant change in the weight of the subjects. However, Combination of focal radiation at 7.5 Gy and PEG-HB-CO at 1000 mg/kg (Group 4) shows the weight of the subject is well maintained throughout 45 days, representing there is not much % Mean Body Weight change in the subjects, further representing the weight is well maintained. There was an average >7.5% increase in Group 4 vs. Group 2 showing long-term weight improvements with addition of a single dose of combination therapy comprising PP-007. [0265] Further figure 10 shows Day 75 Scatter Dot Plots of each treatment group plotted through Kaplan-Meier method. Data are plotted for each individual animal across the entire study. Mean values for each group is shown along with their respective 95% Confidence Intervals. Only the group 4 ( PEG-HB-CO + SARRP) Co-treat group showed mean value of 10^8 BLI. Groups 2 (SARRP) and Grp 4 (PEG-HB-CO + SARRP) showed animals with response < 10^6 and <10^4. Dashed lines were included to highlight these cut-off values. Group 4 displayed a larger percentage of animals in the good responder (<10^6) and High responder (<10^4) groups. The comparison of the group 4 to group 2 good responders (n=7 to n=3, respectively) shows a 57% difference in tumor volume with combination treatment (PEG- HB-CO) versus Radiation therapy alone. The similar comparison of group 4 to group 2 for High responders (n=14 to n=5) exhibits a 64% difference with combination treatment (PEG- HB-CO) versus radiation therapy alone. Example 8: Treatment Protocols [0266] Patient A has lung cancer. In week 1, patient A receives an effective amount of radiation therapy in five fractions. In week 1, patient A also receives a therapeutic dose of PP-007 on day 1, day 3. Patient A repeats this schedule for weeks 2, 3, 4, and 5. Attorney Docket No. PLG-021WO [0267] Patient B has breast cancer. In week 1, patient B receives an effective amount of radiation therapy. In week 1, patient B also receives a therapeutic dose of PP-007 on day1 day 3. Patient repeats this schedule for weeks 2, 3, 4 and 5. Treatment protocol: 2 [0268] Patient A has lung cancer. In week 1, patient A receives an effective amount of about 7.5 Gy focal beam radiation therapy. In week 1, patient A also receives a therapeutic dose of PP-007 of 300 mg/kg or 600 mg/kg administered on day 1, day 3. Patient A repeats this schedule for weeks 2, 3, 4, and 5. [0269] Patient B has breast cancer. In week 1, patient B receives an effective amount of about 7.5 Gy focal beam radiation therapy. In week 1, patient B also receives a therapeutic dose of PP-007 of 300 mg/kg or 600 mg/kg on day1 day 3. Patient repeats this schedule for weeks 2, 3, 4 and 5. Treatment protocol: 3 [0270] Patient A has lung cancer. In week 1, patient A receives an effective amount of about 7.5 Gy focal beam radiation therapy. In week 1, patient A also receives a therapeutic dose of PP-007 of 300 mg/kg or 600 mg/kg administered in the subject on day 1, day 3. Patient A repeats this schedule for weeks 2, 3, 4, and 5. wherein PP-007 is administered in the subject in one or more dosing cycle, wherein the first dose of PP-007 has higher flow rate than the second cycle and the first dose has lower amount of PP-007 than second cycle. [0271] Patient B has breast cancer. In week 1, patient B receives an effective amount of about 7.5 Gy focal beam radiation therapy. In week 1, patient B also receives a therapeutic dose of PP-007 of 300 mg/kg or 600 mg/kg on day1 day 3. Patient repeats this schedule for weeks 2, 3, 4 and 5. wherein PP-007 is administered in the subject in one or more dosing cycle, wherein the first dose of PP-007 has higher flow rate than the second cycle and the first dose have lower amount of PP-007 than second cycle. EMBODIMENTS [0272] The following Embodiments provide certain embodiments disclosed herein. Embodiment 1. A method of treating a cancer in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated H_{B-CO (PEG-HB-} b. treating the subject Attorney Docket No. PLG-021WO wherein the PEG-HB-CO is administered simultaneously or sequentially or in any order before wherein the radiation therapy does not wherein the radiation therapy is selected from focal beam radiation therapy and/or stereotactic wherein the method improves the cancer treatment. Embodiment 2. The method as disclosed in Embodiment 1, wherein the cancer treatment is improved by inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a) and/or, improving one or more incidence of tumor-free survivors and increase in time to progression (ITP). Embodiment 3. A method of reducing the size and/or volume of a tumor in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated