Pharmaceutical composition for activating immunity against tumors and method thereof

Abstract

To provide treatment methods for distant tumors.SOLUTION: An object of the present invention is to provide a pharmaceutical composition for activating immunity against tumors by use with radiation therapy, the pharmaceutical composition including a hydrogen peroxide solution. Another object of the present invention is to provide a method for activating immunity against tumors, the method comprising providing the pharmaceutical composition, administering the pharmaceutical composition to a target site in a patient having the tumor, and irradiating the target site with radiation, the tumor being located remote from the target site in the patient having the tumor.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition and a method for activating immunity against tumors by being used together with radiotherapy. The composition of the present invention is characterized by containing hydrogen peroxide.

Background Art

[0002] Radiotherapy is a method second only to surgical operation as a local treatment method for malignant tumors. It can be applied to elderly patients and can preserve normal organs and tissues. Therefore, it is a treatment method with a rapidly increasing number of treated patients in recent years. However, currently, the high-energy X-rays and electron beams generated by the linear accelerators commonly used in radiotherapy are low-LET (linear energy transfer) radiations, and their biological effects are relatively low. Therefore, the effects of radiotherapy using linear accelerators are poor for tumors such as malignant melanoma, various sarcomas, and glioblastoma multiforme. In addition, not only malignant melanoma and various sarcomas, but also locally advanced cancers that have grown to several centimeters or more have a large number of hypoxic tumor cells and contain a large amount of antioxidant enzymes, so they are radioresistant, and the effects of radiotherapy using linear accelerators are poor. In addition, its effect is limited to the local tumor of the radiotherapy target, and there is also a problem that it cannot contribute to the treatment effect for patients with tumors in distant metastatic foci.

[0003] Also, since the report of Mole et al. in 1953, the antitumor immunity by radiotherapy has been discussed as the abscopal effect. Antitumor immunity includes (1) activation of dendritic cells and T cells by danger signals induced from dying cells, (2) induction of dendritic cells and activation of innate immunity associated with the activation of the type I interferon pathway, (3) induction of chemokines and adhesion factors involved in the migration and infiltration of T cells, (4) diversification of the amino acid sequences of T cell receptors, etc. have been reported, and it is considered that the immunomodulatory mechanism by local radiotherapy is functioning. Therefore, if the abscopal effect can be induced at a high rate, it may be possible to control distant metastasis. However, in actual clinical practice, it is extremely rare to encounter this abscopal effect with radiotherapy alone.

[0004] In recent years, with the development of immune checkpoint inhibitors, attention has been focused on the abscopal effect of radiotherapy, and the combined treatment of radiotherapy and immune checkpoint inhibitors has attracted increasing attention. In basic research, it has been gradually revealed that the combined use of immune checkpoint inhibitors such as anti-PD-L1 antibody and anti-CTLA-4 antibody and radiation can induce the abscopal effect at a high rate, and suppress distant metastasis and prolong survival. However, although multiple large-scale clinical trials have been conducted, the effects are limited and not satisfactory, and it has not been established as a new treatment method. There is a need for a method that can activate more powerful and safer tumor immunity.

[0005] Conventionally, various radiation sensitizers have been developed to enhance the effect of radiotherapy (for example, Patent Document 1). Hydrogen peroxide is also one of them, and it has been reported that its antitumor effect can be improved by sensitizing radiation (Patent Document 1).

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] The therapeutic effect of radiation is limited to the treatment site. Even when using general radiation sensitizers, the local effect is only enhanced, and no satisfactory therapeutic effect on distant metastatic tumors has been reported.

[0009] In addition, combined treatment with radiotherapy and antitumor immunotherapeutic agents typified by immune checkpoint inhibitors has not achieved satisfactory therapeutic effects in medicine and has not become an established treatment method.

