Compositions and methods for immune-mediated cancer treatment

Abstract

To provide compositions and methods for the treatment of immune-mediated cancer. [Solution] Disclosed herein are methods and compositions for enhancing the immune response to a solid tumor in a subject. In some embodiments, the method includes (a) administering a hypoxia-activated bioreducing agent (HABA) to the subject, (b) inducing hypoxia by (i) administering a hypoxia-inducing agent to the subject, or (ii) forming an embolism in one or more blood vessels supplying a solid tumor, and (c) administering an immune checkpoint inhibitor before, simultaneously with, or following step (b) in an amount effective in enhancing the immune response to the solid tumor compared to the immune response in the absence of the immune checkpoint inhibitor. Kits for use in the disclosed methods are also provided.

Description

[Technical Field]

[0001] cross reference This PCT application claims priority to U.S. Provisional Patent Application No. 62 / 244,457, filed on 21 October 2015. This application is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] Background of the Invention While some patients have been shown to exhibit immunity to cancer based on the detection of tumor-specific cytotoxic T cells, these T cells appear to be shielded from the tumor without providing much therapeutic or prophylactic benefit, even if any. Some explain this inconsistency with the theory that tumors evolve various mechanisms to evade immune attack. For a normal immune response to occur, APCs take up tumor-derived antigens and present those antigens to T cells after priming them to express cytotoxicity against tumor cells carrying the antigens. The interaction between APCs and T cells is regulated by various ligand-receptor interactions, including T cell receptors (TCRs) that recognize tumor-associated antigens and form complexes with MHC molecules, and by the co-activator CD28. CTLA4 acts as a negative regulator of T cells, and CD40 acts as a positive regulator for APCs. PD-1 is another negative regulator of cytotoxic T cells and serves as an immune checkpoint. Tumor cells can express a PD-1 ligand called PD-L1, which activates PD-1 and thus suppresses the antitumor activity of T cells. Tumors can also recruit regulatory T cells, which can suppress cytotoxic T cell activity and protect the tumor from immune attack. Efforts have been made to manipulate the immune signaling response to treat cancer, but the effects on response rates and progression-free survival have much room for improvement. Furthermore, excessive reliance on compounds that weaken immunosuppression has risked shifting the balance of the immune system toward autoimmune disorders. For example, observed adverse effects of nivolumab and pembrolizumab include various immune-related conditions such as hepatitis, pneumonia, hypophysitis, and colitis, indicating that further enhancement of systemic immunity may carry potential risks. [Overview of the project] [Means for solving the problem]

[0003] In consideration of the foregoing, there is a need for improved compositions and strategies for cancer treatment, more specifically for cancer treatment that utilizes the immune response in the subject. This disclosure addresses this need and also provides other advantages. Some of the embodiments disclosed herein provide a method for enhancing the immune response against solid tumors by combining the induction of necrosis in the tumor (increasing the immune system's exposure to tumor antigens) with the administration of immune checkpoint inhibitors.

[0004] In one embodiment, the present disclosure provides a method for enhancing an immune response to a solid tumor in a subject, comprising: (a) administering a hypoxia-activated bioreducing agent (HABA) to the subject; (b) inducing hypoxia by (i) administering a hypoxia-inducing agent to the subject; or (ii) forming an embolism in one or more blood vessels supplying the solid tumor; and (c) administering an immune checkpoint inhibitor before, simultaneously with, or following step (b) in an amount effective in enhancing the immune response to the solid tumor compared to the immune response in the absence of the immune checkpoint inhibitor. In some embodiments, the hypoxia-activated bioreducing agent is tirapazamine. In some embodiments, step (b) comprises administering a hypoxia-inducing agent which is an angiolytic agent. In some embodiments, the angiolytic agent is DMXAA, stilbene, or a stilbene derivative. In some embodiments, the stilbene derivative is selected from the group consisting of combretastatin, combretastatin derivatives, cis-3,4',5-trimethoxy-3'-aminostilbene (stilbene 5c), cis-3,4',5-trimethoxy-3'-hydroxystilbene (stilbene 6c), and morpholino-carbamate derivatives that are prodrugs of stilbene 5c. In some embodiments, step (b) includes administering a hypoxia-inducing agent which is an anti-angiogenic agent. In some embodiments, the hypoxia-activating agent and the hypoxia-inducing agent are administered in amounts effective to induce necrosis of at least 75% of the solid tumors. In some embodiments, step (b) includes embolus formation in one or more blood vessels by administering an embolizing agent. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody. In some embodiments, the monoclonal antibody is selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, MEDI4736, and ipilimumab.In some embodiments, the solid tumor is a tumor selected from the group consisting of hepatocellular carcinoma, cholangiocarcinoma, metastatic colorectal cancer of the liver, lung cancer, breast cancer, colorectal cancer, bladder cancer, head and neck cancer, ovarian cancer, and pancreatic cancer. In some embodiments, step (b) includes arterial embolization. In some embodiments, step (b) is performed after step (a). In some embodiments, step (b) includes administering a hypoxia inducer. In some embodiments, step (c) includes administering the immune checkpoint inhibitor two or more times after step (b). In some embodiments, administration of the immune checkpoint modulator is effective in maintaining the solid tumor to a size of less than 50% of its size before treatment for at least six months. In some embodiments, the combination of administration of the HABA, induction of hypoxia, and administration of the immune checkpoint inhibitor has a synergistic effect in treating proliferation disorders in the subject.

[0005] In one embodiment, the present disclosure provides a kit for use in enhancing the immune response to solid tumors in a subject. In some embodiments, the kit comprises (a) a hypoxia-activated bioreducing agent (HABA), (b) a hypoxia-inducing agent or embolizer, and (c) an immune checkpoint inhibitor. In some embodiments, the HABA is tirapazamine. In some embodiments, part (b) is a hypoxia-inducing agent that is an angiogenic agent. In some embodiments, the angiogenic agent is DMXAA, stilbene, or a stilbene derivative. In some embodiments, the stilbene derivative is selected from the group consisting of combretastatin, combretastatin derivatives, cis-3,4',5-trimethoxy-3'-aminostilbene (stilbene 5c), cis-3,4',5-trimethoxy-3'-hydroxystilbene (stilbene 6c), and morpholino-carbamate derivatives that are prodrugs of stilbene 5c. In some embodiments, part (b) is a hypoxia-inducing agent that is an anti-angiogenic agent. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4. In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody. In some embodiments, the monoclonal antibody is selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, MEDI4736, and ipilimumab. In certain embodiments, for example, the following are provided: (Item 1) A method for enhancing the immune response to solid tumors in a subject, (a) Administering hypoxia-activated bioreducing agents (HABAs) to the subject, (b) Inducing hypoxia by (i) administering a hypoxia-inducing agent to the subject, or (ii) forming an embolism in one or more blood vessels supplying the solid tumor, and (c) Prior to step (b), simultaneously with or following step (b), administer an immune checkpoint inhibitor in a dose effective in enhancing the immune response to the solid tumor compared to the immune response in the absence of the immune checkpoint inhibitor. A method that includes this. (Item 2) The method according to item 1, wherein the hypoxia-activating bioreducing agent is tirapazamine. (Item 3) The method according to item 1, wherein step (b) includes administering a hypoxia-inducing agent which is a vasoconstrictor. (Item 4) The method according to item 3, wherein the vasoconstrictor is DMXAA, stilbene, or a stilbene derivative. (Item 5) The method according to item 4, wherein the stilbene derivative is selected from the group consisting of combretastatin, combretastatin derivatives, cis-3,4',5-trimethoxy-3'-aminostilbene (stilbene 5c), cis-3,4',5-trimethoxy-3'-hydroxystilbene (stilbene 6c), and morpholino-carbamate derivatives that are prodrugs of stilbene 5c. (Item 6) The method according to item 1, wherein step (b) includes administering a hypoxia-inducing agent which is an anti-angiogenic agent. (Item 7) The method according to item 1, wherein the hypoxia activator and the hypoxia inducer are administered in an amount effective to induce necrosis of at least 75% of the solid tumors. (Item 8) The method according to item 1, wherein step (b) involves forming an embolus in one or more blood vessels by administering an embolic agent. (Item 9) The method according to item 1, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1, or CTLA-4. (Item 10) The method according to item 9, wherein the immune checkpoint inhibitor is a monoclonal antibody. (Item 11) The method according to item 10, wherein the monoclonal antibody is selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, MEDI4736, and ipilimumab. (Item 12) The method according to item 1, wherein the solid tumor is a tumor selected from the group consisting of hepatocellular carcinoma, cholangiocarcinoma, metastatic colorectal cancer of the liver, lung cancer, breast cancer, colorectal cancer, bladder cancer, head and neck cancer, ovarian cancer, and pancreatic cancer. (Item 13) The method according to item 12, wherein step (b) includes arterial embolization. (Item 14) The method according to item 1, wherein step (b) is performed after step (a). (Item 15) The method according to item 14, wherein step (b) includes administering a hypoxia-inducing agent. (Item 16) The method according to item 1, wherein step (c) includes administering the immune checkpoint inhibitor two or more times after step (b). (Item 17) The method according to item 1, wherein administering the immune checkpoint regulator is effective to maintain the solid tumor at a size less than 50% of its pre-treatment size for at least 6 months. (Item 18) The method according to item 1, wherein the combination of administering the HABA, inducing hypoxia, and administering the immune checkpoint inhibitor exhibits a synergistic effect in treating growth disorders in the subject. (Item 19) A kit for use in enhancing the immune response to solid tumors in a subject, comprising: (a) A hypoxia-activated bioreductive agent (HABA); (b) A hypoxia-inducing agent or an embolizing agent; and (c) An immune checkpoint inhibitor A kit comprising the above components. (Item 20) The kit according to item 19, wherein the HABA is tirapazamine. (Item 21) The kit according to item 19, wherein part (b) is a hypoxia-inducing agent that is a vascular disrupting agent. (Item 22) The kit according to item 21, wherein the vascular disrupting agent is DMXAA, stilbene or a stilbene derivative. (Item 23) The kit according to item 22, wherein the stilbene derivative is selected from the group consisting of combretastatin, a combretastatin derivative, cis-3,4’,5-trimethoxy-3’-aminostilbene (stilbene 5c), cis-3,4’,5-trimethoxy-3’-hydroxystilbene (stilbene 6c), and a morpholino-carbamate derivative which is a prodrug of stilbene 5c. (Item 24) The kit according to item 19, wherein part (b) is a hypoxia-inducing agent which is an anti-angiogenic agent. (Item 25) The kit according to item 19, wherein the immune checkpoint inhibitor is an inhibitor of PD-1, PD-L1 or CTLA-4. (Item 26) The kit according to item 25, wherein the immune checkpoint inhibitor is a monoclonal antibody. (Item 27) The kit according to item 26, wherein the monoclonal antibody is selected from the group consisting of nivolumab, pembrolizumab, atezolizumab, MEDI4736 and ipilimumab.

