Radiosensitizing vaccine

Abstract

To provide a radiosensitizing vaccine for use in cancer immunoradiotherapy that achieves sufficient therapeutic effect against tumors with low doses of external radiation and also exerts therapeutic effect against tumors distant from an external radiation irradiation site.SOLUTION: Provided is a radiosensitizing vaccine for use in cancer immunoradiotherapy, the radiosensitizing vaccine comprising mesoporous silica-radiosensitizer particles in which mesoporous silica and a radiosensitizer are integrated, the immunoradiotherapy comprising administering the radiosensitizing vaccine to a subject with cancer and external radiation irradiation to the cancer, and the radiosensitizing vaccine inducing radiosensitization of the cancer and immune activation in the subject.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to a radiosensitizing vaccine for use in immunoradiotherapy of cancer. [Background technology]

[0002] Cancer treatments include surgery, radiation therapy, chemotherapy, and immunotherapy. Radiation therapy involves irradiating tumors locally with radiation to shrink or eliminate them. Radiation therapy is an excellent treatment method that places minimal physical strain on the patient and allows for the preservation of bodily function and form. However, low-dose external radiation does not provide sufficient therapeutic effect on localized tumors. Furthermore, high-dose external radiation significantly damages the immunogenicity of surrounding tissues and killed cancer cells, resulting in serious adverse events and insufficient therapeutic effect on recurrent or distant metastatic cancer.

[0003] Research is currently being conducted into administering radiosensitizers to patients as a way to enhance the therapeutic effects of external beam radiation therapy. However, because the efficacy of radiosensitizers is difficult to prove through clinical trials, the first and only radiosensitizer approved for human use in the world is the hafnium oxide (HfO2) radiosensitizer, approved in Europe in 2019. While intratumoral administration of HfO2 in combination with radiotherapy has been shown to achieve higher energy deposition and therapeutic efficacy than radiotherapy alone, its pathological complete response rate is less than 20%, and its therapeutic efficacy for recurrent and distant metastatic cancer is poor (Non-Patent Document 1). This is thought to be due to (1) the fact that existing HfO2 radiosensitizers improve therapeutic efficacy by increasing radiation absorption, resulting in insufficient therapeutic efficacy, and (2) the inability of existing HfO2 radiosensitizers to effectively induce systemic anti-tumor immunity after radiation exposure, resulting in insufficient efficacy in preventing cancer recurrence and metastasis.

[0004] Research is also being conducted on radiosensitizers other than hafnium oxide, with copper oxide (CuO) being one of the leading candidates. For example, Non-Patent Document 2 is a paper that studies the effects of a combination of copper oxide nanoparticles, hyperthermia, and external radiation on MCF-7 cells (breast cancer cells), and describes how the combination of these three agents inhibited cell proliferation. Non-Patent Document 3 is also a paper that studies a combination of copper oxide nanoparticles, hyperthermia, and radiation, and describes how microwave irradiation of copper oxide nanoparticles in the tumor microenvironment generates oxygen and upregulates tumor reoxygenation. However, Non-Patent Documents 2 and 3 do not describe the effects of combining copper oxide nanoparticles and radiation.

[0005] Furthermore, Non-Patent Document 4 is a paper that studies the use of copper oxide nanoparticles as a radiosensitizer in cancer radiotherapy, and describes the inhibition of tumor growth by combining the administration of copper oxide nanoparticles with external radiation irradiation. Although Non-Patent Document 4 describes radiosensitization by copper oxide, it does not describe the immunostimulatory effect of radiation irradiation (i.e., vaccine effect).

[0006] Furthermore, Patent Document 1 discloses nanocomposite particles for additively or synergistically enhancing the effects of photothermal therapy for cancer. The nanocomposite particles include core-shell-shell nanoparticles and encapsulated nanorods bound to the core-shell-shell nanoparticles via electrostatic interactions. The nanorods may be copper nanorods, and the nanorods are encapsulated in a mesoporous scaffold, such as a mesoporous silica scaffold. To treat cancer, nanocomposite particles containing an antitumor agent or photosensitizer in the mesoporous scaffold of encapsulated nanorods are administered to a subject, and the subject is then irradiated with light having a wavelength of 750 to 850 nm. While Patent Document 1 discloses nanocomposite particles that may contain copper in the nanorods, it does not disclose the use of copper oxide as a radiosensitizer or immunoradiotherapy using mesoporous silica in combination with a radiosensitizer.

[0007] The mesoporous silica used in Patent Document 1 is amorphous calcined silica (SiO2) with numerous micropores (mesopores) of 2 nm to 50 nm in diameter. Mesoporous silica is an inorganic porous material with adsorptive properties and is non-toxic to living organisms, and has long been used as an additive to health foods and pharmaceuticals. The use of mesoporous silica as an immune adjuvant is disclosed in Patent Documents 2 and 3, jointly filed with the present applicant. Patent Document 2 discloses mesoporous silica particles carrying an immune stimulating factor. Patent Document 3 also discloses mesoporous silica particles that can stimulate cellular adaptive immunity effective against tumor cells without the addition of an immune stimulating substance. However, Patent Documents 2 and 3 do not disclose the combination of mesoporous silica with a radiosensitizer.

[0008] Furthermore, Patent Document 4 discloses an immunotherapy construct comprising a delivery vehicle, at least one adjuvant, and one or more therapeutic agents / compounds that cause antigen release and / or modulate the immunosuppressive tumor microenvironment. In one embodiment of this invention, the delivery vehicle may be mesoporous silica nanoparticles, and the therapeutic agent may be a radiotherapeutic agent, examples of which include: 64 Cu and 67 The radiotherapeutic agent is intended for internal radiation therapy, and there is no mention of radiosensitizers, specifically hafnium oxide or copper oxide, to enhance the therapeutic effect of external radiation. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Special Publication No. 2022-545786 [Patent Document 2] Patent No. 5999639 [Patent Document 3] Patent No. 6868862 [Patent Document 4] Special Publication No. 2022-540867 [Non-patent literature]

[0010] [Non-Patent Document 1] Bonvalot, et al. Lancet Oncol 2019; 20: 1148-1159. [Non-patent document 2] Ghaleh, Hadi Esmaeli Govarchin, et al. Artif Cells Nanomed Biotechnol 2019, Vol. 47, No. 1: 1396-1403. [Non-patent document 3] Chen, Zengzhen, et al., Theranostics. 2020, Vol. 10(10), Issue 9: 4659-4675. [Non-patent document 4] Jiang, Yao-Wen, et al. ACS Biomater. Sci. Eng. 2019, 5, 1569-1579. Summary of the Invention [Problem to be solved by the invention]

[0011] There is a need for a cancer treatment method that can achieve a therapeutic effect on local tumors by low-dose external irradiation and also enables the treatment of distant cancers without direct irradiation. [Means for solving the problem]

[0012] In light of the above-mentioned problems, the present inventors have conducted extensive research and have conceived the idea of ​​creating a radiosensitizing vaccine that simultaneously achieves cancer radiosensitization and immune activation in a subject by nanoscale integration of a radiosensitizer on a porous inorganic adjuvant. They have produced a radiosensitizing vaccine containing mesoporous silica-radiosensitizer particles in which mesoporous silica and a radiosensitizer are integrated. Furthermore, they have found that when the radiosensitizing vaccine of the present invention is administered to a subject and low-dose external irradiation is performed, an abscopal effect is induced, in which not only does the tumor shrink locally but also tumors at distant, unirradiated sites shrink. Induction of the abscopal effect is difficult to achieve using immunotherapy with mesoporous silica particles or radiotherapy with radiosensitizers, and is a surprising effect achieved by the radiosensitizing vaccine of the present invention.

[0013] The mechanism by which the abscopal effect is induced is presumed to be as follows. When the radiosensitizing vaccine of the present invention is administered to a subject, the mesoporous silica-radiosensitizer particles contained in the vaccine increase the tumor's radiosensitivity, resulting in tumor destruction at lower doses than conventional methods. The destroyed tumor promotes the release of neo-cancer antigens, which are then adsorbed and held on the mesoporous silica-radiosensitizer particles while maintaining their immunological activity. The adsorption and holding of neo-cancer antigens on the mesoporous silica-radiosensitizer particles promotes the uptake of neo-cancer antigens into antigen-presenting cells and the activation of T cells, resulting in the creation of a personalized in situ cancer vaccine in the body. Such personalized in situ cancer vaccines are expected to not only treat primary cancers but also prevent recurrence and distant metastasis.