Means for Solving the Problems

[0010] The present inventors have found that a pharmaceutical composition containing a hydrogen peroxide solution, when used in combination with radiation, not only has a radiosensitizing effect but also activates the immunity against tumors. Thereby, the present inventors have discovered that when irradiating a treatment site administered with the pharmaceutical composition containing a hydrogen peroxide solution with radiation, not only the therapeutic effect at the irradiated local site is improved, but also the size of tumors existing remotely from the treatment site is reduced, and the present invention has been completed.

[0011] Furthermore, the present inventors have surprisingly found that the effect of the combined therapy of radiotherapy and antitumor immunotherapeutic agents is improved at each stage by the pharmaceutical composition containing this hydrogen peroxide solution activating tumor immunity.

[0012] An object of the present invention is [1] To provide a pharmaceutical composition for activating immunity against tumors by using it together with radiotherapy, which contains a hydrogen peroxide solution is to provide.

[0013] When radiotherapy is performed using the pharmaceutical composition according to the present invention, the size of tumors existing remotely from the treatment site can be reduced by activating the immunity against tumors.

[0014] [2] In the pharmaceutical composition of [1], the above-mentioned tumor may exist remotely from the target site of the patient having the above-mentioned tumor.

[0015] [3] The pharmaceutical composition according to [1] or [2] may further contain hyaluronic acid or a salt thereof.

[0016] [4] Any of the pharmaceutical compositions from [1] to [3] may contain liposomes, polymer gels, hydrogels or gelatin or salts thereof.

[0017] [5] Any of the pharmaceutical compositions from [1] to [4] may be used in combination therapy with an anti-tumor immunotherapeutic agent.

[0018] [6] The pharmaceutical composition of [5] may have an improved anti-tumor effect by the above combination therapy.

[0019] [7] In the pharmaceutical composition according to [5] or [6], the anti-tumor immunotherapeutic agent may specifically bind to PD-1, PD-L1 or CTLA-4.

[0020] [8] In the pharmaceutical composition of [7], the anti-tumor immunotherapeutic agent may be an immune checkpoint inhibitor.

[0021] [9] In the pharmaceutical composition of [8], the immune checkpoint inhibitor may be a PD-1 inhibitor, a PD-L1 inhibitor or a CTLA-4 inhibitor.

[0022]

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

[0023]

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

[0024]

[12] In the pharmaceutical composition of

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

[0025]

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

[0026]

[14] The pharmaceutical composition according to

[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.

[0027]

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

[0028] Another object of the present invention is

[16] A method for activating immunity against a tumor, the method comprising providing a pharmaceutical composition according to any one of [1] to

[15] ; administering the pharmaceutical composition to a target site of a patient having the tumor; irradiating the target site with radiation, and the tumor is present remotely from the target site of the patient having the tumor. Method is to provide.

[0029] By using the method according to the present invention, immunity against a tumor can be activated and the size of a tumor present remotely from the treatment site can be reduced.

[0030]

[17] The method of

[16] may further comprise administering an anti-tumor immunotherapy agent.

[0031] The method further comprises administering an anti-tumor immunotherapy agent. By using the method including this step, immunity against a tumor can be activated and the anti-tumor effect can be improved by combination therapy with an anti-tumor immunotherapy agent.

Brief Description of the Drawings

[0032]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Mode for Carrying Out the Invention

[0033] 1 Definition For convenience, the specific terms used in this application are collected here. Unless otherwise specified, all technical terms and scientific terms used in this application have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. Unless otherwise clearly stated in the context, the singular forms "a", "an", and "the" include plural references.

[0034] The numerical ranges and parameters shown in the present invention are approximate values. Although the numerical values shown in specific examples are described as accurately as possible, any numerical value inherently contains certain errors that inevitably result from the standard deviation found in each test measurement. Also, the term "about" as used herein generally means within 10%, 5%, 1%, or 0.5% of a given value or range. Alternatively, the term "about" means within an acceptable standard error, as would be considered by a person skilled in the art.