[0006] Incorporation by reference All publications, patents and patent applications cited herein are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of Drawings

[0007] The novel features of the present invention are particularly pointed out in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description which illustrates exemplary embodiments in which the principles of the present invention are utilized, and the accompanying drawings.

[0008] [Figure 1]Figure 1 shows an example diagram illustrating the interactions and regulation between tumor cells, cytotoxic T cells, antigen-presenting cells (APCs), and regulatory T cells. The diagram illustrates how blocking checkpoints PD-1, PD-L1, or CTLA4 can modulate immunosuppressive effects within tumors.

[0009] [Figure 2] Figure 2 illustrates example results of a comparison of left hepatic artery ligation (HAL) with combinations of saline, doxorubicin, or tirapazamine in an HBx transgenic mouse model. HBx transgenic mice carrying tumors were treated with normal saline, tirapazamine (3 mg / kg IV injection via tail vein), or doxorubicin (10 mg / kg), followed by left hepatic artery ligation for 40 minutes. Mouse body weight, bilirubin, and ALT were measured on various days after treatment. Mice were sacrificed on day 7 for histological analysis of hepatocellular carcinoma (HCC) tumors.

[0010] [Figure 3] Figure 3 shows histological images illustrating the tumor necrosis-inducing and inflammatory response-inducing effects of tirapazamine and hepatic artery ligation after HCC treatment. HBx transgenic mice carrying tumors were treated with tirapazamine (3 mg / kg intravenously via tail vein), followed by 40 minutes of left hepatic artery ligation. Mice were sacrificed on day 7 for histological analysis of the HCC tumors. Shown is a composite image of the entire tumor assembled from multiple tissue sections with nearly complete (99%) tumor necrosis. It should also be noted that the edges of the necrotic tumor have a high degree of inflammatory cell infiltration (all are enlarged inset images).

[0011] [Figure 4]Figure 4 shows representative contrast-enhanced MRI scans of two patients who achieved complete response (CR). These patients received tirapazamine at 5 mg / m2 and 10 mg / m2, respectively, followed by arterial embolization. Follow-up MRI scans were performed 6 weeks after the embolization procedure. Dark areas highlighted with arrows are tumors, which do not show evidence of contrast enhancement and are evaluated as complete tumor necrosis or CR.

[0012] [Figure 5] Figure 5 shows tissue images illustrating the tumor necrosis-inducing and inflammatory response-inducing effects of tirapazamine and vasoconstrictors. NCI-H460 cells were subcutaneously injected into BALB / c nude mice to form tumor xenografts. When the tumors grew to a diameter of 10 mm, the mice were treated with tirapazamine (30 mg / kg IP) plus (A) combretastatin A4 (10 mg / kg IV) or (B) DMXAA (20 mg / kg IV) (Chaplin DJ, 2006). The mice were sacrificed 3 weeks after the initial treatment, and the tumors were dissected for H&E staining. Representative histological images are shown, illustrating the area of ​​tumor necrosis and nearby inflammatory infiltration.

[0013] [Figure 6] Figure 6 shows a table describing experimental designs for studies evaluating the pathology and immunohistochemistry of administration of anti-mPD-1, tirapazamine (TPZ), combretastatin A4 phosphate, 5,6-dimethylxantheonone-4-acetic acid (DMXAA), and various combinations thereof, in the treatment of a subcutaneous 3LL syngeneic lung cancer model in C57BL / 6 mice.

[0014] [Figure 7]Figure 7 shows the results of percentage tumor necrosis based on hematoxylin and eosin (H&E) staining of formalin-fixed paraffin-embedded tissue following administration of anti-mPD-1, tirapazamine (TPZ), combretastatin A4 phosphate, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), and various combinations thereof, in a subcutaneous 3LL syngeneic lung cancer model in C57BL / 6 mice. Asterisks (*) indicate statistical significance (P<0.05) compared to the vehicle group, as determined by the Mann-Whitney test.

[0015] [Figure 8] Figure 8 is a table showing the percentage of tumor necrosis in H&E-stained 3LL tumor tissue sections, as depicted in Figure 7. [Modes for carrying out the invention]

[0016] Detailed description of the invention The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which depends in part on how the value was measured or determined, i.e., the limits of the measurement system. For example, “about” could mean a standard deviation of 1 or more than 1 per practice in the art. Alternatively, “about” could mean a range of up to 20%, 10%, 5%, or 1% of a given value. Or, particularly with respect to biological systems or biological processes, the term could mean within one order of magnitude of the value, preferably five times, and more preferably two times. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” should be presumed to mean within an acceptable margin of error for that particular value.

[0017] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, mice, monkeys, humans, farm animals, sport animals, and pets. Tissues, cells, and their offspring obtained in vivo or cultured in vitro are also included.

[0018] The terms “therapeutic agent,” “therapeutic drug,” or “treatment agent” refer to molecules or compounds that are interchangeable and, when administered to a subject, impart some beneficial effect. Beneficial effects include the feasibility of a diagnostic decision; improvement of a disease, symptom, disorder, or condition; reduction or prevention of the onset of a disease, symptom, disorder, or condition; and general countermeasure against a disease, symptom, disorder, or condition.

[0019] As used herein, “treatment,” “to treat,” “to alleviate,” or “to improve” are interchangeable. These terms refer to an approach to obtain beneficial or desired outcomes, including but not limited to therapeutic and / or preventive benefits. Therapeutic benefits mean any therapeutically relevant improvement or effect in one or more diseases, conditions, or symptoms during treatment. Preventive benefits mean that a composition may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more physiological symptoms of a disease, even if the disease, condition, or symptom is not yet present. Typically, preventive benefits include reducing the incidence and / or exacerbation of one or more diseases, conditions, or symptoms during treatment (e.g., between a treated population and an untreated population, or between a treated state and an untreated state in a subject).

[0020] The terms “co-administration,” “administered in combination,” and their grammatical equivalents encompass the administration of two or more drugs to an animal such that both drugs and / or their metabolites are present in the animal at the same time. Co-administration includes the simultaneous administration of separate compositions, the administration of separate compositions at different times (e.g., sequential administration of separate compositions), or the administration of a composition in which both drugs are present.

[0021] The term “effective dose” or “therapeutic effective dose” refers to the amount of a drug sufficient to produce a beneficial or desired effect. The therapeutic effective dose may vary depending on one or more of the following: the subject being treated and the disease state, the subject’s weight and age, the severity of the disease state, and the method of administration, which can be readily determined by those skilled in the art. The effective dose of an active agent may be administered in a single dose or in multiple doses. Ingredients may be described herein as having at least one effective dose, or at least one effective dose such as an amount relevant to a particular target or purpose, for example, any amount described herein.

[0022] A “synergistic” or “synergistic” effect is one in which the effect of one or more of the combination composition is greater than the effect of one or more of the individual components, or greater than the sum of the effects of one or more of the individual components. The synergistic effect may be about 10%, 20%, 30%, 50%, 75%, 100%, 110%, 120%, 150%, 200%, 250%, 350%, or 500%, or greater than about 10%, 20%, 30%, 50%, 75%, 100%, 110%, 120%, 150%, 200%, 250%, 350%, or 500%, or greater than the effect of one of the components alone on a subject, or greater than the additive effect of each component when administered individually. The effect may be any of the measurable effects described herein.

[0023] In one embodiment, the present disclosure provides a method for enhancing the immune response to a solid tumor in a subject. In some embodiments, the method includes (a) administering a hypoxia-activated bioreducing agent (HABA) to the subject; after step (a), (i) administering a hypoxia-inducing agent to the subject; or (ii) inducing hypoxia by embolus formation of one or more blood vessels supplying the solid tumor (wherein HABA and hypoxia are effective in inducing necrosis of the solid tumor); and (c) before step (b), simultaneously with or following step (b), administering an immune checkpoint inhibitor in an amount effective in enhancing the immune response to the solid tumor compared to the immune response in the absence of the immune checkpoint inhibitor.

[0024] Generally, hypoxia-activated bioreducing agents (HABAs) are compounds that are prodrugs inactive in the presence of oxygen and are converted to an active form having increased activity compared to the prodrug form under low-oxygen conditions [e.g., hypoxia]. In some embodiments, the increased activity under low-oxygen conditions is demonstrated by a higher level of tumor cell death within a solid tumor when administration is carried out according to the method herein (e.g., when a localized area of ​​hypoxia is generated in or within a tumor by the method herein) than when the same amount of hypoxia-activated bioreducing agent is administered to a solid tumor or a region containing a solid tumor (where the area of ​​hypoxia is not generated by the method herein). In some embodiments, the increase in activity is at least about 10 times or greater, and the increase can be very large, for example, about 20 to 200 times, or about 50 to 200 times, or 100 to 200 times, or greater. Examples of HABAs include, but are not limited to, tirapazamine, banoxantrone (AQ4N), porphyromycin, apadicon (EO9), 1,2-bis(methylsulfonyl)-1-(2-chloroethyl)-2-[[1-(4-nitrophenyl)ethoxy]carbonyl]hydrazine (KS119), dinitrobenzamide mustard derivatives (e.g., PR104), and 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1, NSC709257). Further examples include, but are not limited to, nitroimidazole, misonidazole, etanidazole, and nimorazole, TG-302, SN30000, mitomycin C (MMC), porphyromycin, RH1, and EO9 (apadicon). In some embodiments, the HABA is tirapazamine. In some embodiments, tirapazamine is structure [ka] It has.

