Pharmaceutical compositions for activating immunity against tumors and methods thereof

Abstract

At present, there is no report on a treatment method for distant tumors. The purpose of the present invention is to provide a pharmaceutical composition for use with radiation therapy to activate immunity against tumors, comprising a hydrogen peroxide solution. Furthermore, another object of the present invention is to provide a method for activating immunity against tumors, the method comprising: a step of providing the pharmaceutical composition; a step for administering the pharmaceutical composition to a target site of a patient suffering from the tumor; and a step for irradiating the target site with radiation, in which the tumor is present at a position distant from the target site in a patient suffering from the tumor.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition and a method for activating immunity against tumors for use together with radiotherapy. The composition of the present invention is characterized in that it contains oxygen peroxide. Background Art

[0002] Radiation therapy is a localized treatment for malignant tumors second only to surgery. Its suitability for elderly patients and its ability to preserve normal organs and tissues have led to a dramatic increase in the number of patients receiving this treatment in recent years. However, the high-energy X-rays and electron beams produced by linear accelerators, currently widely used in this type of radiation therapy, are low-LET (linear energy transfer) radiation, resulting in relatively low biological efficacy. Consequently, radiation therapy using linear accelerators is ineffective for tumors such as malignant melanoma, various sarcomas, and glioblastoma multiforme. Furthermore, radiation therapy using linear accelerators is ineffective for locally advanced cancers, not just malignant melanoma and various sarcomas, but also for locally advanced cancers that grow larger than several centimeters, due to the presence of numerous hypoxic tumor cells and antioxidant enzymes, which render them resistant to radiation. Furthermore, the effects of radiation therapy are limited to the localized area of the tumor being treated, rendering it ineffective for patients with distant metastatic tumors.

[0003] Furthermore, since the report by Mole et al. in 1953, anti-tumor immunity based on radiotherapy has been discussed as an abscopal effect. Regarding anti-tumor immunity, it has been reported that (1) danger signals induced by dying cells activate dendritic cells and T cells, (2) induction of dendritic cells and activation of innate immunity accompanied by activation of the type I interferon pathway, (3) induction of chemokines and adhesion factors related to T cell migration and infiltration, and (4) diversification of the amino acid sequence of T cell receptors. It is believed that the immune regulatory mechanism induced by local radiation exposure is at work. Therefore, if the abscopal effect can be induced efficiently, it may be possible to control distant metastasis. However, in actual clinical practice, it is extremely rare to achieve such an abscopal effect by radiotherapy alone.

[0004] With the recent development of immune checkpoint inhibitors, the abscopal effects of radiation therapy have garnered significant attention, and the combination of radiation therapy and immune checkpoint inhibitors has rapidly gained traction. Basic research has demonstrated that the combined use of immune checkpoint inhibitors, such as anti-PD-L1 and anti-CTLA-4 antibodies, with radiation can effectively induce abscopal effects, inhibit distant metastasis, and prolong survival. However, despite numerous large-scale clinical trials, these results have been limited and unsatisfactory, and their use as a new therapeutic approach has yet to be established. A more potent and safer approach to activating tumor immunity is needed.

[0005] To enhance the effects of radiotherapy (RT), various radiation sensitizers have been developed (e.g., Patent Document 1). Hydrogen peroxide is one example, reported to improve anti-tumor effects by sensitizing the patient to radiation (Patent Document 1).

[0006] Prior art documents Patent Literature Patent Document 1: WO2008 / 041514 Non-patent literature Non-patent document 1: RH Mole, Br J Radiol. 1953 May;26(305):234-41 Summary of the Invention Problems to be solved by the invention The therapeutic effect of radiation is limited to local treatment. Even if conventional radiation sensitizers are used, they can only enhance the local effect. There have been no reports of satisfactory therapeutic effects for distant metastatic tumors.