[0035] The terms "subject" and "patient" are used interchangeably herein and mean an animal, including a human, that can be treated by the synthetic peptides and / or methods of the present invention. The term "subject" or "patient" is intended to refer to both male and female genders unless one gender is specified. Thus, the term "subject" or "patient" includes any mammal that can benefit from the treatment methods of the present disclosure. 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 a human.

[0036] In addition, in this specification, unless otherwise restricted, %(w / v) represents the weight / volume percentage concentration.

[0037] Damage-associated molecular patterns (DAMPs) are biomolecules released in association with cellular stress such as cell death and cell damage, and function as alarms to notify of a cellular crisis. DAMPs are released as an initial reaction to immune cell activation in tumor immunity.

[0038] Hereinafter, embodiments of the present invention will be described. The following embodiments are illustrative, and the scope of the present invention is not limited to those shown in the following embodiments. Note that for the sake of brevity, the disclosure description of similar content will be omitted.

[0039] Method for Activating Immunity Against Tumors The method for activating immunity against tumors according to this embodiment includes the steps of providing a pharmaceutical composition containing a hydrogen peroxide solution, administering the pharmaceutical composition to a target site of a patient having the tumor, and irradiating the target site with radiation, wherein the tumor is present remotely from the target site of the patient having the tumor.

[0040] 3 Pharmaceutical Composition The pharmaceutical composition for activating immunity against tumors according to this embodiment contains a hydrogen peroxide solution. The pharmaceutical composition according to this embodiment can treat tumors by activating immunity against tumors. "Treating a tumor" or "anti-tumor effect" means reducing the size of the tumor and / or suppressing the growth of the tumor.

[0041] 4 Hydrogen Peroxide Solution The hydrogen peroxide solution according to this embodiment is an aqueous solution containing hydrogen peroxide (H2O2; molecular weight 34). The proportion of hydrogen peroxide contained in the pharmaceutical composition according to this embodiment may be 0.01 to 3.5% (w / v), and may be within the 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). "Hydrogen peroxide solution" refers to a solution obtained by dissolving hydrogen peroxide in distilled water such as the Japanese Pharmacopoeia unless otherwise specified.

[0042] 5 Additional Components The pharmaceutical composition according to this embodiment may contain additional components. The additional components include hyaluronic acid, liposomes, polymer gels, hydrogels, and gelatin or salts thereof. Further, the additional components may be pharmaceutically acceptable physiological saline, buffers (for example, phosphate buffer, Tris buffer, and acetate buffer), stabilizers, isotonic agents, and pH adjusters.

[0043] The blending ratio of hyaluronic acid or its salt to hydrogen peroxide may be 1 to 10,000 parts by mass of hyaluronic acid or its salt (as a total amount) with respect to 100 parts by mass of hydrogen peroxide, or may be within the range between two values selected from the group consisting of 1, 5, 10, 50, 100, 150, 500, 1,000, 5,000, and 10,000.

[0044] The hyaluronic acid according to the present embodiment may be extracted from animal tissues or may be produced by a fermentation method. The hyaluronic acid according to the present embodiment is preferably produced by a fermentation method. This is because the hyaluronic acid produced by the fermentation method has high safety and high production stability. Examples of the strain used in the fermentation method include hyaluronic acid-producing microorganisms isolated from nature (for example, Streptococcus), Streptococcus equi FM-100 (FERM BP-9027) described in JP-A-63-123392, and Streptococcus equi FM-300 (FERM BP-2319) described in JP-A-2-234689.

[0045] The hyaluronic acid according to the present embodiment has a mass average molecular weight of about 500,000 to 10,000,000, preferably 500,000 to 8,000,000, and more preferably 500,000 to 5,000,000.

[0046] The mass average molecular weight of hyaluronic acid can be measured by the SEC-MALLS method using a size exclusion chromatogram (SEC) and a multi-angle light scattering detector (MALLS).