[0014] The present invention relates to the following radiosensitizing vaccines. [1] A radiosensitizing vaccine for use in cancer immunoradiotherapy, the radiosensitizing vaccine comprising mesoporous silica-radiosensitizer particles in which mesoporous silica and a radiosensitizer are integrated, the immunoradiotherapy comprising administering the radiosensitizing vaccine to a subject having cancer and external radiation irradiation to the cancer, and the radiosensitizing vaccine inducing radiosensitization of the cancer and immune activation of the subject. [2] A radiosensitizing vaccine according to [1], which further induces the abscopal effect. [3] The radiosensitizing vaccine described in [1], wherein the radiosensitizer is copper oxide (CuO) or hafnium oxide (HfO2). [4] A radiosensitizing vaccine according to [3], wherein when the radiosensitizer is copper oxide (CuO), the molar ratio of copper (Cu) to silicon (Si) (Cu / Si) in the mesoporous silica-radiosensitizer particles is 0.1% to 40%, and when the radiosensitizer is hafnium oxide (HfO2), the molar ratio of hafnium (Hf) to silicon (Si) (Hf / Si) in the mesoporous silica-radiosensitizer particles is 1% to 40%. [5] The radiosensitizing vaccine according to [1], wherein the mesoporous silica-radiosensitizer particles have a particle diameter of 10 nm to 5000 nm. [6] The radiosensitizing vaccine according to [1], wherein the radiation exposure dose is 0.5 to 20 Gy. [7] The radiosensitizing vaccine described in [1], further comprising an immune checkpoint inhibitor. [8] The radiosensitizing vaccine described in [1], wherein the immunoradiotherapy further comprises administration of an immune checkpoint inhibitor. [Effects of the Invention]

[0015] The radiosensitizing vaccine of the present invention, which comprises mesoporous silica-radiosensitizer particles in which mesoporous silica and a radiosensitizer are integrated, can induce radiosensitization and immune activation in vivo by administering the vaccine and locally irradiating tumors with external radiation. It can also induce the shrinkage or disappearance of tumors at distant sites that have not been irradiated, i.e., the abscopal effect. [Brief explanation of the drawings]

[0016] [Figure 1] 1A shows a STEM-EDX image of mesoporous silica (MS)-CuO particles, (b) a TEM image, (c) an XRD pattern, and (d) a graph showing the Cu / Si molar ratio. [Figure 2] SEM image (a), TEM image (b), and XRD pattern (c) of mesoporous silica (MS) particles. [Figure 3] Bar graphs showing the in vitro proliferation rates of MOC2 cells (a, b) and LLC cells (c, d) at HfO2 and CuO concentrations of 0.5 μg / mL (a, c) and 1 μg / mL (b, d), respectively. [Figure 4] Inhibition of treated tumors and distant untreated tumors by a combination of MS-CuO particles and external irradiation. Experimental protocol (a), tumor volume of the left leg (b, c), and right leg (d). L1: No treatment, L2: Radiation only, L3: Intratumoral MS-CuO particle injection (0.1 mg CuO / mouse, i.e., approximately 5 mg / kg body weight) combined with 6 Gy of radiation, L4: Intratumoral HfO2 injection (0.1 mg HfO2 / mouse, i.e., approximately 5 mg / kg body weight) combined with 6 Gy of radiation, L5: Intratumoral MS particle injection combined with 6 Gy of radiation. [Figure 5] Graphs showing the increase in effector memory T cell populations (a, b) and central memory T cell populations (c, d) in mouse splenocytes at the end of an antitumor experiment by a combination of MS-CuO particles and external irradiation. [Figure 6] Graph showing the increase in cytokine levels in mouse splenocytes at the end of the antitumor experiment by the combination of MS-CuO particles and external irradiation: IL-12 (a), IFN-γ (b), and TNF-α (c). [Figure 7]Inhibition of treated tumors and distant untreated tumors by a combination of MS-CuO particles and external irradiation. Experimental protocol (a), tumor volume in the left leg (b), and right leg (c). M1: No treatment, M2: Radiation only, M3: Intratumoral injection of MS-CuO particles (0.1 mg CuO / mouse, i.e., approximately 5 mg / kg body weight) combined with 6 Gy of radiation, M4: Intratumoral injection of HfO2 (0.1 mg HfO2 / mouse, i.e., approximately 5 mg / kg body weight) combined with 6 Gy of radiation. [Figure 8] Graphs showing the increase in effector memory T cell populations (a, b) and central memory T cell populations (c, d) in mouse splenocytes at the end of an antitumor experiment due to the combination of MS-CuO particles and external irradiation. [Figure 9] Graph showing the increase in IL-12 (a), IFN-γ (b), and TNF-α (c) cytokine levels in mouse splenocytes at the end of the antitumor experiment by the combination of MS-CuO particles and external irradiation. [Figure 10] TEM images of mesoporous silica-HfO2-1 particles (a), MS-HfO2-2 particles (b), and MS-HfO2-3 particles (c), and XRD patterns of MS-HfO2-1 particles, MS-HfO2-2 particles, and MS-HfO2-3 particles (d) and a graph showing the Hf / Si molar ratio (e). [Figure 11] TEM images of MS-HfO2-4 particles (a), MS-HfO2-5 particles (b), and MS-HfO2-6 particles (c), and XRD patterns of MS-HfO2-4 particles, MS-HfO2-5 particles, and MS-HfO2-6 particles (d) and a graph showing the Hf / Si molar ratio (d). [Figure 12] Graphs showing the in vitro proliferation rate of MOC2 cells (a, b) and LLC cells (c, d) (HfO2 concentration 100 μg / mL). [Figure 13] STEM-EDX images of MS-HfO2-1 particle (a) and MS-HfO2-4 particle (b). [Figure 14]Inhibition of treated and distant untreated tumors by the combination of MS-HfO2-1 particles and external irradiation. Experimental protocol (a), tumor volume of the left leg (b, c), and right leg (d). L1: No treatment, L2: Radiation only, L4: Intratumoral HfO2 (0.1 mg HfO2 / mouse, i.e., approximately 5 mg / kg body weight) injection combined with 6 Gy of radiation, L5: Intratumoral MS particle injection (2 mg / mouse, i.e., 100 mg / kg body weight) injection combined with 6 Gy of radiation, L6: Intratumoral MS-HfO2-1 (2 mg / mouse, i.e., 100 mg / kg body weight) injection combined with 6 Gy of radiation, L7: Intratumoral HfO2 (0.62 mg HfO2 / mouse, i.e., approximately 31 mg / kg body weight) injection combined with 6 Gy of radiation. [Figure 15] Inhibition of treated tumors and distant untreated tumors by the combination of MS-HfO2-1 particles and radiation. Experimental protocol (a), tumor volumes in the left leg (b) and right leg (c). H1: No treatment, H2: 6 Gy (3 times) irradiation only, H3: Intratumoral MS-HfO2-1 particle injection (0.5 mg / mouse, i.e., approximately 25 mg / kg body weight) combined with 6 Gy (3 times) irradiation, H4: Intratumoral MS-HfO2-1 particle injection (1 mg / mouse, i.e., approximately 50 mg / kg body weight) combined with 6 Gy (3 times) irradiation, H5: Intratumoral MS-HfO2-1 injection (2 mg / mouse, i.e., approximately 100 mg / kg body weight) combined with 6 Gy (3 times) irradiation, H6: 8 Gy (3 times) irradiation only, H7: Intratumoral MS-HfO2-1 (1 mg / mouse) injection combined with 8 Gy (3 times) irradiation. [Figure 16]Inhibition of treated and distant untreated tumors by the combination of MS-HfO2-1 particles and external irradiation. Experimental protocol (a), tumor volume of the left leg (b), and right leg (c). B1: No treatment; B2: 3 Gy (4 doses) irradiation only; B3: intratumoral injection of MS-HfO2-1 particles (2 mg / mouse, i.e., approximately 100 mg / kg body weight) combined with 3 Gy (4 doses) irradiation; B4: 4 Gy (4 doses) irradiation only; B5: intratumoral injection of MS-HfO2-1 particles (2 mg / mouse) combined with 4 Gy (4 doses) irradiation; B6: 5 Gy (3 doses) irradiation only; B7: intratumoral injection of MS-HfO2-1 particles (2 mg / mouse) combined with 5 Gy (3 doses) irradiation. [Figure 17] The combination of MS-HfO2-1 particles and external irradiation increased cytokine levels in mouse splenocytes at the end of the antitumor experiment: TNF-α (a), IL-6 (b), and IL-12 (c). [Figure 18] Inhibition of treated tumors and distant untreated tumors by a combination of MS-HfO2-4 particles, external irradiation, and anti-CTLA-4 administration. Experimental protocol (a), tumor volume of the left leg (b) and right leg (c). A1: No treatment, A2: Intraperitoneal injection of anti-CTLA-4 (0.2 mg / mouse, i.e., approximately 10 mg / kg body weight) and 6 Gy of irradiation, A3: Intratumoral injection of MS-HfO2-4 particles (2 mg / mouse, i.e., approximately 100 mg / kg body weight) combined with intraperitoneal injection of anti-CTLA-4 (0.2 mg / mouse) and 6 Gy of irradiation. [Figure 19] Increase in effector memory T cell populations among CD4+ (a) and CD8+ (b) T cells in spleen cells of mice on day 9 of an antitumor experiment by combining MS-HfO2-4 particles, external irradiation, and anti-CTLA-4 administration. [Figure 20] Photomicrographs of tissue sections from the heart, kidney, lung and liver of a mouse after intratumoral injection of MS-HfO2-1 particles (2 mg / mouse, i.e., approximately 100 mg / Kg body weight). DETAILED DESCRIPTION OF THE INVENTION

[0017] 1. Mesoporous silica - radiosensitizer particles The radiosensitizing vaccine of the present invention comprises mesoporous silica-radiosensitizer particles in which mesoporous silica and a radiosensitizer are integrated.