[0025] In some embodiments, the hypoxic region is a region where the oxygen level is less than about 10%, preferably less than about 5%. In some embodiments, the oxygen level in the hypoxic region is about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%. In some embodiments, an oxygen level of about 10% or lower, preferably about 5% or lower, is sufficient to activate a hypoxia-activated bioreducing agent such as tirapazamine to a level that is at least 10 times more active than in its prodrug form. Those skilled in the art will be familiar with the measurement of oxygen levels in biological systems and will also recognize that oxygen measurements can be expressed in "mmHg" (where, for example, 10% O2 is equal to about 76 mmHg, and 1% O2 is equal to about 7.6 mmHg).

[0026] HABAs can be activated by inducing hypoxia, which is advantageously performed after HABA administration. Any of the various strategies can be used to induce a localized hypoxic region within which the bioreducing agent is activated. In some embodiments, this involves one or more blood vessels supplying the target region. By mechanical embolus formation in blood vessels that supply the targeted region This is achieved, for example, by the administration of an embolizing agent or by a device placed through a catheter by an interventional radiologist to mechanically occlude a blood vessel. In some embodiments, hypoxia is induced by arterial embolization. In some embodiments, one or more hypoxia-inducing agents, such as vasolytic agents (VDAs) and / or anti-angiogenic agents (AAAs), are administered locally or systemically to produce a localized hypoxic area in which previously administered or co-administered HABAs are activated.

[0027] In some embodiments, the vasodilator (VDA) is DMXAA, stilbene, or a stilbene derivative. Examples of stilbene derivatives include, but are not limited to, combretastatin, combretastatin derivatives, cis-3,4',5-trimethoxy-3'-aminostilbene (stilbene 5c), cis-3,4',5-trimethoxy-3'-hydroxystilbene (stilbene 6c), and morpholino-carbamate derivatives, which are prodrugs of stilbene 5c. Further VDAs include, but are not limited to, (5S)-5-(acetylamino)-9,10,11-trimethoxy-6,7-dihydro-5H-dibenzo[a,c]cycloheptene-3-yldihydrogen phosphate (ZD6126) and (N-[2-[4-hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide) (E7010 or ABT-751). These compounds selectively induce deep hypoxia in tumors, even when administered systemically. VDA administration can be considered a type of chemical embolus formation, which uses chemical agents to achieve the same goal of selective embolus formation in tumor-containing areas, in contrast to the direct occlusion of blood vessels in standard embolus formation. The combination of VDA with hypoxia-activating bioreducing agents such as tirapazamine is surprisingly more effective in treating malignant solid tumors than would be expected based on the activity of either agent alone. Their activities are synergistic. For example, VDA administered after tirapazamine enables tirapazamine activation, increasing subsequent tumor cell killing by at least 10 times or more compared to the level of tumor cell killing by either agent alone. This strategy can also be further enhanced in the induction of tumor hypoxia and tirapazamine activation when combined with the embolus formation described below.

[0028] In some embodiments, the hypoxia-inducing agent is an anti-angiogenic agent (AAA). Non-limiting examples of AAAs include bevacizumab, itraconazole, carboxamide triazole, TNP-470, CM101, IFNα, IL-12, platelet factor-4, suramin, SU5416, thrombospongin, VEGFR antagonists, angiostatins, endostatins, 2-methoxyestradiol, tecogalan, tetrathiomolybdate, thalidomide, thrombospongin, prolactin, and α v Examples include β3 inhibitors, linomide, tascinimod, ranibizumab, sorafenib, sunitinib, pazopanib, and everolimus. Further AAAs include, but are not limited to, aflibercept, IMC-1C11, batalanib (PTK-87), N-(2,3-dihydro-3,3-dimethyl-1H-indole-6-yl)-2-[(4-pyridinylmethyl)amino]-3-pyridinecarboxamide (AMG706), 3-(4-bromo-2,6-difluorobenzyloxy)-5-[3-(4-pyrroridine-1-yl-butyl)-ureido]-isothiazol-4-carboxylic acid amide (CP-547,632), N-(4-(3-amino-1H-indazole-4-yl)phenyl)-N'-(2-fluoro-5-methylphenyl)urea (ABT-869), and cediranib (AXD-2171). Monoclonal antibodies such as bevacizumab have a half-life of more than 7 days and act by neutralizing the angiogenic factor VEGF. VEGF deficiency ultimately hinders new angiogenesis in tumors and leads to tumor hypoxia due to oxygen consumption within the tumor. Small molecule compounds such as sorafenib and sunitinib have a half-life of less than 24 hours and act by directly inhibiting the kinase activity of the VEGF receptor.

[0029] In some embodiments, a combination of VDA and AAA is used to induce tumor hypoxia. In some embodiments, VDA induces hypoxia and HABA activation by inducing immediate suppression of tumor blood flow. In some embodiments involving the induction of tumor hypoxia, AAA is used to inhibit the tumor's compensatory hypoxic response, such as the production of VEGF or other angiogenic factors that migrate endothelial progenitor cells from the bone marrow to repair the damaged tumor vascular system. The combination of VDA and AAA such as bevacizumab helps to counteract the compensatory effect of VEGF and enhance the effect of VDA in inhibiting repair processes in tumor blood vessels, thereby keeping the tumor hypoxic.

[0030] In some embodiments, various combinations of these methods are also intended (e.g., embolus formation plus one or more hypoxia-inducing agents), with the overall effect being targeted (local delivery of activated bioreducing agents and effective killing of tumor cells within or at the target site without the side effects typically associated with systemic exposure to bioreducing agents). This enhancement also allows for the use of lower doses of agents while maintaining an appropriate and effective level of tumor cell killing, thereby further reducing toxicity. In some embodiments, components of the combination tumor therapies described herein include one or more anti-angiogenic agents (AAAs), one or more vasolytic agents (VDAs), and hypoxia-activated bioreducing agents (HABAs). The combination of AAAs and VDAs, when administered together, induces prolonged hypoxia in tumor cells and exhibits some effectiveness in killing tumors on their own. However, their activity is significantly enhanced in a synergistic manner when they are administered in combination with hypoxia-activated bioreducing agents (HABAs) as described herein. In some embodiments, the method described herein is a method for enhancing the anticancer activity of AAA and / or VDA by co-administering HABA. In some embodiments, the efficacy is further enhanced by the administration of an immune checkpoint inhibitor.

[0031] In one example of an embolization procedure, embolization involves local treatment used in a tumor or in a region containing a tumor supplied by an identifiable arterial branch, such as the hepatic artery supplying hepatocellular carcinoma. This involves injecting a material (e.g., Lipiodol, gel foam, blood clot, specific beads, etc.) to induce occlusion of the arterial branch supplying the tumor-containing region, thereby depriving tumor cells of adequate blood flow and causing them to die. Dissection of the blood supply sources of the region and surrounding normal organs or tissues determines whether the surrounding organs / tissues may suffer significant damage as a result of the lack of blood supply after embolization. For example, a normal liver is supplied by a dual vessel, the hepatic artery and portal vein, and therefore occlusion of the hepatic artery or a branch thereof is possible without resulting in significant damage to the normal liver. This procedure is generally performed by an interventional radiologist, who, under the guidance of a fluoroscopy device, places a catheter in the femoral artery in the groin and advances the tip of the catheter into the hepatic artery branch supplying the tumor. Once the arterial branches supplying the tumor are identified by injecting a contrast agent, an embolizing agent such as Lipiodol or gel foam is injected to occlude the branches.

[0032] In addition to Lipiodol, other embolic agents include gel forms, blood clots, nanoparticles, or any clinically proven mechanical agent capable of achieving the objective of vascular occlusion. Examples include, but are not limited to, the following. The administration of the embolic agent and the hypoxia-activated bioreducing agent (HABA) may be carried out in any preferred manner. For example, the HABA may be administered before the administration of the embolic agent (e.g., about 1 to 120 minutes prior), so that the subsequent administration of the embolic agent "captures" the HABA in the area. Alternatively, the two agents may be administered together (e.g., using a preparation containing the two agents in a mixture). In some embodiments, the dose of HABA administered is in the range of about 1 mg to about 200 mg, preferably about 5 mg to about 80 mg (e.g., tipalazine) for patients treated in this manner, and the dose of embolic agent administered is in the range of about 5 to 40 ml, preferably about 20 to 30 ml, of lipiodol. To ensure the creation of a hypoxic area or state in the embolic area, a sufficient amount of embolic agent is administered under fluoroscopic guidance to achieve complete occlusion of the intended vascular branch. The administration of the embolic agent is usually carried out by intra-arterial injection. Alternatively, embolus formation may be carried out by other means, such as specific beads, to induce occlusion.

[0033] In some embodiments, the combination of HABA and the induction of hypoxia induces necrosis of solid tumors. In some embodiments, the combination induces at least 50%, 75%, 80%, 85%, 95%, or more necrosis of one or more tumors. The degree of necrosis induced may be the degree achieved a predetermined time after the treatment. For example, at least 50% tumor necrosis may be achieved within 1, 2, 3, or 4 weeks or more after the treatment, or within 1, 2, 3, 4, or 5 months or more after the treatment. In some embodiments, the degree of tumor necrosis induced by the treatment is the maximum level of necrosis observed after the treatment compared to the size of the tumor at the start.