[0047] The hyaluronic acid according to the present embodiment may be used as an aqueous solution or a water-swellable gel.

[0048] The hyaluronic acid according to this embodiment includes non-crosslinked hyaluronic acid and crosslinked hyaluronic acid. The crosslinked hyaluronic acid is a polymer having a three-dimensional network structure. When the crosslinking points of the crosslinked hyaluronic acid are cleaved, linear hyaluronic acid (non-crosslinked hyaluronic acid) is generated. Note that the mass average molecular weight and the branching degree of the hyaluronic acid generated by cleaving the crosslinking points can be measured by GPC-MALLS (multi-angle light scattering) using a gel permeation chromatogram (GPC), a differential refractometer, and a multi-angle laser light scattering detector (MALLS).

[0049] The hyaluronic acid according to this embodiment may be non-crosslinked hyaluronic acid, crosslinked hyaluronic acid, or a combination thereof. The hyaluronic acid according to this embodiment may be composed of different crosslinked hyaluronic acids or may be composed of hyaluronic acids having different molecular weights. The hyaluronic acid according to this embodiment may be a hyaluronate. The hyaluronate may be sodium hyaluronate, potassium hyaluronate, or lithium hyaluronate.

[0050] The proportion of the hyaluronic acid or its salt contained in the pharmaceutical composition according to this embodiment may be 0.1 to 10% (w / v), or may be within the 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).

[0051] Examples of the isotonic agent include sodium chloride, glycerin, glucose, polyethylene glycol, propylene glycol, D-mannitol, fructose, xylitol, sodium dihydrogen phosphate, and sodium phosphate, and preferably sodium chloride. Examples of the pH adjuster include hydrochloric acid and sodium hydroxide. The pH of the pharmaceutical composition according to this embodiment is adjusted to 6 to 8.5, preferably 6.8 to 7.8. The pharmaceutical composition according to this embodiment may contain a buffer solution for maintaining the pH.

[0052] Step of providing a pharmaceutical composition The "step of providing a pharmaceutical composition" according to this embodiment may be a step of preparing a pharmaceutical composition or a step of formulating a pharmaceutical composition. In one embodiment, the step of formulating a pharmaceutical composition includes the step of mixing a hydrogen peroxide solution and hyaluronic acid or a salt thereof.

[0053] Step of administering the pharmaceutical composition to the target site of a patient having a tumor The pharmaceutical composition according to this embodiment is administered to the target site of a patient having a tumor. The method of local administration is not limited. In one embodiment, the target site is a site where the pharmaceutical composition is administered and irradiated with radiation. In another embodiment, it is a site having a tumor that requires treatment where the pharmaceutical composition is administered and irradiated with radiation.

[0054] The tumor according to this embodiment may be present remotely from the target site of a patient having a tumor. In one embodiment, the tumor is present remotely from the target site of a patient having a tumor, and no detectable tumor is present at the target site. In another embodiment, the tumor is present remotely from the target site of a patient having a tumor and is also present at the target site. In yet another embodiment, the tumor includes a first tumor and a second tumor. The first tumor is present remotely from the target site of a patient having a tumor, and the second tumor is present at the target site of a patient having a tumor. The first tumor may be derived from a metastatic cancer, and the second tumor may be derived from a primary cancer.

[0055] "Tumor" refers to cells characterized by uncontrolled growth, including pre-neoplastic hyperplasia, primary cancer, metastatic cancer, neoplasm, and solid tumor. "Tumor" may be caused by "cancer". Examples of "cancer" include, but are 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, thymus cancer, thyroid cancer, uterine cancer, vaginal cancer, and vulvar cancer.

[0056] In certain embodiments, the target tumor is a tumor caused by metastatic cancer and the non-target tumor is a tumor caused by primary cancer. In other embodiments, the target tumor is a tumor caused by primary cancer and the non-target tumor is a tumor caused by metastatic cancer.