[0018] (1) Radiosensitizers In the present invention, a radiosensitizer refers to a reagent that increases the sensitivity of tumors to externally irradiated radiation in radiotherapy. Metals and compounds have been studied as radiosensitizers, and hafnium oxide (HfO2) has been approved in Europe. The radiosensitizer used in the present invention is not particularly limited as long as it can be used to produce mesoporous silica-radiosensitizer particles in which mesoporous silica and a radiosensitizer are integrated and the radiosensitizing effect is maintained even after integration with mesoporous silica. However, metals are preferred from the viewpoint of ease of production, and hafnium oxide (HfO2) and copper oxide (CuO) are preferred because of their high radiosensitizing and immunostimulating effects as mesoporous silica-radiosensitizer particles.

[0019] (2) Mesoporous silica Mesoporous silica is silicon dioxide with mesopores, which are defined by IUPAC as pores with diameters between 2 and 50 nm. If numerous mesopores are regularly arranged, the presence of mesopores can be determined by whether or not peaks appear at positions corresponding to lattice spacings of 2 to 50 nm using powder X-ray diffraction. Such peaks appear in the 2θ range of 0.5 to 3.0 using CuKα X-ray diffraction. If the mesopores are irregularly arranged, they can be confirmed using a transmission electron microscope. Alternatively, pore size can be measured using an automatic specific surface area / pore size distribution analyzer.

[0020] (3) Mesoporous silica-radiosensitizer particles The mesoporous silica-radiosensitizer particles of the present invention are particles in which mesoporous silica and a radiosensitizer are integrated. In the present invention, "mesoporous silica and a radiosensitizer are integrated" means that the radiosensitizer is distributed within the mesoporous silica constituting the particles in a form that allows it to exert its radiosensitizing effect. For example, the radiosensitizer may be distributed uniformly throughout the mesoporous silica constituting the particles, or may be distributed in at least a portion of the particles, such as the surface portion. By integrating the radiosensitizer with mesoporous silica, the tendency for aggregation is reduced compared to the case of the radiosensitizer alone, which may facilitate intravenous injection and uniform distribution within tumor tissue, and is thought to increase the number of cancer types that can be treated. When the radiosensitizer is a metal, the distribution of the radiosensitizer can be confirmed from the element distribution shown in the STEM-EDX image of the mesoporous silica-radiosensitizer particles.

[0021] Furthermore, the amount of radiosensitizer contained in the mesoporous silica-radiosensitizer particles is not particularly limited, as long as it is an amount that can exert both the radiosensitizing effect of the radiosensitizer and the immune activation effect of the mesoporous silica. The amount of radiosensitizer varies depending on the type of radiosensitizer. When the radiosensitizer is copper oxide (CuO), the molar ratio of copper (Cu) to silicon (Si) (Cu / Si) in the mesoporous silica-radiosensitizer particles is preferably 0.1% to 40%, more preferably 1% to 20%. When the radiosensitizer is hafnium oxide (HfO2), the molar ratio of hafnium (Hf) to silicon (Si) (Hf / Si) in the mesoporous silica-radiosensitizer particles is preferably 1% to 40%, more preferably 5% to 20%. When the molar ratio of copper (Cu) to silicon (Si) (Cu / Si) is 0.1% or more, or the molar ratio of hafnium (Hf) to silicon (Si) (Hf / Si) is 1% or more, radiosensitizing effects can be achieved in vivo. Furthermore, when the molar ratio of copper (Cu) to silicon (Si) (Cu / Si) and the molar ratio of hafnium (Hf) to silicon (Si) (Hf / Si) are 40% or less, mesoporous silica-radiosensitizer particles with uniform distribution and good dispersibility of each component can be easily synthesized.

[0022] The mesoporous silica-radiosensitizer particles are preferably spherical. Whether or not a particle is "spherical" can be determined based on the particle's circularity and content when observed under an electron microscope. In the present invention, the circularity of a particle is defined as "the ratio of the difference in diameter between two circles with the smallest distance between them, when the particle's outline is enclosed by a circumscribing circle and an inscribing circle with the same center, to the diameter of the circumscribing circle." When mesoporous silica-radiosensitizer particles are observed under an electron microscope, if the proportion of particles with a circularity of 20% or less is 65% or more, preferably 75% or more, and more preferably 80% or more, the mesoporous silica-radiosensitizer particles can strongly stimulate humoral adaptive immunity and cellular adaptive immunity. Note that the electron microscope may be either a scanning or transmission type, as long as it is possible to observe the mesoporous silica-radiosensitizer particles.

[0023] Mesoporous silica-radiosensitizer particles preferably have mesopores and are hollow (hollow type). Having a hollow interior not only enhances immunostimulatory activity but also increases the internal capacity for adsorbing antigens and other substances, resulting in superior antigen delivery and antigen protection. The "hollow" nature of the particles of the present invention means that, when observed with a transmission electron microscope, the area of ​​bright areas with high electron beam transmittance within the particles accounts for 5% or more of the particle's cross-sectional area, and the outer shell is mesoporous (e.g., Teng J. et al., Chem Mater. 2013; 25: 98-105). The pores in the outer shell may be composed solely of mesopores (5-50 nm), or may be composed of mesopores and pores larger than the mesopores.

[0024] Whether mesoporous silica-radiation sensitizer particles are hollow or not can be confirmed using a transmission electron microscope. If there is no hollow space inside, there is not much difference in the electron beam transmittance between the center and periphery of the particle, so the transmission electron microscope image will either have no difference in brightness between the center and periphery, or the periphery will be brighter. If there is a hollow space inside, the hollow space in the center will be bright because of its high electron beam transmittance, and the periphery will be dark because of its low electron beam transmittance. This peripheral part with low electron beam transmittance is the outer shell.

[0025] The thickness of the outer shell of the hollow mesoporous silica-radiosensitizer particles can be appropriately changed depending on the particle size, etc., but is preferably 10 to 200 nm, more preferably 10 to 80 nm. The reason why the lower limit is 10 nm is that mesopores (2 to 50 nm) must be able to exist in the outer shell.

[0026] For the particles to exhibit an immunostimulatory effect, they must be of a size that allows them to be phagocytosed by cells. Therefore, the particle diameter of the mesoporous silica-radiosensitizer particles is 10 nm to 5,000 nm, preferably 50 nm to 900 nm, more preferably 100 to 500 nm, and even more preferably 170 to 420 nm. In the present invention, the particle size refers to the diameter when the particles are spherical, and refers to the major axis when the particles are irregular in shape.

[0027] (4) Method for producing mesoporous silica-radiation sensitizer particles There are no particular limitations on the method for producing mesoporous silica-radiosensitizer particles, as long as particles in which mesoporous silica and a radiosensitizer are integrated can be obtained. For example, available methods include a method in which a raw material for the radiosensitizer is added to a raw material for the mesoporous silica to form particles, and a method in which mesoporous silica particles are obtained or produced and then the radiosensitizer is added.

[0028] Mesoporous silica particles can be produced by referring to the known method of Radu et al. (J Am Chem Soc. 2004; 126: 13216-7) or the method of Teng et al. (Chem Mater. 2013; 25: 98-105), etc. Spherical mesoporous silica particles can also be produced by referring to the above-mentioned Patent Documents 2 and 3. Specifically, mesoporous silica particles can be produced by a variety of methods, including using tetraethoxysilane, tetramethoxysilane, tetrapropoxysilane, sodium silicate, or the like as raw silicon source materials, and using a block copolymer such as poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) or a surfactant such as a long-chain alkylammonium salt such as hexadecyltrimethylammonium bromide as a mesopore template, making the liquid acidic or basic at 20 to 150°C, and hydrolyzing the silicon source raw materials such as tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate, tetrapropyl orthosilicate, and sodium silicate, followed by removing the template by extraction or combustion.

[0029] In the above-mentioned process for producing mesoporous silica particles, if a metal nitrate or metal alkoxide, which is a raw material for a radiosensitizer, is mixed with the raw silicon source material, it is possible to produce mesoporous silica-radiosensitizer particles that incorporate the metal that serves as the radiosensitizer.

[0030] Alternatively, ammonia is added to a dispersion of mesoporous silica particles produced by the above-mentioned method at 20°C to 150°C while stirring, followed by the addition of a metal nitrate or metal alkoxide, which is the raw material for the radiosensitizer, with very slow stirring, followed by further stirring at room temperature. The resulting solution is heated and further stirred, after which the precipitate is recovered by centrifugation, washed several times with ethanol, dried at 80°C, and heat-treated at 500°C to 800°C for 2 to 5 hours to obtain mesoporous silica-radiosensitizer particles.

[0031] (5) Other ingredients The radiosensitizing vaccine of the present invention may or may not contain other components, so long as it contains mesoporous silica-radiosensitizer particles. Thus, in one embodiment, the radiosensitizing vaccine of the present invention consists of mesoporous silica-radiosensitizer particles. Because the radiosensitizing vaccine of the present invention can be formulated alone, there is no need to add a molecular immunostimulatory substance with low stability. Therefore, the manufacturing process is not complicated, and the obtained formulation has the advantages of excellent storage stability and quality stability.

[0032] In another embodiment, the radiosensitizing vaccine of the present invention contains other components in addition to the mesoporous silica-radiosensitizer particles. The other components contained in the radiosensitizing vaccine are not particularly limited, but include substances that enhance the therapeutic effect against cancer, such as tumor antigens, chemotherapeutic agents, and known pharmacologically acceptable carriers, excipients, and additives. As described below, when the immunoradiotherapy includes treatment with a checkpoint inhibitor, the radiosensitizing vaccine of the present invention may also contain a checkpoint inhibitor.