[0034] In some embodiments, tumor necrosis induces an immune response in the subject to one or more antigens expressed by the tumor. For example, tumor antigens include alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), BCR-abl, p53, immature laminin receptor, TAG-72, HPV E6, and HPV Examples include, but are not limited to, E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivorbin, BAGE, CAGE, GAGE, SAGE, XAGE, NY-ESO-1 / LAGE, PRAME, SSX-2, Melan-A / MART-1, Gp100 / pmel17, TRP-1, TRP-2, P. polypeptide, MC1R, prostate-specific antigen (PSA), β-catenin, BRCA1, BRCA2, CDK4, CML66, fibronectin, MART-2, or TGRbRII.

[0035] The administration of HABA, induction of hypoxia, and administration of immune checkpoint inhibitors may be sequential or simultaneous. In some embodiments, immune checkpoint inhibitors may be administered following HABA and / or induction of hypoxia. For example, immune checkpoint inhibitors may be administered alone before either HABA administration or induction of hypoxia, or both. In some embodiments, checkpoint inhibitors may be administered, followed by HABA administration, and then induction of hypoxia. Immune checkpoint inhibitors may be administered alone after HABA administration and / or induction of hypoxia. Alternatively, immune checkpoint inhibitors may be administered in combination with HABA and / or induction of hypoxia.

[0036] In some embodiments, immune checkpoint inhibitors are administered before, simultaneously with, or immediately following the induction of hypoxia. When administered before the induction of hypoxia, immune checkpoint inhibitors may be administered approximately 5 minutes to approximately 24 hours before or earlier than the induction of hypoxia. In some embodiments, immune checkpoint inhibitors are administered approximately 5, 10, 15, 20, 25, 30, 45, or 60 minutes before the induction of hypoxia. In some embodiments, immune checkpoint inhibitors are administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 12, 18, or 24 hours before the induction of hypoxia. In some embodiments, immune checkpoint inhibitors are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, or 28 days before or earlier than the induction of hypoxia. In some embodiments, immune checkpoint inhibitors are administered as a single dose before the induction of hypoxia. In some embodiments, immune checkpoint inhibitors are administered multiple times (e.g., 2, 3, 4, 5, 10, 15, 25 times or more) before inducing hypoxia. When administered simultaneously, the two drugs may be administered as a combined composition (e.g., in a single liquid suspension) or as separate compositions administered at approximately the same time, via the same or different routes of administration. When administered following the induction of hypoxia, immune checkpoint inhibitors may be administered approximately 5 to 24 hours or later after the induction of hypoxia. In some embodiments, immune checkpoint inhibitors are administered approximately 5, 10, 15, 20, 25, 30, 45, or 60 minutes after the induction of hypoxia. In some embodiments, immune checkpoint inhibitors are administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 12, 18, or 24 hours after the induction of hypoxia. In some embodiments, immune checkpoint inhibitors are administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days or later after hypoxia is induced. In some embodiments, immune checkpoint inhibitors are administered once after hypoxia is induced. In some embodiments, immune checkpoint inhibitors are administered multiple times after hypoxia is induced (e.g., 2, 3, 4, 5, 10, 15, 25 times or more).

[0037] The administration of immune checkpoint inhibitors may follow a set dosing schedule until a specified endpoint, such as a target therapeutic outcome, is achieved. Examples of dosing schedules include administering immune checkpoint inhibitors once or multiple times per day (e.g., 1, 2, 3, or more times); once or multiple times every 1, 2, 3, 4, 5, 6, 7 days or more; once or multiple times every 1, 2, 3, 4 weeks or more; once or multiple times every 1, 2, 3, 4, 5, 6 months or more; or a combination of these (e.g., in a tapering dosing schedule). In some embodiments, immune checkpoint inhibitors are administered multiple times over several weeks after induction of hypoxia, for example, once, 2, 3, 4, 5, 6, 8, 12, 16, 20, 24, or 52 weeks after induction of hypoxia, at a rate of 1, 2, 3, 4, or more times per day.

[0038] Generally, immune checkpoint inhibitors are drugs that completely or partially reduce, inhibit, interfere with, or modulate one or more immune checkpoint proteins. Immune checkpoint proteins modulate the activation or function of T cells. In some embodiments, immune checkpoint inhibitors increase the activity or function of T cells, including but not limited to helper T cells and cytotoxic T cells. In some embodiments, helper T cells include, but are not limited to, Th1, Th2, and Th17 cells. In some embodiments, immune checkpoint inhibitors increase the activity of cytotoxic T cells and helper T cells while reducing the activity of regulatory T cells to increase the immune response to an antigen. In some embodiments, immune checkpoint inhibitors increase T cell proliferation in response to tumor antigens. In some embodiments, immune checkpoint inhibitors include, but are not limited to, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-22, CCL1, CCL2, CCL3, CCL4, CCL5, CCL9, CCL17, CCL19, CCL22, CXCL9, CXCL10, CXCL12, CXCL13, CXCL16, G-CSF, GM-CSF, IFNb, IFNg, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-23, IL-27, MIF, TGFb, TNFa, VEGF, or oncostatin M, which increase the secretion of cytokines by T cells in response to tumor antigens. In some embodiments, immune checkpoint inhibitors increase the activity of cytotoxic T cells or the killing of tumor cells by releasing cytotoxic substances, including but not limited to perforin, granzyme, and glaurisin.Examples of immune checkpoint protein targets include, but are not limited to, adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B7-H4 (also known as VTCN1), B and T lymphocyte attenuators (BTLA, also known as CD272), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene-3 (LAG3), programmed death-1 (PD-1), programmed death-1 ligands (PD-L1 and PD-L2), T cell immunoglobulin domain and mucin domain 3 (TIM-3), T cell activation V-domain Ig suppressor (VISTA), IL-10, or TGF-beta.

[0039] Figure 1 provides an example diagram illustrating the interactions between tumor cells, cytotoxic T cells, antigen-presenting cells (APCs), and regulatory T cells, as well as the modulation of immune checkpoints using various antibodies. For example, Figure 1 illustrates antibodies targeting immune checkpoint receptors, including CTLA4, PD-1, and PD-L1. Anti-PD-1 antibodies and anti-PD-L1 antibodies block the interaction between PD-1 and PD-L1. Blocking these antibodies may enhance the activity of cytotoxic T cells. Administration of anti-PD-L1 or anti-PD-1 to tumor cells may interfere with immune evasion and increase immune-mediated antitumor activity. Anti-CTLA4 antibodies, also depicted, may enhance the immune-mediated cytotoxicity of active cytotoxic T cells by blocking the immunosuppressive effects of CTLA4 and potentially enhancing T cell proliferation. Such antibodies may reduce the activity of regulatory T cells.

[0040] Any of the various immune checkpoint inhibitors may be advantageously utilized. Inhibitors can be small molecules, inhibitory polypeptides (e.g., those found in natural or truncated ligands or receptors), aptamers, or antibodies. In some embodiments, the inhibitor is an antibody, or its antigen-binding fragment that binds to an immune checkpoint protein. In some embodiments, the antibody is a full-length antibody comprising two heavy chain sequences and two light chain sequences. In some embodiments, the antibody is an IgM, IgG, IgE, IgA, or IgD isotype. In some embodiments, the antibody is an IgG subtype, e.g., IgG1, IgG2, IgG3, or IgG4 subtype. In some embodiments, the antibody is a heavy chain, a light chain, or at least one heavy chain and at least one light chain. In some embodiments, the antibody is an antibody fragment, e.g., Fab, Fab' or Fab'2, Fv, Fd, single-chain Fv (scFv), disulfide-linked Fvs (ddFv), V L , V H Camel Ig, V-NAR, VHH, Triple specificity (Fab3), Bispecificity (Fab2), Diabody ((V L -V H )2 or (V H -V L )2), Tribody (trivalent), Tetrabody (tetravalent), Minibody ((scFv-CH3)2), Bispecific Single-Chain Fv (Bis-scFv), IgG Delta C H 2. scFV-Fc or (scFV)2-Fc, fragment or single-chain antibody. In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, atezolizumab, MEDI4736, lambrolizumab, MPDL3280A, BMS-936559, BMS-936558 / MDX-1106, CT-011, pizilizumab, galiximab, AMP-514, MEDI4736, MK-3475, MPDL3280A, IMP321, BMS-986016, IPH2101, MSB0010718C, AUNP12, tremelimumab, and ipilimumab.

[0041] In some embodiments, the immune checkpoint inhibitor is a monoclonal antibody derived from a human or non-human animal (e.g., mouse or rat), where the monoclonal antibody is capable of specifically binding to an immune checkpoint protein. Various methodologies are available for preparing monoclonal antibodies. Generally, monoclonal antibodies are obtained from or derived from a single clone, including any eukaryotic clone, prokaryotic clone, or phage clone. In some embodiments, an antibody comprising one or more heavy and / or light chains derived from a non-human antibody in addition to a portion not derived from the source of the non-human antibody (e.g., in a chimeric antibody or humanized antibody) (a) is capable of competing for binding, (b) retains functional properties, (c) binds to the same epitope, and / or (d) has a binding affinity similar to the corresponding parent non-human antibody.

[0042] In some embodiments, monoclonal antibodies comprising one or more heavy chains and / or light chains derived from human antibodies are provided. Human antibodies include antibodies having a complete human amino acid sequence, such as a variable region and a human constant region of the human heavy and light chains. Antibodies that are non-human antibodies can be made fully human antibodies by replacing non-human amino acid residues with amino acid residues present in human antibodies. Thus, human antibodies include antibodies in which one or more human or non-human amino acid residues are replaced with one or more amino acids present in any other human antibody.