[0057] Step of irradiating the target site with radiation The "step of irradiating the target site with radiation" according to this embodiment is carried out after the "step of administering a pharmaceutical composition to the target site of a patient having a tumor". The pharmaceutical composition according to this embodiment may activate immunity against the tumor by irradiating the target site where the pharmaceutical composition has been administered with radiation.

[0058] The radiation according to this embodiment may be X-rays, electron beams, proton beams, heavy particle beams, α (alpha) rays, β (beta) rays, γ (gamma) rays, or a combination thereof. X-rays or electron beams can be irradiated using a linear accelerator. The radiation dose according to this embodiment may be 1.5 to 4 Gy per time, and may also be within the 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. The irradiation of the radiation according to this embodiment may be performed 2 to 5 times a week, preferably 4 to 5 times a week. The irradiation of the radiation according to this embodiment may be performed over 1 to 5 weeks. The total radiation dose according to this embodiment may be 20 to 70 Gy, and may also be within the range between two values selected from the group consisting of 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, and 70 Gy.

[0059] 9 Step of administering an anti-tumor immunotherapy agent The pharmaceutical composition according to this embodiment may be used in combination therapy with an anti-tumor immunotherapy agent. The method according to this embodiment may further include the step of "administering an anti-tumor immunotherapy agent". In certain embodiments, the step of "administering an anti-tumor immunotherapy agent" may be the step of "administering an anti-tumor immunotherapy agent to the target site", or may be the step of "administering an anti-tumor immunotherapy agent to a site of the patient other than the target site".

[0060] The step of "administering an anti-tumor immunotherapy agent" according to this embodiment may be performed after the step of "irradiating the target site with radiation", or may be performed between the step of "administering the pharmaceutical composition to the target site of a patient having a tumor" and the step of "irradiating the target site with radiation", or may be performed before the step of "administering the pharmaceutical composition to the target site of a patient having a tumor".

[0061] 10 Anti-tumor immunotherapy agent The anti-tumor immunotherapy agent according to this embodiment refers to a substance (e.g., compound, cell (e.g., immune cell), protein (e.g., antibody), and nucleic acid molecule (e.g., DNA and RNA)) that reduces the size of a tumor or suppresses tumor growth by enhancing or assisting the immune function, and includes anti-tumor immunostimulatory chemotherapeutic agents, immune checkpoint inhibitors, and tumor immune activators, as well as combinations thereof.

[0062] In certain embodiments, the anti-tumor immunotherapy agent may be an anti-tumor immunostimulatory chemotherapeutic agent (e.g., gemcitabine).

[0063] In certain embodiments, the anti-tumor immunotherapy agent can specifically bind to PD-1, PD-L1, or CTLA-4. In another embodiment, the anti-tumor immunotherapy agent may be an immune checkpoint inhibitor. In certain embodiments, 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 any antigen-binding fragment of any one of the foregoing.

[0064] In certain embodiments, the anti-tumor immunotherapy agent may be a 41-BB agonist, an OX-40 agonist, a TIGIT inhibitor, a LAG-3 inhibitor, or an IDO inhibitor.

[0065] 11 Other Embodiments A pharmaceutical composition for activating immunity against a tumor when used in combination with radiotherapy, (i) A pharmaceutical composition used in a combination therapy of a hydrogen peroxide solution and an anti-tumor immunotherapy agent, containing a hydrogen peroxide solution, Or, (ii) A pharmaceutical composition used in a combination therapy of a hydrogen peroxide solution and an anti-tumor immunotherapy agent, containing an anti-tumor immunotherapy agent, is also provided.

[0066] There is also provided a pharmaceutical composition containing an antitumor immunotherapeutic agent, which is used in combination therapy with a hydrogen peroxide solution and the antitumor immunotherapeutic agent and is used in combination with radiotherapy to activate immunity against tumors.