[0033] 2. Immunoradiotherapy using radiosensitizing vaccines The radiosensitizing vaccine of the present invention is intended for use in immunoradiotherapy. In the present invention, "immunoradiotherapy" refers to a therapeutic method that involves administering a radiosensitizing vaccine to a cancer-bearing subject and irradiating the cancer with external radiation, in which the administered radiosensitizing vaccine induces radiosensitization and immune activation. The mesoporous silica-radiosensitizer particles contained in the radiosensitizing vaccine of the present invention increase tumor sensitivity to radiation in vivo (inducing radiosensitizing activity), thereby enabling a reduction in the radiation dose required for treatment. Furthermore, when tumors are destroyed by radiation irradiation and the release of neo-cancer antigens and other molecules is promoted, the released neo-cancer antigens are adsorbed and retained on the mesoporous silica portion of the mesoporous silica-radiosensitizer particles while maintaining their immune activity. Next, the mesoporous silica-radiosensitizer particles promote the uptake of neo-cancer antigens into antigen-presenting cells and the activation of T cells, resulting in the formation of a personalized in situ cancer vaccine in vivo. Such personalized in situ cancer vaccines are expected to be effective not only in treating primary cancers but also in preventing recurrence and distant metastasis. Therefore, when the radiosensitizing vaccine of the present invention is used in immunoradiotherapy, it is possible to induce the abscopal effect, in which the effects of radiotherapy, which is supposed to be a local therapy, are also effective on distant lesions. The induction of the abscopal effect is difficult to achieve with radiotherapy using a radiosensitizer, and is a surprising effect achieved by the radiosensitizing vaccine of the present invention.

[0034] The cancer to be treated in the present invention is not particularly limited as long as it is a cancer suitable for radiation therapy, and may be any of solid cancer, cell cancer, and blood cancer. Specific examples include neurogenic tumors including brain tumors, carcinomas such as squamous cell carcinoma or adenocarcinoma (head and neck cancer, skin cancer, esophageal cancer, thyroid cancer, stomach cancer, lung cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, liver cancer, prostate cancer, uterine cancer, ovarian cancer, breast cancer, kidney cancer, bladder cancer, and colorectal cancer), melanoma, bone and soft tissue tumor, lymphoma, leukemia, and myeloma. Furthermore, the radiosensitizing vaccine of the present invention is capable of inducing both radiosensitization and immune activation, and therefore may enable radiation therapy for radiation-resistant cancers that have previously been difficult to treat with radiation therapy.

[0035] The target of treatment with the radiosensitizing vaccine of the present invention is not limited to humans, but includes a wide range of mammals, including primates such as monkeys, dogs, cats, cows, horses, and mice.

[0036] The method of administration of the radiosensitizing vaccine of the present invention is not particularly limited, and examples thereof include oral administration and parenteral administration. Dosage forms suitable for oral administration include solid, semisolid, liquid, or gaseous forms, and specific examples include, but are not limited to, tablets, capsules, powders, granules, solutions, suspensions, syrups, elixirs, and aerosols. Parenteral administration methods include, for example, injection, transdermal administration, rectal administration, and intraocular administration. Injection administration methods include subcutaneous administration, intradermal administration, intravenous administration, intratumoral administration, and intramuscular administration.

[0037] The dose of the radiosensitizing vaccine of the present invention can be appropriately determined by a medical professional from the standpoint of safety and efficacy depending on the type of cancer, tumor size, tumor condition, patient age, severity of symptoms, condition, etc., and the type of radiotherapy to be performed. For example, the vaccine can be administered at a dose of 0.05 to 500 mg / kg body weight, preferably 0.5 to 200 mg / kg body weight, but is not limited thereto.

[0038] The external radiation irradiation administered in conjunction with the administration of the radiosensitizing vaccine of the present invention is not particularly limited, and the type, dose, and frequency of radiation can be similar to those used in conventional radiation therapy. Examples of radiation to be used include medical radiation, specifically X-rays, gamma rays, electron beams, beta rays, or particle beams (π-mesons, neutrons, or other heavy particles). The irradiation method is also not particularly limited, and can include conformal irradiation, stereotactic irradiation that pinpoints the lesion, intensity-modulated irradiation, irradiation using a sealed brachytherapy source, remote gamma irradiation, and irradiation using particle beams.

[0039] The radiation dose used in radiotherapy is selected from doses that are effective in shrinking tumors and have no side effects on the living body. For example, the dose per session can be 0.1 to 100 Gy, with a total dose of 1 to 500 Gy at the end of the treatment period, and the radiation can be administered over a period of 1 week to 6 months. By using the radiosensitizing vaccine of the present invention in combination with external radiation irradiation, it is possible to reduce the radiation dose to a lower level than that used conventionally. Therefore, the external radiation dose per session in radiotherapy in combination with the radiosensitizing vaccine of the present invention is preferably 0.5 to 20 Gy, more preferably 2 to 8 Gy.

[0040] The radiosensitizing vaccine of the present invention is administered to a patient during radiation therapy, but the specific timing of administration is not particularly limited. For example, the radiosensitizing vaccine may be administered before each of multiple repeated external radiation exposures, or the radiosensitizing vaccine may be administered daily while radiation exposure is performed every few days. The frequency with which the set of administration of the radiosensitizing vaccine of the present invention and radiation exposure is performed is not particularly limited, but is usually repeated once a day or less to suppress side effects of radiation therapy. For example, the set can be repeated once a day, at least once a week, at least once every two weeks, or at least once a month.

[0041] The radiation irradiation conditions, type of radiation source, irradiation method, irradiation site and irradiation period, administration route and administration time of the radiosensitizing vaccine, etc. can be appropriately selected by medical professionals.

[0042] 3. Combination use of immune checkpoint inhibitors Furthermore, the radiosensitizing vaccine of the present invention can be used in combination with an immune checkpoint inhibitor. Immune checkpoint inhibitors are drugs used to relieve immunosuppression, and their use is thought to produce a synergistic effect with personalized in situ cancer vaccines in vivo. Examples of immune checkpoint inhibitors include anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-CD40 antibodies, anti-CD137 antibodies, anti-OX40 antibodies, anti-TGF-β antibodies, anti-LAG3 antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-TIM3 antibodies, anti-CD96 antibodies, and anti-TIGIT antibodies. One or more immune checkpoint inhibitors can be used.

[0043] In one embodiment, the immune checkpoint inhibitor is contained in the radiosensitizing vaccine of the present invention together with the mesoporous silica-radiosensitizer particles and is administered simultaneously with the administration of the radiosensitizing vaccine. In another embodiment, a separately formulated immune checkpoint inhibitor is administered to a subject. In this case, the formulated immune checkpoint inhibitor may be administered in a mixture with the radiosensitizing vaccine of the present invention, or may be administered simultaneously or at an interval to the same subject. Furthermore, when the immune checkpoint inhibitor is administered after the administration of the radiosensitizing vaccine of the present invention, the immune checkpoint inhibitor may be administered before or after external radiation irradiation.

[0044] 4. Evaluation methods for radiosensitizing vaccines (1) Radiation sensitization effect The radiosensitizing effect of the radiosensitizing vaccine of the present invention can be evaluated, for example, by the following cell experiment.

[0045] Lewis lung carcinoma (LLC) cells and mouse oral squamous cell carcinoma 2 (MOC2) cells (5 × 10 3 After seeding (0.1 mL of cells / well) and culturing overnight, radiosensitizer alone or radiosensitizing vaccine (mesoporous silica-radiosensitizer particles) was added at a concentration of 0.5-1 μg / mL and cultured for 4 hours. The cells were exposed to different radiation doses (0, 4, 6, or 8 Gy) using an X-ray generator (Faxitron X-Ray Corp.) and cultured for an additional 3 days. Cell proliferation rates were measured using a CCK-8 kit (Dojindo Laboratories). The radiosensitizing effect can be confirmed by the fact that the proliferation rate of cultured cells to which the radiosensitizing vaccine (mesoporous silica-radiosensitizer particles) has been added is lower than that of cultured cells to which the radiosensitizer alone has been added, at the same radiation exposure dose.

[0046] (2) Radiation sensitization and immunostimulatory effects The radiosensitizing effect and immunostimulating effect of the radiosensitizing vaccine of the present invention can be evaluated, for example, by the following animal experiment.

[0047] On day 0, female C57BL / 6 mice (6 weeks old, obtained from CLEA Inc.) were injected subcutaneously into the legs with LLC cells or MOC2 cells (5 x 10 cells in the left leg). 5 cells / mouse, 1 x 10 in the right leg 5 On days 6, 7, and 8, mice were treated as follows: Group 1: no treatment, Group 2: external radiation to the legs only, Group 3: intratumoral injection of a radiosensitizing vaccine (mesoporous silica-radiosensitizer particles) combined with 6 Gy of radiation therapy to the left leg. Tumor volume was calculated as 1 / 2 x major axis x (perpendicular dimension) 2At the end of the experiment, splenocytes were collected from the mouse spleens, mashed, and filtered through a 40 μm cell strainer to obtain a single-cell suspension. Anti-CD16 / CD32 antibodies were used to prevent nonspecific staining. Anti-mouse CD3, anti-mouse CD4, anti-mouse CD8α, anti-mouse CD44, and anti-mouse CD62L (BioLegend) were used to stain the cells for 30 minutes. Flow cytometry analysis was performed to analyze the memory T cell population in the splenocytes. Interleukin-12 (IL-12), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) levels in the spleen were quantified using a mouse ELISA kit (BD Biosciences).