[0007] In addition, combination therapy with radiotherapy and anti-tumor immunotherapeutic agents such as immune checkpoint inhibitors has not yet achieved medically satisfactory therapeutic effects and has not yet become an established treatment method.

[0008] Technical solutions to problems The present inventors have discovered that the combined use of a pharmaceutical composition containing a hydrogen peroxide solution and radiation not only exhibits a radiation-sensitizing effect but also activates anti-tumor immunity. Based on this discovery, the present inventors discovered that irradiating a treatment area to which a pharmaceutical composition containing a hydrogen peroxide solution has been applied not only improves the local therapeutic effect of the irradiated area but also reduces the size of tumors distant from the treatment area, leading to the completion of the present invention.

[0009] The present inventors further unexpectedly discovered that the pharmaceutical composition comprising hydrogen peroxide solution significantly improves the effect of combined therapy of radiation therapy and anti-tumor immunotherapy by activating tumor immunity.

[0010] The object of the present invention is to provide: [1] A pharmaceutical composition for use with radiation therapy to activate immunity against tumors, comprising a hydrogen peroxide solution.

[0011] When the pharmaceutical composition of the present invention is used for radiation irradiation, the size of tumors located far from the treatment site can be reduced by activating immunity against the tumor. [2] In the pharmaceutical composition of [1], the above-mentioned tumor may be present in a location away from the target site in a patient suffering from the above-mentioned tumor. [3] The pharmaceutical composition described in [1] or [2] may further contain hyaluronic acid or a salt thereof. [4] The pharmaceutical composition as described in any one of [1] to [3] may contain liposomes, polymer gels, hydrogels, gelatin, or salts thereof. [5] The pharmaceutical composition as described in any one of [1] to [4] can be used in combination therapy with an anti-tumor immunotherapeutic agent. [6] For example, the pharmaceutical composition of [5] can enhance the anti-tumor effect through the above-mentioned combined therapy. [7] In the pharmaceutical composition described in [5] or [6], the anti-tumor immunotherapeutic agent can specifically bind to PD-1, PD-L1 or CTLA-4. [8] For example, in the pharmaceutical composition of [7], the anti-tumor immunotherapeutic agent may be an immune checkpoint inhibitor. [9] For example, in the pharmaceutical composition of [8], the above-mentioned immune checkpoint inhibitor may be a PD-1 inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor.

[10] For example, in the pharmaceutical composition of [9], the immune checkpoint inhibitor may be atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, or an antigen-binding fragment thereof.

[11] In the pharmaceutical composition of [5] or [6], the anti-tumor immunotherapeutic agent may be an anti-tumor immune-activating chemotherapy drug.

[12] For example, in the pharmaceutical composition of

[11] , the anti-tumor immune activating chemotherapy drug may be gemcitabine.

[13] In the pharmaceutical composition of [5] or [6], the anti-tumor immunotherapeutic agent may be a tumor immune activator.

[14] The pharmaceutical composition as described in

[13] , wherein the tumor immunity activator is a 41-BB agonist, an OX-40 agonist, a TIGIT inhibitor, a LAG-3 inhibitor or an IDO inhibitor.

[15] In the pharmaceutical composition of [5] or [6], the anti-tumor immunotherapeutic agent may be an immune cell, a nucleic acid molecule or a sensitizer.

[0026] Furthermore, another object of the present invention is to provide:

[16] A method for activating immunity against tumors, comprising: A step of providing the pharmaceutical composition as described in any one of [1] to

[15] ; a step of administering the pharmaceutical composition to a target site of a patient suffering from the tumor; and a step of irradiating the target area with radiation; The tumor is present in a location away from the target site in a patient suffering from the tumor.

[0027] By using the methods of the present invention, it is possible to activate immunity against tumors and reduce the size of tumors that exist away from the treatment site.

[17] The method of

[16] may further comprise the step of administering an anti-tumor immunotherapeutic agent.