[0067] There is also provided the use of a hydrogen peroxide solution in the manufacture of a pharmaceutical composition for activating immunity against tumors. There is also provided the use of a hydrogen peroxide solution and an antitumor immunotherapeutic agent in the manufacture of a pharmaceutical composition for activating immunity against tumors.

Example

[0068] The data in the examples are presented as mean ± standard error of the mean (SEM). The means were compared using the Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.00001. "ns" means not significant.

[0069] Example 1 It was confirmed whether HMGB1, a damage-associated molecular pattern (DAMPs) from cancer cells, was produced by the combination of hydrogen peroxide + radiotherapy (RT). KORTUC (hydrogen peroxide + hyaluronic acid) was added to MC38 cells (mouse colorectal cancer cell line), and RT was performed. The concentration of hydrogen peroxide in the medium was 0.0067% (w / v) or 0.0335% (w / v) (10 μM or 50 μM hydrogen peroxide), and the concentration of hyaluronic acid in the medium was 0.0111% (w / v) or 0.0556% (w / v). The dose of RT was 5.0 Gy (1 Gy / min). RT alone for MC38 cells was also performed. The treatment without KORTUC administration or RT was used as a control. MC38 cells and the culture supernatant were collected 24 hours after radiation exposure, and HMGB1 was quantitatively measured by ELISA. The results are shown in Figure 1.

[0070] Both KORTUC administration + RT (RT+KORTUC) and RT released HMGB1 into the culture supernatant, but RT+KORTUC released more HMGB1 into the culture supernatant than RT. From this result, it was suggested that KORTUC administration + RT might induce an immune response.

[0071] Example 2 It was examined whether the combined use of KORTUC (a mixed solution of hydrogen peroxide + hyaluronic acid, the concentration of hydrogen peroxide in the mixed solution was 0.5%, and the concentration of hyaluronic acid was 0.83%) administration + RT also induced an immune response in mice. Further experiments were conducted in mice implanted with MC38 cells. As shown in Fig. 2, MC38 cells were implanted into the left foot (irradiated side site) and right abdomen (non-irradiated side site) of the mice (day 0). Ten days after cell transplantation, the irradiated side site was irradiated with 15 Gy of radiation (RT) or KORTUC was administered to the irradiated side site and irradiated with 15 Gy of radiation (RT+KORTUC). The treatment without KORTUC administration or RT was used as a control.

[0072] Fig. 3A shows a bar graph regarding the number of infiltrating cytotoxic T cells (CD8) in tumor-infiltrating lymphocytes (TIL) infiltrating into the non-irradiated side site measured by flow cytometry. Fig. 3B shows a bar graph regarding the number of infiltrating dendritic cells (DC) in tumor-draining lymph nodes (TDLN) near the irradiated side site measured by flow cytometry.

[0073] In RT+KORTUC, the number of infiltrating cytotoxic T cells (CD8) and DC increased significantly compared with the control and RT (Fig. 3A and 3B, respectively). From this result, it became clear that RT+KORTUC induced an immune response in vivo. In particular, since the number of cytotoxic T cells (CD8), which are immune cells, also increased in the non-irradiated side site in RT+KORTUC, it became clear that RT+KORTUC exerted an effect not only on local tumors but also on distant tumors.

[0074] Example 3 The effect of the combined administration of KORTUC (a mixed solution of hydrogen peroxide and hyaluronic acid, with the concentration of hydrogen peroxide in the mixed solution being 0.5% and the concentration of hyaluronic acid being 0.83%) + RT on tumors was measured over time. MC38 cells were implanted into the left hind limb (irradiated side site) and the right abdomen (non-irradiated side site) of mice (day 0) (see Figure 2). Ten days after transplantation, KORTUC was administered to the irradiated side site (KORTUC alone), 15 Gy of radiation was applied to the irradiated side site (RT alone), or KORTUC was administered to the irradiated side site and 15 Gy of radiation was applied (KORTUC + RT). The treatment with neither KORTUC administration nor radiation administration was used as a control.