[0043] In some embodiments, antibodies obtained from non-human animals can be humanized. In some embodiments, the humanized antibody has non-human amino acid residues, such as mouse, rat, goat, or rabbit amino acid residues in one or more complementarity-determining regions (CDRs) that specifically bind to a desired antigen in the acceptor immunoglobulin molecule and to one or more human amino acid residues in the Fv framework region (FR), which are amino acid residues adjacent to the CDR. In some embodiments, the humanized antibody is created by inserting a suitable CDR segment from a non-human antibody into a human antibody scaffold, where the humanized antibody is capable of specifically binding to an immune checkpoint inhibitor. In some embodiments, the humanized monoclonal antibody includes one or more heavy chain and / or light chain CDR domains from a first non-human antibody, which are used to replace one or more heavy chain and / or light chain CDR domains from a second human antibody. In some embodiments, the first antibody is obtained from a non-human animal and is capable of specifically binding to an immune checkpoint protein. The CDR domain from the first antibody replaces the CDR domain in the second human antibody, where the second human antibody lacks binding specificity to the immune checkpoint protein. This process generates a humanized monoclonal antibody that can specifically bind to the immune checkpoint protein due to the addition of the CDR domain from the first antibody, and the remaining antibody domain is not recognized as foreign by the immune system because it is derived from the human antibody. In some embodiments, one or more heavy CDR domains from the first antibody are added. Humanized antibodies containing chain and / or light chain CDR domains (a) are capable of competing for binding, (b) retain functional properties, (c) bind to the same epitope, and / or (d) have a binding affinity similar to the corresponding first antibody.

[0044] Hypoxia-activated bioreducing agents, hypoxia-inducing agents, and immune checkpoint inhibitors can be formulated as pharmaceutical compositions for administration to subjects by any preferred route of administration. Examples of routes of administration include parenteral (including subcutaneous, intravenous, intra-arterial, intraosseous, intracerebral, intraventricular, intrathecal, intramedullary, intra-articular, intramuscular, or intraperitoneal injection), rectal, topical, and percutaneous. Oral administration (e.g., in capsules, suspensions, or tablets) is also possible, but is not limited to these. Pharmaceutical compositions generally include one or more active agents and one or more pharmaceutically acceptable excipients (solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersions and suspension media, coatings, isotonic agents and absorption enhancers or retarders, but is not limited to these). Aqueous or non-aqueous solvents, solutions and suspensions may also include suspensions and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powders, granules and crystals. Auxiliary active compounds (e.g., preservatives, antimicrobial agents, antiviral agents and antifungal agents) may also be incorporated into the composition.

[0045] Non-limiting examples of cosolvents include hydroxyl groups or other polar groups, e.g., alcohols, e.g., isopropyl alcohol; glycols, e.g., propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers; glycerol; polyoxyethylene alcohol and polyoxyethylene fatty acid esters.

[0046] Auxiliary compounds (e.g., preservatives, antioxidants; biocides such as antimicrobial agents, antiviral agents, and antifungal agents, and antimicrobial agents including biostats) may also be incorporated into the composition. Therefore, a pharmaceutical composition may contain preservatives, antioxidants, and antimicrobial agents. Preservatives may be used to inhibit microbial growth or increase the stability of components, thereby extending the shelf life of the pharmaceutical formulation. Suitable preservatives are known in the art and include, for example, EDTA, EGTA, benzalkonium chloride, or benzoic acid or benzoate, such as sodium benzoate. Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherol, and similar vitamins or provitamins. Antimicrobial agents or antimicrobial compounds directly or indirectly inhibit, reduce, delay, stop, remove, halt, suppress, or prevent contamination by pathogenic or non-pathogenic microbial organisms, or the growth, infectivity, replication, proliferation, or regeneration of pathogenic or non-pathogenic microbial organisms. The classes of antimicrobial agents include antibacterial agents, antiviral agents, antifungal agents, and anthelmintic agents. Antimicrobial agents include drugs and compounds that kill or destroy microbial organisms (cidal), or that inhibit contamination by microbial organisms or the growth, infectivity, replication, proliferation, and regeneration of microbial organisms (static).

[0047] In some embodiments, one or more of the treatment steps may be repeated. For example, the steps of administering an HABA, inducing hypoxia, and administering an immune checkpoint inhibitor may be repeated together once or more times in a single course over a course of treatment. In some embodiments, one step may be repeated in the absence of other steps. For example, after administering an HABA and inducing hypoxia, an immune checkpoint inhibitor may be administered two or more times according to a dosing schedule over a course of treatment. In some embodiments, after administering an HABA and inducing hypoxia, an immune checkpoint inhibitor may be administered three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, fourteen, fifteen, twenty or more times according to a dosing schedule over a course of treatment. In some embodiments, before administering the HABA and inducing hypoxia, an immune checkpoint inhibitor is administered 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 times or more, according to a dosing schedule over the course of treatment. In some embodiments, after administering the HABA and inducing hypoxia, an immune checkpoint inhibitor is administered hourly, daily, weekly, or monthly, according to a dosing schedule over the course of treatment.

[0048] In some embodiments, the methods, compositions, and kits of this disclosure are useful in treating proliferative disorders of subjects, particularly those comprising one or more solid tumors. Examples of proliferative disorders include acanthoma, acinar cell carcinoma, acoustic neuroma, acral lentiginous melanoma, hidradenoma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, mature acute myeloblastic leukemia, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, invasive NK-cell leukemia, and AIDS-related conditions. Cancer, AIDS-associated lymphoma, hydatidiform soft part sarcoma, ameloblastoma, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid cancer, angioimmunoblastic T-cell lymphoma, angiomyolipoma, angiosarcoma, appendiceal cancer, astrocytoma, atypical teratomatoid rhabdomyosarcoma-like tumor, basal cell carcinoma, basaloid carcinoma, B-cell leukemia, B-cell lymphoma, Bellini ductal carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor , bronchioloalveolar carcinoma, Brown tumor, Burkitt lymphoma, cancer of unknown primary site, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, carcinoma of unknown primary site, carcinosarcoma, Castleman disease, central nervous system germ cell tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chordoma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon Hmm, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, fibrinogenic small round cell tumor, diffuse large B-cell lymphoma, germinal dysplastic neuroepithelial tumor, embryonic carcinoma, endodermal sinus tumor, endometrial cancer, endometrial uterine cancer, endometrial tumor, enteropathy-associated T-cell lymphoma, ependymoblastoma, ependymoma, epithelioid sarcoma, erythroleukemia, esophageal cancer, sensory neuroblastoma, Ewing family tumor, Ewing family sarcoma, Ewing's sarcomaSarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic cholangiocarcinoma, extramammary Paget's disease, fallopian tube cancer, fetal fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, ganglioglioma, gangliocytoma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, germ cell tumor, gestational trophoblastic carcinoma, gestational trophoblastic tumor, bone tumor Cellular tumors, glioblastoma multiforme, glioma, cerebral gliomatosis, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, heart cancer, hemangioblastoma, hemangioepidermal cell tumor, angiosarcoma, hematological malignancies, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast-ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammation Symptomatic breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi's sarcoma, Kaposi's sarcoma, kidney cancer, Kratzkin's tumor, Krukenberg's tumor, laryngeal cancer, malignant lentigo melanoma, leukemia, oral cancer, liposarcoma, lung cancer, luteal malformation, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma, macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous Histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant Triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, mediastinal tumor, medullary thyroid carcinoma, medulloblastoma, medullary epithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin (Metastatic Squamous Neck Cancer with Occult Primary), metastatic urothelial carcinoma, metastatic colon Rectal cancer, Müllerian mixed tumor, monocytic leukemia, oral cancer, myxoid neoplasm, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, myelodysplastic disorder, myelodysplastic syndrome, myeloid leukemia, myelosarcoma, myeloproliferative disorder, myxoma, nasal cavity cancer, nasopharyngeal cancer, neoplasm, schwannoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, ocular oncology, oligoastrocytoma, oligodendroglioma, oncocytoma, optic nerve meningioma, oral cancer, oral cancer cancer), oropharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, low-grade ovarian tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, penile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, polygermoma, progenitor T lymphoblastic lymphoma, primary central nervous system lymphoma, Primary exudative lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, pseudomyxoma peritonei, rectal cancer, renal cell carcinoma, airway cancer containing the NUT gene on chlorosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannomatosis, sebaceous gland carcinoma, secondary neoplasm, seminoma, serous neoplasm, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, signet ring cell carcinoma, skin cancer, small blue round cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue Sarcoma, somatostatin-producing tumors, sooty vegetative warts, spinal cord tumors, vertebral tumors (Spinal tumors), spleen Perivisceral zone lymphoma, squamous cell carcinoma, gastric cancer, superficial spreading melanoma, supratentorial primitive neuroectodermal tumor, surface epithelial stromal tumor, synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prelymphocytic leukemia, teratoma, terminal lymphatic cancer, testicular cancer, theca cell tumor, throat cancer Examples of cancers treated include, but are not limited to, thymic carcinoma, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urachal carcinoma, urethral cancer, urogenital neoplasms, uterine sarcoma, uveal melanoma, vaginal cancer, Berner-Morrison syndrome, verrucous carcinoma, optic tract glioma, vulvar cancer, Waldenström macroglobulinemia, Warsin tumor, Wilms tumor, or any combination thereof. In some embodiments, solid tumors are treated. Examples of solid tumors include, but are not limited to, lung cancer, breast cancer, colorectal cancer, bladder cancer, head and neck cancer, ovarian cancer, and pancreatic cancer. In some embodiments, a combination of HABA administration and induction of hypoxia and administration of immune checkpoint inhibitors is synergistic in treating proliferative disorders in the subject.

[0049] The effectiveness of the treatment of cancer can be measured, in particular, by any preferred metric. In some embodiments, therapeutic effectiveness is measured based on the effect of treating proliferative disorders such as cancer. Generally, with respect to the treatment of proliferative disorders (e.g., cancer, whether benign or malignant), the therapeutic effectiveness of the methods and compositions of the present invention can be measured by the extent to which the methods and compositions promote inhibition of tumor cell proliferation, inhibition of tumor angiogenesis, eradication of tumor cells, and / or reduction in the size of at least one tumor to which a person is treated for a proliferative disorder. Several parameters to be considered in determining therapeutic effectiveness are discussed herein. Appropriate combinations of parameters for a particular situation can be established by a clinician. The progress of the methods of the present invention in treating cancer (e.g., reduction in tumor size, or eradication of cancer cells) can be verified using any preferred method, e.g., methods currently used in clinics to track tumor size and cancer progression. In some embodiments, the primary effectiveness parameter used to evaluate the treatment of cancer is preferably reduction in tumor size. The size of the tumor may be determined using any preferred technique, such as dimensional measurement, or by using available computer software, such as FreeFlight software developed at Wake Forest University, which allows for accurate estimation of tumor volume. The size of the tumor may also be determined by visualization of the tumor, such as using CT, ultrasound, SPECT, spiral CT, MRI, or photography. In embodiments where the tumor is surgically removed after the completion of the treatment period, the presence and size of the tumor tissue may be determined by gross analysis of the tissue to be removed and / or by pathological analysis of the tissue removed.