[0029] The above method further comprises the step of administering an anti-tumor immunotherapeutic agent. By using the above method comprising this step, immunity against tumors can be activated, and the anti-tumor effect can be enhanced by combined therapy with an anti-tumor immunotherapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A bar graph showing the amount of HMGB1, a damage-associated molecular pattern (DAMP), produced from cancer cells.

[0031] Figure 2 Shown are the mouse with the locations of MC38 cell implantation and radiation therapy (RT) indicated, along with the experimental schedule.

[0032] Figure 3 middle, Figure 3 A is a bar graph showing the number of cytotoxic T cells (CD8) infiltrating in tumor-infiltrating lymphocytes (TIL) infiltrating the non-irradiated side, as measured by flow cytometry. Figure 3 B is a bar graph showing the number of dendritic cells (DCs) infiltrating the tumor-draining lymph nodes (TDLN) near the irradiated site, measured using flow cytometry.

[0033] Figure 4 middle, Figure 4 A is a graph showing the change in tumor size over time at the irradiated side (n=6). Figure 4 B is a graph showing the changes in tumor size over time at the non-irradiated side (n=6).

[0034] Figure 5 Figure 2 is a graph showing the changes in tumor size over time in non-irradiated areas (n=5). DETAILED DESCRIPTION

[0035] 1 Definition For convenience, specific terms used in this application are collectively described here. Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as commonly understood by those of ordinary skill in the art to which this invention belongs. Unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" include plural references.

[0036] The numerical ranges and parameters shown in this invention are approximate, but the numerical values shown in the specific examples are reported as accurately as possible. However, any numerical value inherently includes certain errors that inevitably arise from the standard deviation observed in the respective experimental measurements. In addition, the term "about" as used in this specification generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within the standard error range that is considered acceptable by those skilled in the art.

[0037] The terms "subject" and "patient" are used interchangeably in this specification to refer to animals, including humans, that can be treated by the synthetic peptides and / or methods of the present invention. Unless a particular gender is specifically specified, the term "subject" or "patient" is intended to include both male and female genders. Therefore, the term "subject" or "patient" includes any mammal that can benefit from the treatment methods disclosed herein. Examples of "subjects" or "patients" include, but are not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, goats, cows, horses, dogs, cats, birds, and chickens. In an exemplary embodiment, the patient is human.

[0038] In addition, in this specification, unless otherwise specified, % (w / v) represents weight / volume percentage concentration.

[0039] Damage-associated molecular patterns (DAMPs) are biological substances released in response to cellular stresses such as cell death and cell damage, acting as alarms to warn cells of danger. In tumor immunity, DAMPs are released as an initial response by immune cells.

[0040] The following embodiments of the present invention are described. The following embodiments are for illustration only, and the scope of the present invention is not limited to the contents shown in the following embodiments. In addition, to avoid tedious repetition, the detailed description of the same contents will be omitted.

[0041] 2 Methods for activating immunity against tumors The method for activating immunity against tumors according to this embodiment includes: providing a pharmaceutical composition containing a hydrogen peroxide solution; administering the pharmaceutical composition to a target site of a patient suffering from the tumor; and irradiating the target site with radiation; wherein the tumor is located at a location distant from the target site of the patient suffering from the tumor.

[0042] 3 Pharmaceutical Composition The pharmaceutical composition for activating anti-tumor immunity according to this embodiment comprises a hydrogen peroxide solution. The pharmaceutical composition according to this embodiment can treat tumors by activating anti-tumor immunity. "Treating tumors" or "anti-tumor effect" means reducing tumor size and / or inhibiting tumor growth.

[0043] 4 Hydrogen peroxide solution The hydrogen peroxide solution of this embodiment is an aqueous solution containing hydrogen peroxide (H₂O₂; molecular weight 34). The proportion of hydrogen peroxide in the pharmaceutical composition of this embodiment can be 0.01 to 3.5% (w / v), or can be within a range between two values selected from the group consisting of 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, and 3.5% (w / v). Unless otherwise specified, "hydrogen peroxide solution" means a solution obtained by dissolving hydrogen peroxide in distilled water as specified in the Japanese Pharmacopoeia, etc.