H_{B-CO (PEG-HB-} b. treating the subject wherein the administration of PEG-HB-CO and radiation therapy to the subject exhibits s improvement in tumor volume reduction. Embodiment 4. The method as disclosed in Embodiment 3, wherein reducing the size and/or volume of a tumor comprising inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a). Embodiment 5. A method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a_{. administering a dose of PEG-HB-} b. Attorney Docket No. PLG-021WO wherein the administration of PEG-HB-CO and radiation therapy to the subject improves tumor-free survivors and/or improves anticancer activity. Embodiment 6. The method as disclosed in Embodiment 5, wherein tumor free survivors improves increase in time to progression (ITP) and/or inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a). Embodiment 7. A method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. treating the subject and wherein the cancer treatment is improved by inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a) and/or improving one or more incidence of tumor-free survivors, increase in time to progression (ITP). Embodiment 8. The method as disclosed in any of the preceding Embodiment, wherein the method improves the incidence of partial tumor regression is in the range of about 30% to about 50%, or in the range of about 30% to about 40%. Embodiment 9. The method as disclosed in Embodiment 8, wherein the partial tumor regression is in the range of about 30% to about 40%. Embodiment 10. The method as disclosed in Embodiment 9, wherein the method improves the incidence partial tumor regression by 40%. Embodiment 11. The method as disclosed in preceding Embodiment, wherein the method improves the incidence of complete tumor regression in the range of about 15% to about 40%, or about 20% to about 30%. Embodiment 12. The method as disclosed in Embodiment 11, wherein the method improves the incidence of complete tumor regression in the range of about 20% to about 30%. Attorney Docket No. PLG-021WO Embodiment 13. The method as disclosed in Embodiment 12, wherein the method improves the incidence of complete tumor regression by about 20%. Embodiment 14. The method as disclosed in preceding Embodiment, wherein the administration of PEG-HB-CO and radiation therapy increases the tumor-free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. Embodiment 15. The method as disclosed in preceding Embodiments, wherein the subject administered with combination therapy of the PEG-HB-CO and the radiation therapy exhibits at least 50% to about 70% improvement in reduction of tumor volume compared to the subject treated either with PEG-HB-CO or radiation therapy alone. Embodiment 16. The method as disclosed in Embodiment 15, wherein the subject administered with combination therapy of the PEG-HB-CO and the radiation therapy exhibits at least 55% to about 65% improvement in reduction of tumor volume compared to the subject treated either with PEG-HB-CO or radiation therapy alone. Embodiment 17. The method as disclosed in Embodiment 16, wherein the subject administered with combination therapy of the PEG-HB-CO and the radiation therapy exhibits at least 57% to about 64% improvement in reduction of tumor volume compared to the subject treated either with PEG-HB-CO or radiation therapy alone. Embodiment 18. The method as disclosed in any preceding Embodiment, wherein the method reduces or alleviates hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression. Embodiment 19. The method as disclosed in any preceding Embodiment, wherein the hypoxia- inducible factor 1-alpha (HIF-1a) mediated gene expression provides an EC50 value in the range of about 0.5 mg/ml to about 2.0 mg/ml, or in the range of about 0.5 mg/ml to about 1.0 mg/ml. Embodiment 20. The method as disclosed in Embodiment 19, wherein the hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression provides an EC50 value of about 0.52 mg/ml. Embodiment 21. The method as disclosed in any preceding Embodiment, wherein the method improves the increases in time to progression (ITP) about 160%, about 200% and about 216%. Attorney Docket No. PLG-021WO Embodiment 22. The method as disclosed in any preceding Embodiment, wherein the method improves the increase in time to progression (ITP) at least by 10% to 75 % or at least about 10% to about 50% or about 50% to about 75% compared to the increase in time to progression (ITP) when the subject treated either with PEG-HB-CO or radiation therapy alone. Embodiment 23. The method as disclosed in any preceding Embodiment, wherein the subject is selected from animal model