[0050] Preferably, tumor growth stabilizes as a result of the treatment (i.e., one or more tumors do not increase in size by more than 1%, 5%, 10%, 15%, or 20%, and / or metastasize). In some embodiments, the tumors stabilize for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more. In some embodiments, the tumors stabilize for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. In some embodiments, the tumors stabilize for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more. Preferably, the size of the tumors shrinks by at least about 5% (e.g., at least about 10%, 15%, 20%, or 25%). More preferably, the tumor size is reduced by at least about 30% (e.g., at least about 35%, 40%, 45%, 50%, 55%, 60%, or 65%). Even more preferably, the tumor size is reduced by at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, or 95%). Most preferably, the tumor is completely removed or reduced to below the detection level. In some embodiments, the subject remains tumor-free (e.g., in remission) for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more following the treatment. In some embodiments, the subject remains tumor-free for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more following the treatment. In some embodiments, the subject remains tumor-free for at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more after the procedure.

[0051] When the tumor is subjected to surgical excision following the completion of the treatment period, the effectiveness of the present invention in reducing the size of the tumor can be determined by measuring the percentage of necrotic (i.e., dead) excised tissue. In some embodiments, the treatment is therapeutically effective when the percentage of necrotic excised tissue is greater than about 20% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), more preferably about 90% or more (e.g., about 90%, 95%, or 100%). Most preferably, the percentage of necrotic excised tissue is about 100%, i.e., no tumor tissue is present or undetectable.

[0052] Many secondary parameters may be used to determine the effectiveness of the methods of the present invention. Examples of secondary parameters include, but are not limited to, the detection of new tumors, the detection of tumor antigens or tumor markers (e.g., CEA, PSA, or CA-125), biopsy, surgical downstaging (i.e., conversion of the surgical stage of the tumor from unresectable to resectable), PET scans, survival rates, disease progression-free survival rates, time to disease progression, and quality of life assessments such as clinical utility response assessments, all of which can indicate the overall progression (or regression) of cancer in humans. Biopsy is particularly useful for detecting the eradication of cancer cells within tissue. Radioimmunoassay (RAID) is used to locate and stage tumors using serum levels of markers (antigens) ("tumor markers" or "tumor-associated antigens") produced by and / or associated with tumors, and can be useful as a pre-treatment diagnostic definitive, a diagnostic indicator of recurrence after treatment, and an indicator of treatment effectiveness after treatment. Examples of tumor markers or tumor-associated antigens that can be evaluated as indicators of therapeutic efficacy include, but are not limited to, carcinembryonic antigen (CEA), prostate-specific antigen (PSA), CA-125, CA19-9, ganglioside molecules (e.g., GM2, GD2, and GD3), MART-1, heat shock proteins (e.g., gp96), sialyl Tn (STn), tyrosinase, MUC-1, HER-2 / neu, c-erb-B2, KSA, PSMA, p53, RAS, EGF-R, VEGF, MAGE, and gp100. Other tumor-associated antigens are known in the art. RAID technology, combined with endoscopic detection systems, also efficiently identifies small tumors from surrounding tissue (see, for example, U.S. Patent No. 4932412).

[0053] In some embodiments, cancer treatment in a human patient is demonstrated by one or more of the following results: (a) complete disappearance of the tumor (i.e., complete response), (b) a reduction in tumor size of approximately 25% to approximately 50% compared to the tumor size before treatment for at least 4 weeks after the completion of the treatment period, (c) a reduction in tumor size of at least approximately 50% compared to the tumor size before treatment for at least 4 weeks after the completion of the treatment period, and (d) a reduction of at least 2% (e.g., approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) in specific tumor-associated antigen levels at approximately 4 to 12 weeks after the completion of the treatment period compared to the tumor-associated antigen levels before treatment. A reduction of at least 2% in tumor-associated antigen levels is preferred, but any reduction in tumor-associated antigen levels is evidence of cancer treatment in the patient. For example, in the case of unresectable, locally advanced pancreatic cancer, treatment can be demonstrated by a reduction of at least 10% in CA19-9 tumor-associated antigen levels at 4–12 weeks after completion of the treatment period compared to pre-treatment CA19-9 levels. Similarly, in the case of locally advanced rectal cancer, treatment can be demonstrated by a reduction of at least 10% in CEA tumor-associated antigen levels at 4–12 weeks after completion of the treatment period compared to pre-treatment CEA levels.

[0054] With regard to the assessment of quality of life, for example, the Clinical Benefit Response Criteria, the therapeutic benefit of the treatment according to the present invention is pain intensity, analgesic consumption and / or This can be demonstrated in terms of the Karnovski Performance Scale score. The Karnovski Performance Scale allows for the classification of patients according to their functional deficits. The Karnovski Performance Scale is scored on a scale of 0 to 100. Generally, lower Karnovski scores predict a poorer prognosis for survival. Therefore, cancer treatment in human patients is demonstrated, either alternatively or additionally, by (a) a reduction of at least 50% (e.g., at least 60%, 70%, 80%, 90%, or 100%) in patient-reported pain intensity compared to pre-treatment pain intensity, for example, during any four consecutive weeks within 12 weeks after the completion of treatment; (b) a reduction of at least 50% (e.g., at least 60%, 70%, 80%, 90%, or 100%) in patient-reported analgesic consumption compared to pre-treatment analgesic consumption, for example, during any four consecutive weeks within 12 weeks after the completion of treatment; and / or (c) an increase of at least 20 points (e.g., at least 30, 50, 70, or 90 points) in patient-reported Karnovski Performance Scale score compared to pre-treatment Karnovski Performance Scale score, for example, during any four consecutive weeks within 12 weeks after the completion of treatment.

[0055] Treatment of proliferative disorders in human patients (e.g., cancer, whether benign or malignant) is preferably demonstrated by one or more of the aforementioned results (in any combination), but alternative or additional results from the referenced and / or other studies may demonstrate the effectiveness of the treatment.

[0056] In some embodiments, tumor size is preferably reduced without significant adverse events in the subject. Adverse events are classified, or "graded," by the National Cancer Institute's (NCI) Cancer Therapy Evaluation Program (CTEP), with Grade 0 representing the least harmful side effect and Grade 4 representing the most serious adverse event. The NCI Toxicity Scale (published April 1999) and the Common Toxicity Criteria Manual (updated August 1999) are available through the NCI, for example, through the NCI Internet website www.ctep.info.nih.gov, or sponsored by the NCI Cancer Therapy and Diagnostics Division. This information is available in the Principal Investigator's Handbook (updated March 1998) for participants in clinical trials of investigational drugs. Preferably, the methods described herein relate to the least severe adverse events, e.g., grade 0, grade 1, or grade 2, as graded by CTEP / NCI. However, while tumor size reduction is desirable, it is not desired because the actual size of the tumor may not shrink despite the eradication of tumor cells (e.g., by necrosis). Eradication of cancer cells is sufficient to clearly understand the therapeutic effect. Similarly, any reduction in tumor size is also sufficient to clearly understand the therapeutic effect.

[0057] The detection, monitoring, and rating of various cancers in humans are based on Cancer Facts and Figures 2001, American Cancer Society, New York, NY, and the national Further details are provided in the previous patent application WO01 / 24684. Therefore, clinicians can use standard tests to determine the effectiveness of various embodiments of the method of the present invention in treating cancer. However, in addition to tumor size and extent, clinicians may also consider quality of life and patient survival in evaluating the effectiveness of the treatment.

[0058] In some embodiments, administration of HABA, induction of hypoxia, and administration of immune checkpoint inhibitors provide improved therapeutic efficacy through treatment with either drug alone, treatment with simultaneous delivery of both drugs, and / or treatment with both drugs in reverse order. Improved efficacy can be measured using any preferred method, including but not limited to those described herein. In some embodiments, improved therapeutic efficacy is an improvement of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, 100%, 110%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 1000%, 10000%, or more, using an appropriate measure (e.g., reduction in tumor size, duration of stable tumor size, duration of metastatic events, duration of disease-free survival). Improved efficacy can also be expressed as a fold improvement, such as at least approximately 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 1000x, 10000x or more, using an appropriate measure (e.g., reduction in tumor size, duration of stable tumor size, duration of metastatic events, duration of disease-free survival).

[0059] In one embodiment, the Disclosure provides a kit for enhancing the immune response to a solid tumor in a subject, including by any use described herein. The kit may contain one or more compositions described herein in any combination. In some embodiments, the kit includes (a) a hypoxia-activated bioreducing agent (HABA), (b) a hypoxia-inducing agent or embolizer, and (c) an immune checkpoint inhibitor. Non-limiting examples of HABAs, hypoxia-inducing agents, embolizers, and immune checkpoint inhibitors are provided above, for example, with respect to various methods of the Disclosure. In some embodiments, the kit further includes instructions for use by a clinician, healthcare provider, or patient, such as printed materials or packaging. The pharmaceutical compositions and other materials in the kit may be contained in any suitable container, may be in a ready-to-use form, or may require a combination with other reagents in the kit or reagents supplied by the user (e.g., dilution of concentrated compositions or reshaping of lyophilized compositions). [Examples]

[0060] The following examples are provided for illustrative purposes to illustrate various embodiments of the invention and are not intended to limit the invention in any way. These examples, together with the methods described herein, represent preferred embodiments and are illustrative, and are not intended to limit the scope of the invention. Modifications and other uses therein, which are encompassed within the spirit of the invention as defined by the claims, will be recalled by those skilled in the art.