[0044] 5 additional ingredients The pharmaceutical composition of this embodiment may contain additional ingredients. Additional ingredients include hyaluronic acid, liposomes, polymer gels, hydrogels, and gelatin, or salts thereof. Additionally, additional ingredients may include pharmaceutically acceptable physiological saline, buffers (e.g., phosphate buffer, Tris buffer, and acetate buffer), stabilizers, isotonic agents, and pH adjusters.

[0045] The mixing ratio of hyaluronic acid or its salt to hydrogen peroxide may be 1 to 10,000 parts by mass per 100 parts by mass of hydrogen peroxide, or may be within a range between two values selected from the group consisting of 1, 5, 10, 50, 100, 150, 500, 1,000, 50,000, and 10,000.

[0046] The hyaluronic acid of the present embodiment can be extracted from animal tissue or manufactured by fermentation. The hyaluronic acid of the present embodiment is preferably manufactured by fermentation. This is because the hyaluronic acid manufactured by fermentation is safe and has high manufacturing stability. As the bacterial strain used in the fermentation method, hyaluronic acid-producing microorganisms (e.g., Streptococcus) isolated from nature, Streptococcus equi FM-100 (Micro-industry research bacteria No. 9027) recorded in Japanese Unexamined Patent Publication No. 63-123392, and Streptococcus equi FM-300 (Micro-industry research bacteria No. 2319) recorded in Japanese Unexamined Patent Publication No. 2-234689 can be cited.

[0047] The hyaluronic acid of this embodiment has a mass average molecular weight of approximately 500,000 to 10 million, preferably 500,000 to 8 million, and more preferably 500,000 to 5 million.

[0048] The mass average molecular weight of hyaluronic acid can be measured by the SEC-MALLS method using size exclusion chromatography (SEC) and a multi-angle light scattering detector (MALLS).

[0049] The hyaluronic acid of this embodiment can be used as an aqueous solution or a water-swellable gel.

[0050] The hyaluronic acid in this embodiment includes non-crosslinked hyaluronic acid and cross-linked hyaluronic acid. Cross-linked hyaluronic acid is a polymer with a three-dimensional network structure. When the crosslinks in the cross-linked hyaluronic acid are severed, linear hyaluronic acid (non-cross-linked hyaluronic acid) is generated. Furthermore, the mass average molecular weight and degree of branching of the hyaluronic acid generated by severing the crosslinks can be measured using GPC-MALLS (Multi-Angle Light Scattering) using gel permeation chromatography (GPC), a differential refractometer, and a multi-angle laser light scattering detector (MALLS).

[0051] The hyaluronic acid of this embodiment can be non-crosslinked hyaluronic acid, crosslinked hyaluronic acid, or a combination thereof. The hyaluronic acid of this embodiment can be composed of different crosslinked hyaluronic acids, or can be composed of hyaluronic acids of different molecular weights. The hyaluronic acid of this embodiment can also be a hyaluronate. The hyaluronate can be sodium hyaluronate, potassium hyaluronate, or lithium hyaluronate.

[0052] The ratio of hyaluronic acid or its salt contained in the pharmaceutical composition of this embodiment may be 0.1-10% (w / v), or may be within a range between two values selected from the group consisting of 0.1, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10% (w / v).

[0053] Examples of isotonic agents include sodium chloride, glycerol, glucose, polyethylene glycol, propylene glycol, D-mannitol, fructose, xylitol, sodium dihydrogen phosphate, and sodium phosphate, preferably sodium chloride. Examples of pH adjusters include hydrochloric acid and sodium hydroxide. The pH of the pharmaceutical composition of this embodiment is adjusted to 6 to 8.5, preferably 6.8 to 7.8. The pharmaceutical composition of this embodiment may contain a buffer to maintain pH.