suitable for cancer research, and human suffering from cancer. Embodiment 24. The method as disclosed in Embodiment 23, wherein the animal model suitable for cancer research is selected from guinea pig, mouse and rat. Embodiment 25. The method as disclosed in any preceding Embodiment, wherein the method alleviates at least one symptom associated with tumor and/or cancer treatment selected from lung cancer, breast cancer, melanoma, renal cell carcinoma, squamous cell non-small cell lung cancer (NSCLC), non-squamous NSCLC, colon-rectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, head and neck. Embodiment 26. The method as disclosed in Embodiment 25, wherein the method treats lung cancer, breast cancer, head and neck. Embodiment 27. The method as disclosed in any preceding Embodiment, wherein the method reduces or alleviate HIF-1a mediated gene expression in suitable cell line selected from the group comprising, A549 lung cancer cell line, MCF7 breast cancer cell line, and human HNSCC cell lines. Embodiment 28. The method as disclosed in any preceding Embodiment, wherein the therapeutic effective amount PEG-HB-CO is administered in subject intravenously and/or subcutaneously at a dose selected in the range of about 300 mg/kg, to about 1500 mg/kg. Embodiment 29. The method as disclosed in Embodiment 28, wherein the therapeutic effective amount PEG-HB-CO is administered in subject intravenously and/or subcutaneously at a dose selected in the range of about 500 mg/kg, to about 1200 mg/kg. Embodiment 30. The method as disclosed in Embodiment 29, wherein the therapeutic effective amount PEG-HB-CO is administered in subject intravenously and/or subcutaneously at a dose Attorney Docket No. PLG-021WO wherein the PEG-HB-CO is administered in the subject intravenously and/or subcutaneously at a dose of about 1000 mg/kg. Embodiment 31. The method as disclosed in any preceding Embodiment, wherein the radiation therapy is focal beam radiation therapy. Embodiment 32. The method as disclosed in Embodiment 31, wherein the subject is treated with focal radiation of about 5 Gy to about 10 Gy. Embodiment 33. The method as disclosed in Embodiment 32, wherein the subject is treated with focal radiation of about 7.5 Gy. Embodiment 34. The method as disclosed in any preceding Embodiment, wherein the subject is administered with PEG-HB-CO and simultaneously treated with radiation therapy. Embodiment 35. The method as disclosed in any preceding Embodiment, wherein the subject is administered with PEG-HB-CO and sequentially treated with radiation therapy PEG-HB-CO is administered in any order, before or after treating the subject with radiation therapy. Embodiment 36. The method as disclosed in any preceding Embodiment, wherein the reduction of tumor improves the increase in time to progression (ITP) at least about 73% compared to the subject treated either with PEG-HB-CO or radiation therapy alone. Embodiment 38. The method as disclosed in any preceding Embodiment, wherein the treatment maintains the body weight of the subject during the treatment compared to the subject administered with PEG-HB-CO or radiation therapy alone. Embodiment 39. The method as disclosed in any preceding Embodiment, wherein the PEG- HB-CO is administered in dose dependent manner, wherein PEG-HB-CO dose is dependent on percentage of tumor hypoxia. Embodiment 40. The method as disclosed in Embodiment 38, wherein the PEG-HB-CO dose in the dose dependent manner are selected from about 0.1 mg/mL, to about 11 mg/mL. Embodiment 41. The method as disclosed in any preceding Embodiment, wherein the treatment is performed in at least one cycle. Attorney Docket No. PLG-021WO Embodiment 42. The method as disclosed in Embodiment 40, wherein the treatment is performed in more than one cycle. Embodiment 43. A method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. treating the subject wherein the administration of PEG-HB-CO is performed simultaneously, sequentially, or in wherein wherein the reduction of tumor improves the increase in time to progression (ITP) at least by 10% to 75 % or at least about 10% to about 50% or about 50% to about 75% compared to the increase in time to progression (ITP) when the subject is treated either with PEG-HB-CO or radiation therapy alone. Embodiment 44. The method disclosed in any of the preceding Embodiments, wherein the composition of PEG-HB-CO comprises bovine hemoglobin and poly(ethylene glycol), wherein bovine hemoglobin is bound to carbon monoxide, wherein the composition comprises stabilizing agent. Embodiment 45. A method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the combination therapy