[0061] (Example 1) The combination of tirapazamine and hepatic artery ligation in a hepatocellular carcinoma model.

[0062] Materials and Methods: All HBx transgenic mice were raised and tracked in a facility free of specific pathogens. Subsequently, tails of individual mice were collected for genotyping at weaning at 3 weeks of age. Hepatocellular carcinoma (HCC) spontaneously developed in >95% of male HBx transgenic mice at 17–18 months of age. Transgenic mice with tumors 0.5–2 cm in diameter were used in this study.

[0063] Treatment of tumor-bearing HBx mice with tirapazamine and hepatic artery ligation: HBx transgenic mice were subjected to left hepatic artery ligation for 40 minutes, after which the ligation was removed. During the study of drug effects, 0.9% saline, doxorubicin (10 mg / kg), or TPZ (3 mg / kg) was injected into the tail vein of each mouse for 7 minutes prior to hepatic artery ligation. After the completion of injection, a midline laparotomy was performed to expose the left lobe of the liver and the hepatic hilum, and the left hepatic artery or common hepatic artery was dissected for temporary ligation. All mice were anesthetized during the experiment by intraperitoneal injection of 400 mg / kg avertin (Sigma-Aldrich, St. Louis, MO, USA). A series of serum samples were collected within the first week at ARKRA. Liver tissue was collected to assess ALT and total bilirubin using a Y Spotchem EZ Chemistry Analyzer SP-4430 (Arkray, Inc., Kyoto, Japan). Liver tissue was collected for HE staining after 1 or 7 days.

[0064] Tumor necrosis histological examination and morphometric analysis: For each mouse, liver tissues containing whole tumors and adjacent non-tumor areas were fixed overnight in 10% formaldehyde and embedded in paraffin. Liver tissue sections (3 μm thick) transecting whole tumors were subjected to hematoxylin and eosin (H&E) staining for histological analysis. The percentage of necrosis in each tumor was quantified by dividing the necrosis area by the total tumor area in five transecting sections covering the whole tumor using ImageJ software (version 1.46r, National Institutes of Health, Bethesda, MD, USA). Detailed results will be further discussed below.

[0065] (Example 2) Combination of tirapazamine, combretastatin A4 and DMXAA in a lung cancer model.

[0066] Materials and methods: Human lung cancer NCI-H460 cells were purchased from ATCC and used in this study. Cells were passaged within 5 passages before implanting into mice. After the animal acclimation period, approximately 1.5×10 6 cells in 200 μL of serum-free medium / matrigel (50:50 v / v) were subcutaneously injected into each mouse under anesthesia with 3 - 4% isoflurane. BALB / c nude mice, male, approximately 4 - 5 weeks old, weighing 18 - 20 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., China and housed in the animal facility at 3 - 5 mice / cage for each treatment group. Mice were allowed free access to food (irradiated, Shanghai SLAC Laboratory Animal Co., Ltd., China) and water (municipal tap water filtered by a Mol Ultrapure Water System).

[0067] Mice from each group were euthanized 18 days after the initial medication by CO2 asphyxiation followed by cervical dislocation. Tumors were collected, weighed, and photographed. Formalin-fixed tumors were used for histopathological work. After formalin fixation, the tumors were embedded in paraffin to obtain FFPE blocks. One block was prepared for each tumor mass from one mouse. The FFPE blocks were incised to a thickness of approximately 4 μm and processed for H&E staining, histopathological examination, and quantification of tumor necrosis. Detailed results are discussed further below.

[0068] (Example 3) Induction of tumor necrosis in vivo, and conversion of tumors into cancer vaccines.

[0069] The ideal cancer vaccine to address the problems of tumor heterogeneity and genomic instability is each patient's own tumor. However, tumors themselves are not typically highly immunogenic, and the degree of anti-tumor immunity is generally insufficient, even in the presence of immune checkpoint inhibitors alone. The immune response can be enhanced by inducing tumor necrosis, associated with a potent inflammatory response that leads to the presentation of tumor-associated antigens to T cells and increases the population of tumor-specific T cells. One approach to inducing tumor necrosis is applicable, for example, in intrahepatic cancers, which are organs with dual blood supply from the portal vein and hepatic artery, allowing for embolus formation of the hepatic artery supplying the tumor without significant damage to the normal liver. The strategy to induce tumor necrosis is to combine tirapazamine with a hypoxia activator and transarterial embolization (TAE) to create tumor hypoxia that activates tirapazamine to induce tumor necrosis. The efficacy of the combination was confirmed in an HBx transgenic mouse model.

[0070] The animal model used in this study is a transgenic mouse expressing HBx, a hepatitis B virus oncogene that has been shown to induce tumor transformation of hepatocytes. HBx transgenic mice spontaneously develop hepatocellular carcinoma at approximately 18 months of age. The tumor lesions do not exhibit baseline tumor necrosis and are ideal for investigating the extent of tumor necrosis induced by the combination of tirapazamine and hepatic artery ligation (HAL). In the HBx transgenic mouse model, the effective dose of tirapazamine in combination with transient HAL was initially established as 3 mg / kg in the first titration test. Next, the efficacy of tirapazamine in combination with HAL was investigated and compared with doxorubicin, a chemotherapeutic agent commonly used in TACE. In a small group test, the effects of saline (n=1), doxorubicin (10 mg / kg, n=1), and tirapazamine (3 mg / kg, n=1) were compared to treating HBx transgenic mice with palpable HCC along with transient left HAL. Mice were sacrificed one day after treatment with tirapazamine and transient left HAL. ALT levels were significantly higher in mice treated with tirapazamine than in mice treated with doxorubicin. Histopathological examination on day 1 post-treatment showed that tirapazamine induced over 99% necrosis in HCC within the HAL area, compared to only about 5% necrosis in HCC treated with doxorubicin. These results indicated that tirapazamine, when combined with HAL, is far more effective than doxorubicin in inducing tumor necrosis.

[0071] The same study was conducted to examine histopathological changes seven days after treatment. The number of mice used in each group was saline (n=2), doxorubicin (10 mg / kg, n=2), and tirapazamine (3 mg / kg, n=3). Tumor blood flow was monitored with an oxyFlo sensor and showed that in HCC treated with either tirapazamine or doxorubicin, blood flow decreased by 30% in the HAL, which was sufficient to induce tumor hypoxia.

[0072] Mouse body weight analysis did not show statistically significant changes, although the average body weight of mice treated with doxorubicin appeared to be slightly lower (Figure 2A). Serum total bilirubin levels remained within the normal range across all three groups throughout the 7-day period (Figure 2B). Serum ALT levels peaked on day 1 in the group treated with tirapazamine and transient left HAL (Figure 2C). Subsequently, ALT levels decreased and returned to normal around day 3. Doxorubicin and transient HAL also induced an increase in ALT on day 1, but to a lower degree than tirapazamine and left HAL, and ALT levels in both groups returned to normal on day 2. During dissection on day 7, tumor necrosis was evident in the pale color of HCC treated with tirapazamine and transient HAL, but not in HCC from mice treated with saline or doxorubicin (Figure 2D). Histopathological analysis by H&E staining confirmed that HCCs in the left HAL region had 90–99% necrosis after treatment with tirapazamine and left HAL. Pathological changes were little to no in HCCs treated with saline or doxorubicin in combination with left HAL (Figure 2E). A tumor nodule located in the right lobe served as an internal control, as these mice frequently had multiple HCCs. Tumor necrosis did not occur in any of the right lobes in any of the animals. No evidence of necrosis was observed in the tumor-free portion of the left lobe of the liver, indicating that the combination of tirapazamine and left HAL did not cause any significant damage to the normal liver of the same lobe by day 7. Despite possible transient damage on day 1, as suggested by elevated ALT, it was completely recovered by day 7.

[0073] The research results show that the combination of tirapazamine and transarterial embolization (TAE) is superior to TAE alone or to the combination of doxorubicin and TAE in inducing tumor necrosis. Doxorubicin is currently used as a chemotherapeutic component of TACE (transarterial chemoembolization) for the treatment of intermediate-stage HCC. The data described above indicates that using tirapazamine in combination with TAE is far superior to using TACE in combination with doxorubicin in achieving maximum tumor necrosis to induce immunity against HCC.

[0074] Next, the same model was used to examine the extent of tumor necrosis after treatment and to investigate whether the tumor necrosis induced by this approach was accompanied by a significant inflammatory response that promoted phagocytosis of necrotic debris by macrophages / dendritic cells acting as antigen-presenting cells. In the higher-powered figure (Figure 3), the percentage of tumor necrosis was determined by collecting multiple histological sections and evaluating the overall percentage of tumor necrosis after treatment with tirapazamine and HAL. We observed that over 99% of the entire tumor became necrotic. The peripheral regions of the necrotic tumors were examined in detail for inflammatory cell infiltration. The inset in Figure 3 shows that the peripheral regions of the necrotic tumors had very strong inflammatory infiltration, which is consistent with the theory that necrosis triggers an inflammatory response.

[0075] (Example 4) Induction of tumor necrosis using a combination of tirapazamine and transarterial embolization (TAE) in patients with hepatocellular carcinoma who are suitable for embolus formation.

[0076] To investigate the clinical tolerability and efficacy of tirapazamine in combination with transcatheter arterial embolization (TAE) in humans, a Phase I dose-defining study was initiated at a major US medical center to examine tolerability, preliminary efficacy, and determine the recommended Phase II dose (RP2D) of tirapazamine in combination with TAE. Enrolled HCC patients were Child-Pugh class A, not candidates for surgery, had up to four tumor lesions, none exceeding 10 cm in size, and were suitable for embolization. Results from the first 12 evaluable patients were available for analysis.