[0048] The abscopal effect was confirmed by the inhibition of tumor growth in both the treated leg (left leg) and the untreated distant leg (right leg). Additionally, T cell activation was confirmed by the increase in cytokine (IL-12, IFN-γ, and TNF-α) levels in mouse spleen cells. Furthermore, when the T cell population in mouse spleen cells was examined at the end of the antitumor experiment, a larger effector memory T cell population (CD4 + and CD8 + CD44 in high CD62L - ) and central memory T cell populations (CD4 + and CD8 + CD44 in high CD62L high ) can be predicted for the generation of personalized in situ cancer vaccines.

[0049] (3) Safety The in vivo safety of the radiosensitizing vaccine of the present invention can be evaluated, for example, by the following animal experiment.

[0050] Cancer cells (e.g., MOC2 cells) were injected subcutaneously into the legs of female C57BL / 6 mice (6 weeks old, obtained from CLEA Inc.) (1 × 10 6On the 10th day after injection, the mice were intratumorally injected with the radiosensitizing vaccine, and saline was injected as a control. On the 11th day, the heart, spleen, kidneys, lungs, and liver were harvested, fixed in 10% neutral buffered formalin solution, embedded in paraffin, and histological sections were prepared. The sections were stained with hematoxylin and eosin (HE) and observed under a microscope. If no significant differences are observed in tissue sections of the heart, spleen, kidney, lung, and liver between mice injected with the radiosensitizing vaccine and mice injected with saline under microscopic observation, it will be clear that the radiosensitizing vaccine is not significantly toxic and is safe.

[0051] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to the following examples. Other terms and concepts in the present invention are based on the meanings of terms commonly used in the relevant field, and the techniques used to carry out the present invention, except for those whose sources are particularly specified, can be easily and reliably carried out by a person skilled in the art based on known literature, etc. Furthermore, various analyses were carried out according to the methods described in the instruction manuals, catalogs, etc. of the analytical instruments, reagents, and kits used. The contents of the technical documents, patent publications and patent application specifications cited in this specification are to be referred to as the contents of the present invention. [Example]

[0052] Example 1: Preparation of mesoporous silica-CuO particles 0.4 g of triethanolamine (TEA) (Sigma-Aldrich) and 0.8 g of cetyltrimethylammonium p-toluenesulfonate (CTAT) (Sigma-Aldrich) were added to 20 g of ultrapure water and stirred at 75 °C for 30 minutes to dissolve. Next, 1.5 mL of tetraethyl orthosilicate (TEOS) (Fujifilm Wako Pure Chemical Industries, Ltd.) and 0.16 g of copper nitrate (Fujifilm Wako Pure Chemical Industries, Ltd.) were added to the solution. After 4.5 hours, the precipitate was collected, washed three times with ultrapure water and once with ethanol, and dried at 75 °C for 2 hours. The resulting powder was heat-treated at 550 °C for 5 hours to obtain mesoporous silica-CuO particles (hereafter often abbreviated as "MS-CuO particles"), which integrate the radiation sensitizer CuO and mesoporous silica.

[0053] The synthesized MS-CuO particles were evaluated using a transmission electron microscope (TEM) (JEOL) and powder X-ray diffraction analysis using CuKα radiation (Rigaku Corporation). The Cu / Si molar ratio of the synthesized MS-CuO particles was determined by dissolving the particles in NaOH and HCl, followed by analysis using inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Hitachi High-Tech Corporation). The results are shown in Figure 1.

[0054] As is clear from the element distribution (Cu, Si, O from left) in the STEM-EDX image of the MS-CuO particles (Figure 1a), all elements, Cu, Si, and O, were uniformly distributed in the MS-CuO particles, indicating that CuO was uniformly distributed in the mesoporous silica. The particle size of the MS-CuO particles was approximately 70-80 nm in diameter (Figure 1b), there were no impurities in the amorphous phase (Figure 1c), and the Cu / Si molar ratio was approximately 0.04 (Figure 1d).

[0055] Reference Example 1: Synthesis of mesoporous silica (MS) particles First, carbonaceous particles were synthesized to serve as templates. Glucose (0.5 M, Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a heat-resistant bottle (80 mL) in a stainless steel autoclave, sealed, and heated at 180°C for 4 hours. The carbonaceous particles were then collected by centrifugation at 4400 rpm for 20 minutes. The collected particles were washed three times with ultrapure water and twice with ethanol, and then dried at 80°C for 4 hours.

[0056] Next, mesoporous silica (MS) particles were synthesized using the co-templating method. Cetyltrimethylammonium p-toluenesulfonate (CTAT) (Sigma-Aldrich) and triethanolamine (TEA) (Sigma-Aldrich) were added to ultrapure water and dissolved by stirring at 70 °C. Next, carbonaceous particles (0.5 wt%) serving as templates were dispersed in the solution using ultrasonic waves. Tetraethoxysilane (TEOS) (Fujifilm Wako Pure Chemical Corporation) was added dropwise and vigorously stirred for 3 minutes. The molar ratio of the components in the reaction mixture was 1.00 TEOS: 0.06 CTAT: 0.026 TEA: 80 HO. Stirring was continued at 70 °C for 2 hours. The precipitate was collected by centrifugation, washed with deionized water and ethanol, and dried at 80 °C. The resulting product was heat-treated at 550 °C for 5 hours to remove the carbonaceous particles and CTAT serving as templates, yielding MS particles.

[0057] The synthesized MS particles were observed using a scanning electron microscope (SEM) and a transmission electron microscope (TEM), and the micrographs are shown in Figure 2a and b. They were also evaluated by powder X-ray diffraction analysis using CuKα radiation (Rigaku Corporation), and the resulting XRD pattern is shown in Figure 2c.

[0058] The obtained MS particles had a diameter of approximately 400-600 nm and a hollow structure with pores of 3-15 nm in the shell layer (Fig. 2a and b). o From the broad peak and Si-O band, it was confirmed that the MS particles were formed of amorphous silica.

[0059] Example 2: Cellular evaluation of mesoporous silica-CuO particles Lewis lung carcinoma (LLC) cells and mouse oral squamous cell carcinoma 2 (MOC2) cells (5 × 10 3 Cells were seeded in a 0.1 mL solution (cells / well) and cultured overnight. Next, HfO2 or MS-CuO particles prepared in Example 1 were added at concentrations of 0.5 to 1 μg / mL, respectively, and the cells were cultured for 4 hours. The cells were exposed to different radiation doses (0, 4, 6, or 8 Gy) using an X-ray generator (Faxitron X-Ray Corp.) and cultured for an additional 3 days. The cell proliferation rate was evaluated using a CCK-8 kit (Dojindo Laboratories). The results are shown in Figure 3. The target used was HfO2, a known radiation sensitizer.

[0060] The cell proliferation rate decreased with increasing HfO2 or CuO concentration and X-ray dose. Cells (LLC and MOC2) treated with MS-CuO particles and X-rays showed a decreased cell proliferation rate compared to cells treated with HfO2 particles (Figure 3a-d). MOC2 cells treated with HfO2 particles at a HfO2 concentration of 1 μg / mL showed proliferation rates of 81%, 76%, and 65% when exposed to 4 Gy, 6 Gy, and 8 Gy of X-rays, respectively (Figure 3b). On the other hand, MOC2 cells treated with MS-CuO particles at a CuO concentration of 1 μg / mL showed proliferation rates of 60%, 56%, and 53% when exposed to 4 Gy, 6 Gy, and 8 Gy of X-rays, respectively (Figure 3b). LLC cells treated with 1 μg / mL HfO2 showed proliferation rates of 80%, 71%, and 65% when exposed to 4, 6, and 8 Gy of X-rays, respectively (Fig. 3b). LLC cells treated with 1 μg / mL MS-CuO particles showed proliferation rates of 69%, 54%, and 48% when exposed to 4, 6, and 8 Gy of X-rays, respectively (Fig. 3d).

[0061] The above results confirmed that MS-CuO has a significant inhibitory effect on the proliferation of MOC2 and LLC cells, but no such high inhibitory effect on cell proliferation was observed with HfO2 at the same concentration.