of the PEG-HB-CO and the radiation therapy inhibits growth of the tumor in the subject compared to the subject treated either with PEG-HB-CO or radiation wherein the method exhibits s at least 50% to about 70% improvement in reduction of tumor _{volume compared to the subject treated either with PEG-HB-} Attorney Docket No. PLG-021WO wherein the method reduces or alleviates hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, providing an EC50 value in the range of about 0.5 mg/ml to about 2.0 mg/ml, wherein the method improves the incidence of partial tumor regression in the range of about wherein the method improves the incidence of complete tumor regression in the range of about wherein wherein the administration of PEG-HB-CO and radiation therapy increases the tumor-free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG- HB-CO or radiation therapy alone. Embodiment 46. The method as Embodiment in any preceding Embodiments, wherein the method improves the cancer treatment. Embodiment 47. A method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, the method comprising: a_{. culturing the cells capable to express hypoxia-inducible factor 1-alpha (HIF-} b. c_{. treating with PEGylated HB-CO (PEG-HB-} d. incubating the assay plate e. determining the HIF- reduction in HIF-1a or f. collect the sample from step (d) in suitable apparatus and treated with fixation buffer g. h. separating the supernatant in suitable apparatus i. j. k. Treating the sample of step (j) with Phycoerythrin conjugated mouse anti-human H_IF- l. Incubating for suitable time Attorney Docket No. PLG-021WO m. Analyze sample to read phycoerythrin fluorescent. Embodiment 48. A method as disclosed in Embodiment 48, wherein the cell line is A549 or MCF7. Embodiment 49. A method as disclosed in Embodiment 48, wherein the PEGylated HB-CO (PEG-HB-CO) is added in 1:10 dilution. Embodiment 50. A method as disclosed in Embodiment 48, wherein the assay plate is incubated for suitable time for suitable period in normoxia or hypoxia. Embodiment 51. A method as disclosed in Embodiment 48, wherein the assay plate is incubated for suitable time for suitable period in normoxia Embodiment 52. A method as disclosed in Embodiment 48, wherein the assay plate is incubated at temperature 30⁰C to 40⁰C. Embodiment 53. A method as disclosed in Embodiment 48, wherein the assay plate is incubated Embodiment 54. A method as disclosed in Embodiment 48, wherein the determination of the HIF-1a expression is performed by the Luciferase assay. Embodiment 55. A method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, the method comprising: a_{. culturing the cells capable to express hypoxia-inducible factor 1-alpha (HIF-} b. c_{. treating with PEGylated HB-CO (PEG-HB-} d. incubating the assay plate e. collect the sample from step (d) in suitable apparatus and treated with fixation buffer f. g. h. i. j. treating the sample of step (i) with Phycoerythrin conjugated mouse anti-human H_IF- k. l. analyzing the sample to read phycoerythrin fluorescent. Embodiment 56. The method as disclosed in Embodiment 55, wherein the PEGylated HB-CO (PEG-HB-CO) is added in 4.0, 0.4 mg/ml PEG-HB-CO or media diluent. Attorney Docket No. PLG-021WO Embodiment 57. The method disclosed in Embodiment 55, wherein the suitable apparatus is microfuge tube. Embodiment 58. The method disclosed in Embodiment 55, wherein the incubation time in step (f) is 25⁰C to 40⁰C for 5 minutes to 30 minutes. Embodiment 59. The method as disclosed in Embodiment 55, wherein the incubation time in step (f) is 37⁰C for 10 minutes. Embodiment 60. The method as disclosed in Embodiment 55, wherein the incubation time in step (h) and (k) on ice for 5 minutes to 30 minutes. Embodiment 61. The method as disclosed in Embodiment 55, wherein the incubation time in step (h) on ice for 10 minutes. Embodiment 62. The method as disclosed in Embodiment 55, wherein the cell line is U266 human multiple myeloma cells. BRIEF DESCRIPTION OF THE DRAWINGS: [0273] Figure 1: Relative Luciferace activity on A549 cells. [0274] Figure 2: Dose-Dependent effects of PEGylated bovine hemoglobin in A549 HIF-1a- LUC reporter assay. [0275] Figure 3: Carbon Monoxide Delivery: MCF7 NrF2-LUC assay. [0276] Figure 4: Reduction of HIF-1 alpha levels in U266 myeloma cells treated with SG (PEG-Hb-CO). [0277] Figure 5: Primary BLI Signal by Group with Std. Error by Mean [0278] Figure 6: Primary BLI Signal by Median [0279] Figure 7: % Progression Free Survival by Group [0280] Figure 8: Individual BLI Growth Curves by Group [0281] Figure 9: % Mean Body Weight Change by Group [0282] Figure 10: BLI Representative Images [0283] Figure 11: Day 75 Scatter Dot Plots of each treatment group