[0077] In the first two cohorts, tirapazamine was administered at 5 mg / m². 2 and 10 mg / m² 2 It is administered by systemic IV injection, which is the same dose of 250-330 mg / m² used in the previous Phase 3 study. 2 This is significantly less than [the other dose]. The dose was selected based on a dose equivalent to 1 / 10 of the toxic dose estimated from the strong results demonstrated in mouse studies of tirapazamine and left hepatic artery ligation (Example 1) and animal model studies. Three patients were enrolled in one of the cohorts. The procedure was tolerated in all cases, with no toxicity or dose-limiting toxicity (DLT) observed. Subsequently, in rat toxicology studies, the route of administration was switched to intra-arterial (IA) administration via the hepatic artery, based on the profile of good tolerability with intra-arterial (IA) administration, followed by hepatic artery ligation. The initial clinical dose for the IA route was 5 mg / m². 2 This represents 50% of the IV dose that has been shown to be tolerable. Next, the dose for the IA cohort was set at 10 mg / m² for three patients in each cohort. 2 The dose was increased to [value]. The procedure was tolerated in all patients in which no toxicity or dose-limiting toxicity (DLT) was observed.

[0078] The preliminary efficacy of the first 12 patients was analyzed and illustrated in Figure 4. Efficacy outcomes were measured based on the revised Response Evaluation Criteria in Solid Tumors (RECIST) criteria, so that only the size of the surviving tumor was measured. Transcontrast MRI was used for interpretation. A preliminary efficacy analysis showed robust activity in 6 / 12 evaluable patients without contrast-enhanced lesions (as shown in Figure 4), indicating that all tumor tissue had become necrotic, i.e., achieving complete response (CR) according to the revised RECIST after a single dose of tirapazamine with transarterial embolization (TACE). Three additional patients had over 30% necrosis or partial response (PR). The overall response rate (CR+PR) was 83%. This result is significantly better than the historical control group, which reported a 50% CR+PR in a meta-analysis of over 10,000 patients who had previously undergone transarterial chemoembolization (TACE).

[0079] (Example 5) Tumor reduction in untreated lesions in patients with other tumor lesions treated with tirapazamine and arterial embolization.

[0080] In a Phase I clinical study involving 12 patients, several patients had multiple tumor lesions that could not be treated in a single procedure. One patient had three lesions with maximum diameters of 15 mm, 26 mm, and 10 mm, respectively. Due to vascular supply limitations, the intervening radiologist chose to treat the 26 mm and 10 mm lesions because they were located close to each other. The third lesion, 15 mm, was left untreated in the first procedure. When follow-up MRI was performed at week 6, it was found that the two treated lesions had achieved complete response (CR), and the third lesion, which was 15 mm at baseline, had shrunk to a maximum diameter of only 9 mm. One possible explanation consistent with these findings is that treatment of the two lesions induced anti-tumor immunity, which reduced the size of the untreated lesion. In other words, the two treated lesions potentially acted as a cancer vaccine, strengthening the patient's immune system to control the remaining tumor lesions.

[0081] (Example 6) Induction of tumor necrosis in non-hepatic tissue using tirapazamine and an vasoconstrictor.

[0082] Another approach to inducing tumor necrosis is for application in various solid tumors that are unacceptable to embolus formation procedures. In this example, the approach to inducing hypoxia in solid tumors involves the use of vasolytic agents, such as DMXAA (5,6-dimethylxanthenone-4-acetic acid, also known as ASA404 or bazimezan), or stilbene derivatives including combretastatin A4, combretastatin A4 phosphate, or cis-3,4',5-trimethoxy-3'aminostilbene (stilbene 4a). These compounds have been demonstrated to induce tumor hypoxia by blocking tumor blood flow (Chaplin DJ, 2006) (Tozar GM, 2005). In this example, the combination of tirapazamine with combretastatin A4 or DMXAA was investigated, for example, in a lung cancer xenograft model to test their ability to induce tumor necrosis.

[0083] A lung cancer mouse model was created by subcutaneously injecting NCI-H460 human lung cancer cells into BALB / c nude mice to form tumor xenografts. Combinations of tirapazamine with two vasoconstrictors, or combretastatin A4 or DMXAA, were then tested. The average tumor volume was approximately 480-550 mm³. 3 When the tumors reached approximately 1 cm in diameter, were solid, and were increasing in size, mice with tumors were administered tirapazamine (30 mg / kg ip) and either combretastatin A4 (10 mg / kg iv) or DMXAA (20 mg / kg iv) in two doses once a week to ensure that the combination was capable of inducing tumor necrosis. Tirapazamine was administered first, followed by the injection of combretastatin A4 or DMXAA 3–5 minutes later. Both vasodilators caused almost immediate blockade of the tumor vascular system. Animals were observed for any abnormalities up to 3 weeks after the first dose.

[0084] The treated mice were sacrificed at the end of the third week, and the tumors were collected for histological examination by H&E staining. Most tumors showed approximately 50–70% tumor necrosis, while control tumors treated with normal cerebrospinal fluid had less than 20% necrosis. Representative histopathological images are shown in Figure 5. Key findings include the extensive area of ​​tumor necrosis after treatment and the presence of strong inflammatory infiltration around the necrotic tumors. These results are consistent with previous findings in HCC that tumor necrosis is associated with a strong inflammatory response and therefore can enhance anti-tumor immunity.

[0085] When autologous tumor "vaccination" is achieved by necrotizing tumors induced by a combination of tirapazamine and either TAE or an vasolytic agent, the induced T cell population may still be ineffective in the long term in ultimate tumor control due to the immunosuppressive effect of the tumor microenvironment. Therefore, we propose using checkpoint inhibitors in combination as maintenance therapy to eliminate the immunosuppressive effect within the tumor.

[0086] (Example 7) A combination of HABA, hypoxia inducers, and immune checkpoint inhibitors administered in a lung cancer model.

[0087] In a study investigating the effects of administering immune checkpoint inhibitors in combination with hypoxia-activating bioreducing agents and hypoxia-inducing agents, anti-mPD-1, tirapazamine (TPZ), combretastatin A4 phosphate, 5,6-dimethylxanthenone-4-acetic acid (DMXAA), and various combinations thereof were administered as treatment for a subcutaneous 3LL syngeneic lung cancer model in C57BL / 6 mice.

[0088] C57BL / 6 male mice were purchased from Shanghai Laboratory Animal Center (SLAC (Shanghai, China, SCXK 2012-0002), age: 6-8 weeks, weight: 18-22g). Four mice were placed in each cage and kept in a temperature- and humidity-controlled SPF room. The temperature was maintained at 20.5-24.5°C. Humidity was maintained at 40-75%. The light cycle consisted of 12 hours of light and 12 hours of darkness. The mice were kept in polycarbonate cages (325mm x 210mm x 180mm). The cage floor material was corn cob, which was changed twice a week. Throughout the study, the mice were given free access to irradiated dry granular food and sterile drinking water.

[0089] 3LL tumor cells were maintained in vitro as monolayer cultures in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum and L-glutamine (2 mM) at 37°C in an atmosphere of 5% CO2 air. The tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. 3LL tumor cells growing in the exponential growth phase were collected and counted for implantation.

[0090] C57BL / 6 mice are given 3LL tumor cells (2 × 10⁶ cells) in 0.05 mL of phosphate-buffered saline (PBS) for tumor development. 5 A tumor was implanted in the lower right abdomen. Treatment began 9 days after implantation, at which point the average volume of the tumor was approximately 455.64 mm³. 3 The tumor size in the 3LL syngeneic model was approximately 4000 mm² 11 days after treatment with the test product. The treatment administration in each study group is shown in Figure 6 ("N" represents the number of animals, "iv" represents intravenous injection, and "ip" represents intraperitoneal injection). The dosage volume was adjusted based on body weight (0.1 mg / 10 g). 3 When this was reached, the tumor was surgically excised and fixed in 10% formalin for H&E staining and immunomarker staining with F4 / 80.

[0091] All tumor tissue was trimmed as quickly as possible and placed in 10% neutral buffered formalin. The fixed tissue was processed into blocks (or more) by dehydration, cleaning, and infiltration. This procedure can be performed using an automated tissue processor. To prepare formalin-fixed paraffin-embedded specimens, the following steps were then performed: Melted paraffin was poured into the embedding mold. The processed tissue was placed in the mold with the applicable embedding surface facing down (to avoid bubble formation). The paraffin mold containing the tissue was placed on a cryostage. The paraffin block was then cut into 5 μm thick sections using a microtome. Appropriate sections were placed in a 45°C distilled water bath. The spread sections were withdrawn from the water bath via a slide, and the slide was then air-dried.

[0092] For hematoxylin and eosin (H&E) staining, paraffin slides were placed in a 60°C oven for approximately 2 hours, then cooled to room temperature. The paraffin slides were then dewaxed, H&E stained, dehydrated, cleared, and prepared as specimens. Cover slips were applied using permanent mounting medium. Finally, the slides were examined under a microscope and photographed. The percentage of tumor necrosis was calculated by dividing the area of ​​tumor necrosis by the total area of ​​the tumor slide.

[0093] Figure 7 shows tumor necrosis rates, as indicated by H&E staining, after treatment with test samples including anti-mPD-1, tirapazamine (TPZ), combretastatin A4 phosphate, 5,6-dimethylxanthenon-4-acetic acid (DMXAA), and various combinations thereof. Asterisks (*) indicate statistical significance (P<0.05) compared to the vehicle group by the Mann-Whitney test. Combinations of TPZ, combretastatin A4 phosphate, and anti-mPD-1 showed a significant increase in tumor necrosis rates compared to the vehicle group. Figure 8 provides the data depicted in Figure 7.

[0094] Preferred embodiments of the present invention are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by analogy only. Many variations, alterations, and substitutions are now conceivable to those skilled in the art without departing from the present invention. It should be understood that various alternative forms of the embodiments of the present invention described herein may be used in carrying out the present invention. The following claims define the scope of the present invention, and methods and structures within the scope of these claims, as well as their equivalents, are intended to be covered thereby.

Claims

[Claim 1] The invention as shown in the drawings.

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