[0062] Example 3: Evaluation of mesoporous silica-CuO particles using mice (LLC cells) On day 0 (d0), LLC cells were injected subcutaneously into the legs of female C57BL / 6 mice (6 weeks old, obtained from CLEA Inc.) (5 × 10 cells in the left leg). 5 cells / mouse, 1 x 10 in the right leg 5 On days 6, 7, and 8 (d6, d7, and d8), the mice were treated as follows: L1: no treatment; L2: external irradiation to the leg only; L3: intratumoral injection of MS-CuO (0.1 mg CuO / mouse, i.e., approximately 5 mg / kg body weight) in the left leg combined with 6 Gy of irradiation; L4: intratumoral injection of HfO2 (0.1 mg HfO2 / mouse, i.e., approximately 5 mg / kg body weight) in the left leg combined with 6 Gy of irradiation. As a control, a combination of intratumoral injection of MS particles (prepared in Reference Example 1) in the left leg combined with 6 Gy of irradiation (L5 group) was also examined. Specifically, mice subcutaneously injected with LLC cells as described above were irradiated with 6 Gy of radiation in the left leg on days 6, 7, and 8 (d6, d7, and d8), and MS particles (2 mg / mouse, i.e., 100 mg / kg body weight) were injected into the tumor in the left leg on days 9, 10, and 13 (d9, d10, and d13). The tumor volume was calculated as 1 / 2 × major axis × (vertical dimension) 2 The total number of cells was calculated as 1. At the end of the experiment, splenocytes were collected from the mouse spleens, mashed, and filtered through a 40 μm cell strainer to obtain a single-cell suspension. Anti-CD16 / CD32 antibodies were used to prevent nonspecific staining. Anti-mouse CD3, anti-mouse CD4, anti-mouse CD8α, anti-mouse CD44, and anti-mouse CD62L (BioLegend) were used to stain the cells for 30 minutes. Flow cytometry analysis was performed to analyze the memory T cell population in the splenocytes. Interleukin-12 (IL-12), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) levels in the spleen were quantified using a mouse ELISA kit (BD Biosciences). The results are shown in Figures 4 to 6.

[0063] Mice in the group receiving intratumoral MS-CuO injection combined with external irradiation (L3) showed significant tumor growth inhibition in both the treated leg (left leg, Figure 4b, c) and the untreated remote leg (right leg, Figure 4d) compared with mice in the untreated group (L1), the group receiving external irradiation only (L2), the group receiving intratumoral HfO injection combined with irradiation (L4), and the group receiving intratumoral MS particle injection combined with irradiation (L5). For tumors in the left leg, the tumor size in the L3 group on day 16 after LLC injection was 14%, 40%, 48%, and 62% of the tumor sizes in the L1, L2, L4, and L5 groups, respectively. For tumors in the right leg, the tumor size in the L3 group on day 16 after LLC injection was 50%, 37%, 38%, and 73% of the tumor size in the L1, L2, L4, and L5 groups, respectively.

[0064] To further investigate the mechanism of this abscopal effect, we examined the T cell population of mouse spleen cells at the end of the antitumor experiment. We found that the mice in the MS-CuO and external irradiation group (L3) had a larger effector memory T cell population (CD4 + and CD8 + CD44 in high CD62L - , Fig. 5a and b) and the central memory T cell population (CD4 + and CD8 + CD44 in high CD62L high , and Fig. 5c and d). In addition, the levels of IL-12 (Fig. 6a), IFN-γ (Fig. 6b), and TNF-α (Fig. 6c) in mouse splenocytes were highest in the group treated with MS-CuO and external irradiation (L3), demonstrating a significant increase in cytokine levels compared to the untreated group (L1) and the group treated with external irradiation only (L2).

[0065] Therefore, we confirmed the radiosensitizing and immunostimulating effects of MS-CuO on LLC cells in vivo, and further confirmed a significant proliferation inhibitory effect not only on the treated side but also on the untreated side. On the other hand, such a high proliferation inhibitory effect was not observed with HfO2 or MS particles alone.

[0066] Example 4: Evaluation of mesoporous silica-CuO particles using mice (MOC2 cells) Female C57BL / 6 mice (6 weeks old, obtained from CLEA Inc.) were injected subcutaneously into the legs with MOC2 cells (5 × 10 cells in the left leg). 5 cells / mouse, 1 x 10 in the right leg 5 Mice were treated on days 6, 7, and 8 (d6, d7, and d8) as follows: M1: no treatment; M2: external irradiation to the left leg only; M3: intratumoral injection of MS-CuO particles (0.1 mg CuO / mouse, i.e., approximately 5 mg / kg body weight) in the left leg combined with 6 Gy of irradiation; M4: intratumoral injection of HfO2 (0.1 mg HfO2 / mouse, i.e., approximately 5 mg / kg body weight) in the left leg combined with 6 Gy of irradiation. Tumor volume was calculated as 1 / 2 × major axis × (vertical dimension). 2 The total number of cells was calculated as 1. At the end of the study, splenocytes were collected from the mouse spleens, mashed, and filtered through a 40 μm cell strainer to obtain a single-cell suspension. Anti-CD16 / CD32 antibodies were used to prevent nonspecific staining. Anti-mouse CD3, anti-mouse CD4, anti-mouse CD8α, anti-mouse CD44, and anti-mouse CD62L (BioLegend) were used to stain the cells for 30 minutes. Flow cytometry analysis was performed to analyze the memory T cell population in the splenocytes. Interleukin-12 (IL-12), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) levels in the spleen were quantified using a mouse ELISA kit (BD Biosciences). The results are shown in Figures 7 to 9.

[0067] Mice in the group receiving intratumoral MS-CuO particle injection combined with external irradiation (M3) showed clear tumor growth inhibition in both the treated leg (left leg) and the untreated distant leg (right leg) compared with untreated mice in the group receiving external irradiation only (M1), mice in the group receiving external irradiation only (M2), and mice in the group receiving intratumoral HfO2 injection combined with external irradiation (M4) (Figure 7). To further explore the mechanism of this abscopal effect, we examined the T cell population in mouse spleen cells at the end of the antitumor experiment. We found that mice in the group receiving MS-CuO particle injection combined with external irradiation (M3) had a larger effector memory T cell population (CD4 + and CD8 + CD44 in high CD62L - , Fig. 8a and b) and the central memory T cell population (CD4 + and CD8 + CD44 in high CD62L high , and Fig. 8c and d). In addition, the levels of IL-12 (Fig. 9a), IFN-γ (Fig. 9b), and TNF-α (Fig. 9c) in mouse splenocytes were highest in the group treated with MS-CuO particles in combination with external irradiation (M3), demonstrating a significant increase in cytokine levels compared to the untreated group (M1) and the group treated with external irradiation alone (M2).

[0068] Therefore, we confirmed the radiosensitizing and immunostimulatory effects of MS-CuO particles on MOC2 cells in vivo, and further confirmed a significant proliferation inhibitory effect not only on the treated side but also on the untreated side. On the other hand, no such proliferation inhibitory effect was observed with HfO2 at the same concentration.

[0069] Summary of Examples 1 to 4 Mesoporous silica-copper oxide particles (MS-CuO particles) demonstrated a stronger antitumor effect than MS particles without CuO. Furthermore, MS-CuO particles achieved the highest levels of central memory T cells and effector memory T cells in the spleens of mice treated with the same dose of commercially available HfO2, while the same dose of commercially available HfO2 failed to produce radiosensitizing or immunostimulatory effects. They also achieved the highest levels of IL-12, IFN-γ, and TNF-α in the spleens of mice treated with MS-CuO.

[0070] Example 5: Preparation of mesoporous silica-HfO2 particles 1 0.6 g of triethanolamine (TEA) (Sigma-Aldrich) and 2 g of cetyltrimethylammonium chloride (CTAC) (Tokyo Chemical Industry Co., Ltd.) were added to 20 g of ultrapure water and stirred at 75 °C for 30 minutes to dissolve. Next, 2 mL of tetraethyl orthosilicate (TEOS) (Fujifilm Wako Pure Chemical Corporation) and 0.22 g of hafnium(IV) chloride (City Chemical LLC) were added to the solution. After 4.5 hours, the precipitate was collected, washed three times with ultrapure water and once with ethanol, and dried at 75 °C for 2 hours. The resulting powder was heat-treated at 550 °C for 5 hours to obtain mesoporous silica-HfO ​​particles (hereafter often abbreviated as "MS-HfO particles"), which are a combination of the radiosensitizer HfO and mesoporous silica. MS-HfO2 particles were produced in the same manner as in the synthesis of MS-HfO2-1 particles, except that the amount of TEA was changed to 0.4 g and 0.2 g, respectively, to obtain MS-HfO2-2 particles and MS-HfO2-3 particles (see Table 1 below).

[0071] [Table 1]

[0072] The synthesized MS-HfO2 particles were observed using a transmission electron microscope (TEM) (JEOL) and characterized by powder X-ray diffraction analysis using CuKα radiation (Rigaku Corporation). The Hf / Si molar ratio of the synthesized MS-HfO2 particles was analyzed by dissolving the particles in NaOH and HCl, followed by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Hitachi High-Tech Corporation). The results are shown in Figure 10.

[0073] The MS-HfO2-1, MS-HfO2-2, and MS-HfO2-3 particles all had spherical morphologies with diameters of approximately 40 nm, 20 nm, and 10 nm, respectively (Fig. 10a-c). The MS-HfO2-1, MS-HfO2-2, and MS-HfO2-3 particles were composed of amorphous silica and hafnium oxide and contained no impurities (Fig. 10d). The Hf / Si molar ratios of the MS-HfO2-1, MS-HfO2-2, and MS-HfO2-3 particles were approximately 0.13, 0.15, and 0.20, respectively (Fig. 10e).

[0074] Example 6: Preparation of mesoporous silica-HfO2 particles 2 MS-HfO2-4, MS-HfO2-5, and MS-HfO2-6 particles were synthesized in two steps. In the first step, mesoporous silica (MS) particles were synthesized as follows.