Claims

Attorney Docket No. PLG-021WO CLAIMS We Claim: 1. A method of treating a cancer in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated H_{B-CO (PEG-HB-} b. treating the subject wherein the PEG-HB-CO is administered simultaneously or sequentially or in any order before or after the wherein the radiation therapy does not wherein the radiation therapy is selected from focal beam radiation therapy and/or _{stereotactic radiation surgery} wherein the method improves the cancer treatment. 2. The method as claimed in claim 1, wherein the cancer treatment is improved by inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a) and/or improving one or more incidence of tumor-free survivors, increase in time to progression (ITP). 3. A method of reducing the size and/or volume of a tumor in a subject in need thereof, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated H_{B-CO (PEG-HB-} b. treating the subject wherein the administration of PEG-HB-CO and radiation therapy to the subject exhibits improvement in tumor volume reduction. 4. The method as claimed in claim 3, wherein reducing the size and/or volume of a tumor comprising inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a). 5. A method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a_{. administering a dose of PEG-HB-} b. Attorney Docket No. PLG-021WO wherein the administration of PEG-HB-CO and radiation therapy to the subject improves tumor-free survivors and/or improves anticancer activity. 6. The method as claimed in claim 5, wherein tumor free survivors improves increase in time to progression (ITP) and/or inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF-1a). 7. A method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. treating the subject and wherein the cancer treatment is improved by inhibiting tumor growth by improving one or more incidence selected from partial tumor regression, and complete tumor regression, and by reducing tumor volume and hypoxia inducible factor 1 alpha (HIF- 1a) and/or improving one or more incidence of tumor-free survivors, increase in time to progression (ITP). 8. The method as claimed in any of the preceding claims, wherein the method improves the incidence of partial tumor regression is in the range of about 30% to about 50%, or in the range of about 30% to about 40%. 9. The method as claimed in claim 8, wherein the partial tumor regression is in the range of about 30% to about 40%. 10. The method as claimed in claim 9, wherein the method improves the incidence partial tumor regression by 40%. 11. The method as claimed in any of the preceding claims, wherein the method improves the incidence of complete tumor regression in the range of about 15% to about 40%, or about 20% to about 30%. 12. The method as claimed in claim 11, wherein the method improves the incidence of complete tumor regression in the range of about 20% to about 30%. 13. The method as claimed in claim 12, wherein the method improves the incidence of complete tumor regression by about 20%. 14. The method as claimed in any of the preceding claims, wherein the administration of PEG-HB-CO and radiation therapy increases the tumor-free survivors by at least 50% Attorney Docket No. PLG-021WO compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. 15. The method as claimed in any of the preceding claims, wherein the subject is administered with a combination therapy of the PEG-HB-CO and the radiation therapy exhibit at least 50% to about 70% improvement in reduction of tumor volume compared to the subject treated either with PEG-HB-CO or radiation therapy alone. 16. The method as claimed in claim 15, wherein the subject is administered with the combination therapy of the PEG-HB-CO and the radiation therapy exhibit at least 55% to about 65% improvement in reduction of tumor volume compared to the subject treated either with PEG-HB-CO or radiation therapy alone. 17. The method as claimed in claim 16, wherein the subject is administered with the combination therapy of the PEG-HB-CO and the radiation therapy exhibit at least 57% to about 64% improvement in reduction of tumor volume compared to the subject treated either with PEG-HB-CO or radiation therapy alone. 18. The method as claimed in any preceding claim, wherein the method reduces or alleviates hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression. 