[0054] 6. Step of Providing a Pharmaceutical Composition The "step of providing a pharmaceutical composition" in this embodiment may be a step of preparing the pharmaceutical composition or a step of preparing the pharmaceutical composition. In one embodiment, the step of preparing the pharmaceutical composition includes the step of mixing a hydrogen peroxide solution with hyaluronic acid or a salt thereof.

[0055] 7. Step of administering the pharmaceutical composition to the target site of a patient with a tumor The pharmaceutical composition of this embodiment is administered to a target site in a patient suffering from a tumor. The method of local administration is not limited. In one embodiment, the target site is the site where the pharmaceutical composition is administered and irradiated. In another embodiment, the target site is the site where the pharmaceutical composition is administered and irradiated and where the tumor is located.

[0056] The tumor of this embodiment may be present in a patient with a tumor at a location remote from the target site. In one embodiment, the tumor is present in a patient with a tumor at a location remote from the target site, while no detectable tumor is present at the target site. In another embodiment, the tumor is present in a patient with a tumor at a location remote from the target site and is also present at the target site. In yet another embodiment, the tumor comprises a first tumor and a second tumor, the first tumor being present in a patient with a tumor at a location remote from the target site, and the second tumor being present at the target site in the patient with a tumor. The first tumor may be derived from a metastatic cancer, and the second tumor may be derived from a primary cancer.

[0057] "Tumor" refers to cells characterized by uncontrolled growth, including pre-neoplastic hyperproliferation, primary cancer, metastatic cancer, neoplasms, and solid tumors. "Tumor" can be caused by "cancer". "Cancer" includes, but is not limited to, lymphoma, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, brain cancer, breast cancer, triple-negative breast cancer, central or peripheral nervous system cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gallbladder cancer, gastrointestinal cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, nasopharyngeal cancer, nasal cavity cancer, oropharyngeal cancer, oral cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pituitary cancer, prostate cancer, retinoblastoma, sarcoma, salivary gland cancer, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer, vaginal cancer, and vulvar cancer.

[0058] In one embodiment, the target tumor is a tumor arising from a metastatic cancer and the non-target tumor is a tumor arising from a primary cancer. In another embodiment, the target tumor is a tumor arising from a primary cancer and the non-target tumor is a tumor arising from a metastatic cancer.

[0059] 8 Steps for irradiating the target area The "step of irradiating the target site" of this embodiment is performed after the "step of administering the pharmaceutical composition to the target site of a patient suffering from a tumor." The pharmaceutical composition of this embodiment can activate anti-tumor immunity by irradiating the target site to which the pharmaceutical composition has been administered.

[0060] The radiation in this embodiment may be X-rays, electron beams, proton beams, heavy particle beams, alpha rays, beta rays, gamma rays, or a combination thereof. X-rays or electron beams may be administered using a linear accelerator. The radiation dose in this embodiment may be 1.5 to 4 Gy per dose, or may be within a range between two values selected from the group consisting of 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, and 4 Gy. Radiation irradiation in this embodiment may be performed 2 to 5 times per week, preferably 4 to 5 times per week. Radiation irradiation in this embodiment may be performed for 1 to 5 weeks. The total radiation dose in this embodiment may be 20 to 70 Gy, or may be within a range between two values selected from the group consisting of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, and 70 Gy.

[0061] 9. Steps for administering anti-tumor immunotherapy agents The pharmaceutical composition of this embodiment can be used in combination therapy with an anti-tumor immunotherapeutic agent. The method of this embodiment may further comprise "administering the anti-tumor immunotherapeutic agent." In one embodiment, "administering the anti-tumor immunotherapeutic agent" may be "administering the anti-tumor immunotherapeutic agent to the target site" or "administering the anti-tumor immunotherapeutic agent to a site in the patient other than the target site."