[0075] 1.03 g of cetyltrimethylammonium p-toluenesulfonate (CTAT) (Sigma-Aldrich) and 0.45 g of triethanolamine (TEA) (Sigma-Aldrich) were added to 54 g of ultrapure water and dissolved by stirring at 70 °C. Next, 8.37 mL of tetraethoxysilane (TEOS) (Fujifilm Wako Pure Chemical Industries, Ltd.) was added dropwise to the solution, and the mixture was stirred vigorously for 2 hours to obtain a white precipitate. The precipitate was collected by centrifugation, washed with ultrapure water and ethanol, and dried at 80 °C. The synthesized powder itself was heat-treated at 550 °C for 5 hours to obtain MS particles.

[0076] In the second step, MS-HfO2 particles were synthesized. To synthesize MS-HfO2-4 particles, 100 mg of MS particles were added to 40 mL of ethanol and dispersed by ultrasonication for 30 minutes. While stirring the dispersion, 200 μL of ammonia (Fujifilm Wako Pure Chemical Industries, Ltd.) was added over 10 minutes at room temperature. Next, 280 μL of n-butoxyhafnium (Sigma-Aldrich) was added over 15 minutes and stirred for another 30 minutes at room temperature. The resulting solution was heated to 45°C and stirred for 24 hours. The precipitate was collected by centrifugation and washed several times with ethanol. The resulting product was dried at 80°C and subjected to heat treatment at 500°C for 2 hours to obtain MS-HfO2-4 particles.

[0077] The synthesis of MS-HfO2-5 and MS-HfO2-6 particles was carried out in substantially the same manner as MS-HfO2-4 particles, except that different amounts of ammonia and n-butoxyhafnium were used (see Table 2 below).

[0078] [Table 2]

[0079] The synthesized MS-HfO2 particles were observed using a transmission electron microscope (TEM) (JEOL) and characterized by powder X-ray diffraction analysis using CuKα radiation (Rigaku Corporation). The Hf / Si molar ratio of each synthesized MS-HfO2 particle was determined by dissolving the particles in NaOH and HCl, followed by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Hitachi High-Tech Corporation). The results are shown in Figure 11.

[0080] The MS-HfO2-4, MS-HfO2-5, and MS-HfO2-6 particles all had a spherical morphology and a diameter of approximately 100 nm (Fig. 11a-c). The MS-HfO2-4, MS-HfO2-5, and MS-HfO2-6 particles were composed of amorphous silica and hafnium oxide and contained no impurities (Fig. 11d). The Hf / Si molar ratios of the MS-HfO2-4, MS-HfO2-5, and MS-HfO2-6 particles were approximately 0.35, 0.39, and 0.55, respectively (Fig. 11e).

[0081] Example 7: Cellular evaluation of mesoporous silica-HfO2 particles Lewis lung carcinoma (LLC) cells and mouse oral squamous cell carcinoma 2 (MOC2) cells (5 × 10 3 Cells (0.1 mL / well) were seeded and cultured overnight. Next, 100 μg / mL HfO2 or MS-HfO2 particles were added and cultured for 4 hours. The cells were exposed to different radiation doses (0, 4, 6, or 8 Gy) using an X-ray generator (Faxitron X-Ray Corp.) and cultured for an additional 3 days. Cell proliferation was assessed using a CCK-8 kit (Dojindo Laboratories). The results are shown in Figure 12.

[0082] The cell proliferation rate decreased with increasing HfO2 concentration and X-ray dose. Cells (LLC and MOC2) treated with MS-HfO2 particles and X-rays showed slightly decreased (or equivalent) cell proliferation rates compared to cells treated with HfO2 particles (Figure 12a-b). The proliferation rates of MOC2 cells treated with 8 Gy of X-rays were 56%, 53%, 53%, 56%, 54%, 56%, and 55% when treated with HfO2, MS-HfO2-1 particles, MS-HfO2-2 particles, MS-HfO2-3 particles, MS-HfO2-4 particles, MS-HfO2-5 particles, and MS-HfO2-6 particles, respectively (Figure 12a). The proliferation rates of LLC cells treated with 8 Gy of X-rays were 60%, 57%, 60%, 60%, 52%, 61%, and 57% when treated with HfO2, MS-HfO2-1 particles, MS-HfO2-2 particles, MS-HfO2-3 particles, MS-HfO2-4 particles, MS-HfO2-5 particles, and MS-HfO2-6 particles, respectively (Figure 12b).

[0083] The results of the cell experiments above confirmed that mesoporous silica-hafnium oxide (MS-HfO2) exerted a tumor growth inhibitory effect on MOC2 and LLC cells that was roughly equivalent to or slightly stronger than that of commercially available HfO2, which is known as a radiosensitizer. Furthermore, MS-HfO2-1 and MS-HfO2-4 particles showed particularly strong radiosensitizing effects.

[0084] Example 8: Characterization of mesoporous silica-HfO2 particles The elemental distribution of the MS-HfO2-1 and MS-HfO2-4 particles was analyzed using STEM-EDX images, and the results are shown in Figure 13.

[0085] As is clear from the element distribution (Si, O, Hf from left) shown in the STEM-EDX images, all of the elements Hf, Si, and O were uniformly distributed in the MS-HfO2-1 and MS-HfO2-4 particles.

[0086] Example 9: Evaluation of mesoporous silica-HfO2 particles using mice 1 (LLC cells) On day 0 (d0), LLC cells were injected subcutaneously into the legs of female C57BL / 6 mice (6 weeks old, obtained from CLEA Inc.) (5 × 10 cells in the left leg). 5 cells / mouse, 1 x 10 in the right leg 5 On days 6, 7, and 8 (d6, d7, and d8), mice were treated as follows: L1: no treatment; L2: external irradiation to the leg only; L4: intratumoral injection of HfO2 (0.1 mg HfO2 / mouse, i.e., approximately 5 mg / kg body weight) in the left leg combined with 6 Gy of irradiation; L6: intratumoral injection of MS-HfO2-1 (2 mg / mouse, i.e., 100 mg / kg body weight) in the left leg combined with 6 Gy of irradiation; L7: intratumoral injection of HfO2 (0.62 mg HfO2 / mouse, i.e., approximately 31 mg / kg body weight) in the left leg combined with 6 Gy of irradiation. The amount of HfO2 contained in MS-HfO2-1 injected intratumorally in group L6 was the same as the amount of HfO2 injected intratumorally in group L7 (i.e., 0.62 mg of HfO2 / mouse). Furthermore, as a control, a combination of intratumoral injection of MS particles (prepared in Reference Example 1) and 6 Gy of radiation exposure in the left leg (group L5) was also examined. Specifically, mice subcutaneously injected with LLC cells as described above were irradiated with 6 Gy of radiation in the left leg on days 6, 7, and 8 (d6, d7, and d8), and MS particles (2 mg / mouse, i.e., 100 mg / kg body weight) were injected intratumorally in the left leg on days 9, 10, and 13 (d9, d10, and d13). Tumor volume was calculated as 1 / 2 × major axis × (vertical dimension) 2 The results are shown in Figure 14.

[0087] Mice in the group receiving intratumoral MS-HfO2-1 injection combined with external irradiation (L6) showed significant tumor growth inhibition in both the treated leg (left leg, Figure 14b, c) and the untreated distant leg (right leg, Figure 14d) compared with mice in the untreated group (L1), the group receiving external irradiation only (L2), the groups receiving intratumoral HfO2 injection combined with external irradiation (L4, L7), and the group receiving intratumoral MS particle injection combined with external irradiation (L5). For tumors in the left leg, the tumor size in the L6 group on day 16 after LLC injection was 17%, 48%, 58%, 58%, and 74% of the tumor sizes in the L1, L2, L4, L7, and L5 groups, respectively. For tumors in the right leg, the tumor size in the L6 group on day 16 after LLC injection was 53%, 39%, 41%, 67%, and 78% of the tumor size in the L1, L2, L4, L7, and L5 groups, respectively.

[0088] Therefore, the radiosensitizing and immunostimulatory effects of MS-HfO2-1 on LLC cells in vivo were confirmed, and a significant proliferation inhibitory effect was also confirmed not only in the treated side but also in the untreated side (L6 group).On the other hand, such a high proliferation inhibitory effect was not confirmed when the same concentration of HfO2 alone (L7 group) or MS particles alone (L5 group) were used.

[0089] Example 10: Evaluation of mesoporous silica-HfO2 particles in mice 2 This experiment was conducted to elucidate the effect of MS-HfO2-1 dose and radiation dose on tumor growth in treated and untreated distant tumors.

[0090] On day 0 (d0), female C57BL / 6 mice (6 weeks old, obtained from CLEA Inc.) were injected subcutaneously into the legs with MOC2 cells (1 × 10 cells in the left leg). 6 cells / mouse, 2 x 10 in the right leg 5On days 4, 7, and 11 (d4, d7, and d11), mice were treated as follows: H1: no treatment; H2: external irradiation of 6 Gy (3 times) to the left leg only; H3: intratumoral injection of MS-HfO2-1 (0.5 mg / mouse, i.e., approximately 25 mg / kg body weight) to the left leg combined with external irradiation of 6 Gy (3 times); H4: intratumoral injection of MS-HfO2-1 (1 mg / mouse, i.e., approximately 50 mg / kg body weight) to the left leg combined with external irradiation of 6 Gy (3 times); H5: intratumoral injection of MS-HfO2-1 (2 mg / mouse, i.e., approximately 100 mg / kg body weight) to the left leg combined with external irradiation of 6 Gy (3 times); H6: external irradiation of 8 Gy (3 times) to the left leg only; and H7: intratumoral injection of MS-HfO2-1 (1 mg / mouse) to the left leg combined with external irradiation of 8 Gy (3 times). Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-12 (IL-12) levels in the spleen were quantified using a mouse ELISA kit (BD Biosciences). The results are shown in Figures 15 and 17.