19. The method as claimed in any preceding claim, wherein the hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression provides an EC50 value in the range of about 0.5 mg/ml to about 2.0 mg/ml, or in the range of about 0.5 mg/ml to about 1.0 mg/ml. 20. The method as claimed in claim 19, wherein the hypoxia-inducible factor 1-alpha (HIF- 1a) mediated gene expression provides an EC50 value of about 0.52 mg/ml. 21. The method as claimed in any preceding claim, wherein the method improves the increases in time to progression (ITP) about 160%, about 200% and about 216%. 22. The method as claimed in any preceding claim, wherein the method improves the increase in time to progression (ITP) at least by 10% to 75 % or at least about 10% to about 50% or about 50% to about 75% compared to the increase in time to progression (ITP) when the subject treated either with PEG-HB-CO or radiation therapy alone. 23. The method as claimed in any preceding claims, wherein the subject is selected from animal model suitable for cancer research, and human suffering from cancer. 24. The method as claimed in claim 23, wherein the animal model suitable for cancer research is selected from guinea pig, mouse and rat. 25. The method as claimed in any preceding claims wherein the method alleviates at least one symptom associated with tumor and/or cancer treatment selected from lung cancer, Attorney Docket No. PLG-021WO breast cancer, melanoma, renal cell carcinoma, squamous cell non-small cell lung cancer (NSCLC), non-squamous NSCLC, colon-rectal cancer, castration-resistant prostate cancer, ovarian cancer, gastric cancer, hepatocellular carcinoma, pancreatic carcinoma, head and neck. 26. The method as claimed in claim 25, wherein the method treats lung cancer, breast cancer, head and neck. 27. The method as claimed in any preceding claims, wherein the method reduces or alleviates HIF-1a mediated gene expression in suitable cell line selected from the group comprising, A549 lung cancer cell line, MCF7 breast cancer cell line, and human HNSCC cell lines. 28. The method as claimed in any preceding claims, wherein the therapeutic effective amount PEG-HB-CO is administered in subject intravenously and/or subcutaneously at a dose selected in the range of about 300 mg/kg, to about 1500 mg/kg. 29. The method as claimed in claim 28, wherein the therapeutic effective amount PEG-HB- CO is administered in subject intravenously and/or subcutaneously at a dose selected in the range of about 500 mg/kg, to about 1200 mg/kg. 30. The method as claimed in claim 29, wherein the therapeutic effective amount PEG-HB- CO is administered in subject intravenously and/or subcutaneously at a dose selected wherein the PEG-HB-CO is administered in the subject intravenously and/or subcutaneously at a dose of about 1000 mg/kg. 31. The method as claimed in any preceding claim, wherein the radiation therapy is focal beam radiation therapy. 32. The method as claimed in any preceding claim, wherein the radiation therapy does not substantially affect the healthy tissues. 33. The method as claimed in claim 32, wherein the subject is treated with focal radiation of about 5 Gy to about 10 Gy. 34. The method as claimed in claim 33, wherein the subject is treated with focal radiation of about 7.5 Gy. 35. The method as claimed any preceding claim, wherein the subject is administered with PEG-HB-CO and simultaneously treated with radiation therapy. 36. The method as claimed in any preceding claim, wherein the subject is administered w_{ith PEG-HB-CO and sequentially treated with radiation therapy PEG-} Attorney Docket No. PLG-021WO HB-CO is administered in any order, before or after treating the subject with radiation therapy. 37. The method as claimed in any preceding claim, wherein the reduction of tumor improves the increase in time to progression (ITP) at least about 73% compared to the subject treated either with PEG-HB-CO or radiation therapy alone. 38. The method as claimed in any preceding claim, wherein the treatment maintains the body weight of the subject during the treatment compared to the subject administered with PEG-HB-CO or radiation therapy alone. 39. The method as claimed in any precedent claim, wherein the PEG-HB-CO is administered in dose dependent manner, wherein PEG-HB-CO dose is dependent on percentage of tumor hypoxia. 40. The method as claimed in claim 39, wherein the PEG-HB-CO dose in the dose dependent manner are selected from about 0.1 mg/mL, to about 1 mg/mL. 41. The method as claimed in any preceding claims, wherein the treatment is performed in at least one cycle. 42. The method as claimed in claim 41, wherein the treatment is performed in more than one cycle. 