[0062] The "step of administering an anti-tumor immunotherapy agent" of this embodiment may be performed after the "step of irradiating the target site with radiation", or between the "step of administering the pharmaceutical composition to the target site of a patient suffering from a tumor" and the "step of irradiating the target site with radiation", or before the "step of administering the pharmaceutical composition to the target site of a patient suffering from a tumor".

[0063] 10 Anti-tumor immunotherapy agents The anti-tumor immunotherapeutic agent of this embodiment refers to a substance (e.g., a compound, a cell (e.g., an immune cell), a protein (e.g., an antibody), and a nucleic acid molecule (e.g., DNA and RNA)) that reduces tumor size or inhibits tumor growth by enhancing or assisting immune function, including anti-tumor immune-activating chemotherapy drugs, immune checkpoint inhibitors, tumor immune activators, and combinations thereof.

[0064] In one embodiment, the anti-tumor immunotherapeutic agent may be an anti-tumor immune-activating chemotherapy drug (eg, gemcitabine).

[0065] In one embodiment, the anti-tumor immunotherapeutic agent may specifically bind to PD-1, PD-L1, or CTLA-4. In another embodiment, the anti-tumor immunotherapeutic agent may be an immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor may be a PD-1 inhibitor, a PD-L1 inhibitor, or a CTLA-4 inhibitor. In another embodiment, the immune checkpoint inhibitor may be selected from atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, and antigen-binding fragments of any of the foregoing.

[0066] In one embodiment, the anti-tumor immunotherapeutic agent may be a 41-BB agonist, an OX-40 agonist, a TIGIT inhibitor, a LAG-3 inhibitor, or an IDO inhibitor.

[0067] 11 Other Implementation Methods The present invention also provides a pharmaceutical composition for use with radiotherapy to activate immunity against tumors, (i) comprising a hydrogen peroxide solution for use in a combination therapy with a hydrogen peroxide solution and an anti-tumor immunotherapeutic agent, or (ii) comprising an anti-tumor immunotherapeutic agent, for use in combination therapy with a hydrogen peroxide solution and the anti-tumor immunotherapeutic agent.

[0068] The present invention also provides a pharmaceutical composition comprising an anti-tumor immunotherapeutic agent for use in combination therapy with a hydrogen peroxide solution and the anti-tumor immunotherapeutic agent together with radiation therapy to activate immunity against tumors.

[0069] The present invention also provides a use of a hydrogen peroxide solution in the manufacture of a pharmaceutical composition for activating immunity against tumors. Furthermore, the present invention also provides a use of a hydrogen peroxide solution and an anti-tumor immunotherapeutic agent in the manufacture of a pharmaceutical composition for activating immunity against tumors.

[0070] Example The data in the examples are expressed as mean ± standard error of the mean (SEM), and the means were compared using Student's t-test. P<0.05, P<0.01, P<0.001, P<0.00001. “ns” indicates not significant.

[0071] Example 1 To confirm whether the combined use of hydrogen peroxide + radiation therapy (RT) can cause cancer cells to produce HMGB1 (a damage-associated molecular pattern (DAMP)). KORTUC (hydrogen peroxide + hyaluronic acid) was added to MC38 cells (mouse colon cancer cell line), and RT was performed. The concentration of hydrogen peroxide in the culture medium was 0.0067% (w / v) or 0.0335% (w / v) (10μM or 50μM hydrogen peroxide), and the concentration of hyaluronic acid in the culture medium was 0.0111% (w / v) or 0.0556% (w / v). The dose of RT was 5.0Gy (1Gy / min). In addition, MC38 cells were only subjected to RT. The control group was treated without KORTUC or RT. 24 hours after irradiation, MC38 cells and culture supernatant were collected, and HMGB1 was quantitatively measured by ELISA. The results are as follows Figure 1 shown.