[0075] Figure 4A is a graph plotting the tumor size over time at the irradiated side site (n = 6), and Figure 4B is a graph plotting the tumor size over time at the non-irradiated side site (n = 6). At the irradiated side site, a decrease in tumor size over time was observed in both RT alone and KORTUC + RT. At the non-irradiated site, an inhibitory effect on tumor growth was observed in KORTUC + RT. Although the antitumor effect on the irradiated side was equivalent between RT alone and KORTUC + RT, RT alone did not suppress tumor growth on the non-irradiated side, while KORTUC + RT significantly suppressed tumor growth. Therefore, it is considered that this is not due to the effect of radiosensitization by KORTUC at the irradiated site, but rather due to the induction of tumor immunity by KORTUC.

[0076] Example 4 The effects of the combined use of KORTUC (a mixed solution of hydrogen peroxide and hyaluronic acid, the concentration of hydrogen peroxide in the mixed solution is 0.5%, and the concentration of hyaluronic acid is 0.83%) administration + RT + an anti-tumor immunotherapeutic agent were measured over time. MC38 cells were implanted into the left foot (irradiated side site) and the right abdomen (non-irradiated side site) of mice (day 0) (see Figure 2). As the anti-tumor immunotherapeutic agent, an anti-PD-1 antibody (aPD1), an immune checkpoint inhibitor, was used. Ten days after transplantation, the anti-PD-1 antibody was administered intraperitoneally (aPD1 alone), or the anti-PD-1 antibody was administered intraperitoneally and the irradiated side site was irradiated with 15 Gy of radiation (RT + aPD1), or the anti-PD-1 antibody was administered intraperitoneally and KORTUC was further administered to the irradiated side site and irradiated with 15 Gy of radiation (KORTUC + RT + aPD1). The treatment with no KORTUC administration, no anti-PD-1 antibody administration, and no radiation irradiation was used as a control.

[0077] Figure 5 is a graph plotting the size of tumors in the non-irradiated site over time (n = 5). As shown in Figure 5, a decrease in tumor size was observed over time in KORTUC + RT + aPD1. First, comparing the results of KORTUC + RT in Figure 4B and the results of KORTUC + RT + aPD1 in Figure 5, it became clear that KORTUC + RT + aPD1 had a higher anti-tumor effect on distant tumors than KORTUC + RT.

[0078] It was clarified that the combined use of KORTUC (hydrogen peroxide) administration and RT induced an immune response and exerted an anti-tumor effect on distant tumors, especially those not related to RT irradiation or local injection. In addition, it was clarified that the combined use of KORTUC (hydrogen peroxide), the administration of an anti-tumor immunotherapeutic agent, and RT induced a stronger immune response and exerted a greater anti-tumor effect on distant tumors than the combined use of KORTUC (hydrogen peroxide) administration and RT.

Claims

1. A pharmaceutical composition comprising a hydrogen peroxide solution for use in conjunction with radiation therapy to stimulate immunity against tumors.

2. The pharmaceutical composition of claim 1 , wherein the tumor is located remotely from the target site in a patient having the tumor.

3. The pharmaceutical composition of claim 1 , further comprising hyaluronic acid or a salt thereof.

4. The pharmaceutical composition according to claim 1 , comprising a liposome, a polymer gel, a hydrogel, or gelatin, or a salt thereof.

5. The pharmaceutical composition according to any one of claims 1 to 4, which is used in combination therapy with an antitumor immunotherapeutic agent.

6. The pharmaceutical composition of claim 5 , wherein the combination therapy improves antitumor effects.

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

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

11. The pharmaceutical composition of claim 5 , wherein the anti-tumor immunotherapeutic agent is an anti-tumor immunostimulating chemotherapeutic agent.

12. The pharmaceutical composition of claim 11, wherein the anti-tumor immunostimulatory chemotherapeutic drug is gemcitabine.

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

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

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

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