43. A method of treating and/or alleviating one or more symptoms of cancer, the method comprising the steps: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. treating the subject wherein the administration of PEG-HB-CO is performed simultaneously, sequentially, wherein wherein the reduction of tumor improves the increase in time to progression (ITP) at least by 10% to 75 % or at least about 10% to about 50% or about 50% to about 75% compared to the increase in time to progression (ITP) when the subject is treated either with PEG-HB-CO or radiation therapy alone. 44. The method claimed in any of the preceding claims, wherein the composition of PEG- HB-CO comprises bovine hemoglobin and poly(ethylene glycol), wherein bovine hemoglobin is bound to carbon monoxide, wherein the composition comprises a stabilizing agent. Attorney Docket No. PLG-021WO 45. A method of inhibiting tumor growth in a subject in need thereof, wherein the subject is treated with combination therapy, the method comprising: a. administering to the subject a therapeutically effective amount of PEGylated HB- C_{O (PEG-HB-} b. wherein the combination therapy of the PEG-HB-CO and the radiation therapy inhibits growth of the tumor in the subject compared to the subject treated either with PEG-HB- wherein the method exhibits at least 50% to about 70% improvement in reduction of tumor volume compared to the subject treated either with PEG-HB-CO or radiation wherein the method reduces or alleviates hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, providing an EC50 value in the range of about 0.5 mg/ml to wherein the method improves the incidence of partial tumor regression in the range of wherein the method improves the incidence of complete tumor regression in the range wherein wherein the administration of PEG-HB-CO and radiation therapy increases the tumor- free survivors by at least 50% compared to tumor-free survivors obtained by administering PEG-HB-CO or radiation therapy alone. 46. The method as claimed in any preceding claims, wherein the method improves the cancer treatment. 47. A method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, the method comprising: a_{. culturing the cells capable to express hypoxia-inducible factor 1-alpha (HIF-} b. c_{. treating with PEGylated HB-CO (PEG-HB-} d. incubating the assay plate Attorney Docket No. PLG-021WO e. determining the HIF- reduction in HIF-1a or f. collect the sample from step (d) in suitable apparatus and treated with fixation buffer g. h. separating the supernatant in suitable apparatus i. j. k. Treating the sample of step (j) with Phycoerythrin conjugated mouse anti-human H_IF- l. Incubating for suitable time m. Analyze sample to read phycoerythrin fluorescent. 48. A method as claimed in claim 48, wherein the cell line is A549 or MCF7. 49. A method as claimed in claim 48, wherein the PEGylated HB-CO (PEG-HB-CO) is added in 1:10 dilution. 50. A method as claimed in claim 48, wherein the assay plate is incubated for suitable time for suitable period in normoxia or hypoxia. 51. A method as claimed in claim 48, wherein the assay plate is incubated for suitable time for suitable period in normoxia 52. A method as claimed in claim 48, wherein the assay plate is incubated at temperature 30⁰C to 40⁰C. 53. A method as claimed in claim 48, wherein the assay plate is incubated at least for 3 54. A method as claimed in claim 48, wherein the determination of the HIF-1a expression is performed by the Luciferase assay. 55. A method of treating and/or alleviating hypoxia-inducible factor 1-alpha (HIF-1a) mediated gene expression, the method comprising: a_{. culturing the cells capable to express hypoxia-inducible factor 1-alpha (HIF-} b. c_{. treating with PEGylated HB-CO (PEG-HB-} Attorney Docket No. PLG-021WO d. incubating the assay plate e. collect the sample from step (d) in suitable apparatus and treated with fixation buffer f. g. h. i. j. treating the sample of step (i) with Phycoerythrin conjugated mouse anti-human H_IF- k. l. analyzing the sample to read phycoerythrin fluorescent. 56. The method as claimed in claim 55, wherein the PEGylated HB-CO (PEG-HB-CO) is added in 4.0, 0.4 mg/ml PEG-HB-CO or media diluent. 57. The method as claimed in claim 55, wherein the suitable apparatus is microfuge tube. 58. The method as claimed in claim 55, wherein the incubation time in step (f) is 25⁰C to 40⁰C for 5 minutes to 30 minutes. 59. The method as claimed in claim 55, wherein the incubation time in step (f) is 37⁰C for 10 minutes. 60. The method as claimed in claim 55, wherein the incubation time in step (h) and (k) on ice for 5 minutes to 30 minutes. 61. The method as claimed in claim 55, wherein the incubation time in step (h) on ice for 10 minutes. 62. The method as claimed in claim 55, wherein the cell line is U266 human multiple myeloma cells.