[0072] Both KORTUC administration plus RT (RT+KORTUC) and RT released HMGB1 into the culture supernatant, but RT+KORTUC released more HMGB1 into the culture supernatant than RT. This result suggests that KORTUC administration plus RT may trigger an immune response.

[0073] Example 2 To investigate whether the combined use of KORTUC (a mixture of hydrogen peroxide and hyaluronic acid, with a hydrogen peroxide concentration of 0.5% and a hyaluronic acid concentration of 0.83%) and RT would induce an immune response in mice, further experiments were conducted on mice implanted with MC38 cells. Figure 2 As shown, MC38 cells were implanted into the left leg (irradiated site) and right abdomen (non-irradiated site) of mice (day 0). Ten days after cell implantation, the irradiated site was irradiated with 15 Gy of radiation (RT), or KORTUC was administered to the irradiated site and then irradiated with 15 Gy of radiation (RT+KORTUC). A control group was treated without KORTUC or RT.

[0074] Figure 3 A is a bar graph showing the number of cytotoxic T cells (CD8) infiltrating in tumor-infiltrating lymphocytes (TIL) infiltrating the non-irradiated site, as measured by flow cytometry. Figure 3 B is a bar graph showing the number of dendritic cells (DCs) infiltrating the tumor-draining lymph nodes (TDLN) near the irradiated site, measured using flow cytometry.

[0075] Compared with the control group and RT, the infiltration numbers of cytotoxic T cells (CD8) and DCs in RT+KORTUC were significantly increased (see Figure 3 A and Figure 3 B). This result indicates that RT+KORTUC triggers an immune response in vivo. In particular, since RT+KORTUC increases cytotoxic T cells (CD8), a type of immune cell, even in non-irradiated areas, it suggests that RT+KORTUC is effective not only against local tumors but also against distant tumors.

[0076] Example 3 The effect of KORTUC (a mixture of hydrogen peroxide and hyaluronic acid, with a hydrogen peroxide concentration of 0.5% and a hyaluronic acid concentration of 0.83%) and RT on tumors was measured over time. MC38 cells were implanted in the left leg (irradiated site) and right abdomen (non-irradiated site) of mice (day 0) (refer to Figure 2 Ten days after transplantation, KORTUC was administered to the irradiated area (KORTUC alone), and the irradiated area was irradiated with 15 Gy of radiation (RT alone), or KORTUC was administered to the irradiated area and irradiated with 15 Gy of radiation (KORTUC+RT). A control group was used in which neither KORTUC nor radiation was administered.

[0077] Figure 4 A is a graph showing the change in tumor size over time at the irradiated side (n=6). Figure 4 B is a graph showing the changes in tumor size over time at the non-irradiated site (n = 6). In the irradiated site, tumor size decreased over time with both RT alone and KORTUC+RT. In the non-irradiated site, KORTUC+RT demonstrated an inhibitory effect on tumor growth. While the anti-tumor effects on the irradiated site were comparable between RT alone and KORTUC+RT, RT alone failed to inhibit tumor growth in the non-irradiated site, while KORTUC+RT significantly suppressed tumor growth. This suggests that this effect is not due to the radiosensitization of KORTUC at the irradiated site, but rather to the induction of tumor immunity by KORTUC.

[0078] Example 4 The combined effects of KORTUC (a mixture of hydrogen peroxide and hyaluronic acid, with a hydrogen peroxide concentration of 0.5% and a hyaluronic acid concentration of 0.83%), RT, and an anti-tumor immunotherapy agent were measured over time. MC38 cells were implanted in the left leg (irradiated site) and right abdomen (non-irradiated site) of mice (day 0) (refer to Figure 2 ). Anti-PD-1 antibodies (aPD1), which are immune checkpoint inhibitors, were used as anti-tumor immunotherapeutic agents. Ten days after transplantation, anti-PD-1 antibodies were administered intraperitoneally (aPD1 alone), or anti-PD-1 antibodies were administered intraperitoneally and the irradiated area was irradiated with 15 Gy (RT+aPD1), or anti-PD-1 antibodies were administered intraperitoneally and KORTUC was administered to the irradiated area followed by 15 Gy of irradiation (KORTUC+RT+aPD1). A control group was used in which KORTUC, anti-PD-1 antibodies, and irradiation were not performed.

[0079] Figure 5 Figure 2 is a graph showing the changes in tumor size over time in non-irradiated areas (n=5). Figure 5 As shown, a decrease in tumor size over time was observed in KORTUC+RT+aPD1. Figure 4 The results of KORTUC+RT of B are similar to Figure 5 The results of KORTUC+RT+aPD1 show that compared with KORTUC+RT, KORTUC+RT+aPD1 has a higher anti-tumor effect on distant tumors.

[0080] It was shown that the combined use of KORTUC (hydrogen peroxide) and RT can induce an immune response, particularly exerting anti-tumor effects on distant tumors unrelated to RT irradiation or local injection. Furthermore, the combined use of KORTUC (hydrogen peroxide) and an anti-tumor immunotherapeutic agent with RT induces a stronger immune response than the combined use of KORTUC (hydrogen peroxide) and RT, leading to a more potent anti-tumor effect on distant tumors.

Claims

1. A pharmaceutical composition for use with radiotherapy to activate immunity against tumors, comprising a hydrogen peroxide solution.

2. The pharmaceutical composition according to claim 1, wherein The tumor is present in a patient having the tumor at a location distant from the target site.

3. The pharmaceutical composition according to claim 1 or 2, further comprising hyaluronic acid or a salt thereof. 4 . The pharmaceutical composition according to claim 1 , comprising liposomes, polymer gels, hydrogels, gelatin, or salts thereof.

5. The pharmaceutical composition according to any one of claims 1 to 4, for use in combination therapy with an anti-tumor immunotherapeutic agent.

6. The pharmaceutical composition according to claim 5, wherein The anti-tumor effect is improved by the combined therapy.

7. The pharmaceutical composition according to claim 5 or 6, wherein The anti-tumor immunotherapeutic agent specifically binds to PD-1, PD-L1 or CTLA-4.

8. The pharmaceutical composition according to claim 7, wherein The anti-tumor immunotherapeutic agent is an immune checkpoint inhibitor.

9. The pharmaceutical composition according to claim 8, wherein The immune checkpoint inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor.

10. The pharmaceutical composition according to claim 9, wherein The immune checkpoint inhibitor is atezolizumab, avelumab, durvalumab, ipilimumab, nivolumab, pembrolizumab, or an antigen-binding fragment thereof.

11. The pharmaceutical composition according to claim 5 or 6, wherein The anti-tumor immunotherapeutic agent is an anti-tumor immune activating chemotherapy drug.

12. The pharmaceutical composition according to claim 11, wherein The anti-tumor immune activation chemotherapy drug is gemcitabine.

13. The pharmaceutical composition according to claim 5 or 6, wherein The anti-tumor immunotherapeutic agent is a tumor immune activator.

14. The pharmaceutical composition according to claim 13, wherein The tumor immune activator is a 41-BB agonist, an OX-40 agonist, a TIGIT inhibitor, a LAG-3 inhibitor or an IDO inhibitor.

15. The pharmaceutical composition according to claim 5 or 6, wherein The anti-tumor immunotherapeutic agent is an immune cell, a nucleic acid molecule or a sensitizer.

16. A method for activating immunity against tumors, the method comprising: The step of providing a pharmaceutical composition according to any one of claims 1 to 15; a step of administering the pharmaceutical composition to a target site in a patient suffering from the tumor; as well as a step of irradiating the target area with radiation; wherein the tumor is present at a location distant from a target site in a patient suffering from the tumor.

17. The method of claim 16, further comprising the step of administering an anti-tumor immunotherapeutic agent.

Images