[0091] Mice in groups H3–H7 treated with intratumoral injection of MS-HfO2-1 (0–2 mg / mouse, i.e., 0–approximately 100 mg / kg body weight) in combination with external irradiation (6–8 Gy) showed significant tumor growth inhibition in the treated (left) leg compared with mice in the untreated group H1. However, tumor sizes were similar across different MS-HfO2-1 doses and radiation doses (Figure 15b, H2–H7). Mice in group H5 treated with intratumoral injection of MS-HfO2-1 (2 mg / mouse) in combination with external irradiation (6 Gy) showed the smallest tumor volume in the untreated (right) leg (Figure 15c, H5).

[0092] In summary, the highest growth inhibitory effect was observed in both the treated and untreated sides when the dose of MS-HfO2-1 was 100 mg / kg and the radiation dose was 6 Gy.

[0093] Example 11: Evaluation of mesoporous silica-HfO2 particles in mice 3 Experiments were conducted to further elucidate the effect of MS-HfO2-1 dose and radiation dose on tumor growth of treated and untreated distant tumors.

[0094] On day 0 (d0), female C57BL / 6 mice (6 weeks old, obtained from CLEA Inc.) were injected subcutaneously into the legs with MOC2 cells (1 × 10 cells in the left leg). 6 cells / mouse, 2 x 10 in the right leg 5 On days 4, 6, 8, and 11 (d4, d6, d8, and d11), mice were treated as follows: B1: no treatment; B2: 3 Gy (4 doses) of radiation to the left leg only; B3: intratumoral injection of MS-HfO2-1 (2 mg / mouse, i.e., approximately 100 mg / kg body weight) to the left leg combined with 3 Gy (4 doses); B4: 4 Gy (4 doses) of radiation to the left leg only; B5: intratumoral injection of MS-HfO2-1 (2 mg / mouse) to the left leg combined with 4 Gy (4 doses). On days 4, 6, and 8 (d4, d6, and d8), mice were treated as follows: B1: no treatment; B2: 3 Gy (4 doses) of radiation to the left leg only; B3: intratumoral injection of MS-HfO2-1 (2 mg / mouse, i.e., approximately 100 mg / kg body weight) to the left leg combined with 3 Gy (4 doses); B4: 4 Gy (4 doses) of radiation to the left leg only; B5: intratumoral injection of MS-HfO2-1 (2 mg / mouse) to the left leg combined with 4 Gy (4 doses). B6: 5 Gy (3 doses) of radiation to the left leg only; B7: 5 Gy (3 doses) of radiation to the left leg combined with intratumoral injection of MS-HfO2-1 (2 mg / mouse) and 5 Gy (3 doses). Tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-12 (IL-12) levels in the spleen were quantified using mouse ELISA kits (BD Biosciences). The results are shown in Figures 16 and 17.

[0095] Mice in groups B3–B7 treated with intratumoral injection of MS-HfO2-1 (2 mg / mouse) in combination with external irradiation (3–5 Gy) showed significant tumor growth inhibition in the treated (left) leg compared with untreated mice (B1). However, the tumor sizes were similar for all groups treated with different doses of MS-HfO2-1 and different radiation doses (Figure 16b, B2–B7). Mice in groups treated with intratumoral injection of MS-HfO2-1 (0–1 mg / mouse, i.e., 0–approximately 50 mg / kg body weight) in combination with external irradiation (3–5 Gy) in the untreated (right) leg (Figure 16c).

[0096] Furthermore, combining the results of Examples 10 and 11, among the groups treated with a combination of intratumoral injection of MS-HfO2-1 (2 mg / mouse) and external irradiation (3, 4, 5, and 8 Gy), the group treated with a combination of intratumoral injection of MS-HfO2-1 (2 mg / mouse) and external irradiation (6 Gy) showed the smallest tumor volume in the untreated distal leg (right leg) (Fig. 15c). Furthermore, among the groups treated with a combination of intratumoral injection of MS-HfO2-1 (0 to 1 mg / mouse) and external irradiation (3, 4, 5, and 8 Gy), the group treated with a combination of intratumoral injection of MS-HfO2-1 (2 mg / mouse) and external irradiation (6 Gy) showed the highest IL-6 and IL-12 levels and the second highest TNF-α levels (Fig. 17). 15 to 17, the combination of MS-HfO2-1 (2 mg / mouse) and external radiation (6 Gy) exhibited the highest antitumor effect.

[0097] Example 12: Interdisciplinary study of mesoporous silica-HfO2 particles, external radiation, and immune checkpoint inhibitors On day 0 (d0), LLC cells were injected subcutaneously into the legs of female C57BL / 6 mice (6 weeks old, obtained from CLEA Inc.) (5 × 10 cells in the left leg). 5 cells / mouse, 1 x 10 in the right leg 5On days 7, 8, 9, and 11 (d7, d8, d9, and d11), mice were treated as follows: A1: no treatment; A2: intraperitoneal injection of anti-CTLA-4 (BioXcell, 0.2 mg / mouse, i.e., approximately 10 mg / kg body weight); A3: intratumoral injection of MS-HfO2-4 (2 mg / mouse, i.e., approximately 100 mg / kg body weight) and intraperitoneal injection of anti-CTLA-4 (0.2 mg / mouse). On days 8, 10, 14, and 17 (d8, d10, d14, and d17), mice in groups A2 and A3 were irradiated with 6 Gy of radiation to the left leg. Tumor volume was calculated as 1 / 2 × major axis × (vertical dimension). 2 The total number of cells was calculated as 1. At the end of the study, splenocytes were collected from the spleens, mashed, and filtered through a 40 μm cell strainer to obtain a single-cell suspension. Anti-CD16 / CD32 antibodies were used to prevent nonspecific staining. Anti-mouse CD3, anti-mouse CD4, anti-mouse CD8α, anti-mouse CD44, and anti-mouse CD62L (BioLegend) were used to stain the cells for 30 minutes. Flow cytometry analysis was performed to analyze the memory T cell population in the splenocytes. The results are shown in Figures 18 and 19.

[0098] In mice in the group (A3) receiving intratumoral injection of MS-HfO2-4, external irradiation, and anti-CTLA-4, growth of both the treated tumor (left leg tumor) and the untreated distant tumor (right leg tumor) was clearly inhibited compared with the untreated group (A1) and the group (A2) receiving external irradiation and anti-CTLA-4 (Figure 18). The mice in the group (A3) receiving intratumoral injection of MS-HfO2-4, external irradiation, and anti-CTLA-4 showed significantly higher CD8+ T cell proliferation than the untreated group (A1) and the group (A2) receiving external irradiation and anti-CTLA-4. + The effector memory T cell population (CD44 high CD62L - ) is the highest, and CD4 + The effector memory T cell population (CD44 high CD62L - ) was the second highest (Figure 19). Therefore, in the group (A3) using MS-HfO2-4, CD8+ An increase in the effector memory T cell population was confirmed among T cells.

[0099] Example 13: Safety evaluation of mesoporous silica-HfO2 particles MOC2 cells were injected subcutaneously into the legs of female C57BL / 6 mice (6 weeks old, obtained from CLEA Inc.) (1 × 10 6 On day 10 after injection, the mice were injected intratumorally with MS-HfO2-1 (2 mg / mouse, i.e., approximately 100 mg / kg body weight), and controls were injected with saline. On day 11, the heart, spleen, kidneys, lungs, and liver were harvested, fixed in 10% neutral buffered formalin solution, embedded in paraffin, and histological sections were prepared. The sections were stained with hematoxylin and eosin (HE) and observed under a microscope. The results are shown in Figure 20.

[0100] As can be seen from the micrographs in Figure 20, no significant differences were observed in the tissue sections of the heart, spleen, kidney, lung, and liver between the mice injected with MS-HfO2-1 particles and those injected with saline, indicating that MS-HfO2-1 particles do not have significant toxicity.

[0101] Summary of Examples 5 to 13 In cell experiments, mesoporous silica-hafnium oxide particles (MS-HfO2 particles) demonstrated a slightly stronger radiosensitizing effect than the same amount of commercially available HfO2, and a stronger antitumor effect than MS particles without HfO2. Furthermore, after examining the dosage, timing, and radiation dose of MS-HfO2 particles, the highest growth inhibitory effect was observed in both the treated and untreated sides when MS-HfO2 was administered at a dose of 2 mg / mouse (approximately 100 mg / kg body weight) and a radiation dose of 6 Gy. Furthermore, a combination of MS-HfO2, an anti-CTLA-4 antibody (an immune checkpoint inhibitor), and external radiation irradiation demonstrated a significant tumor growth inhibitory effect. [Industrial Applicability]

[0102] The use of the radiosensitizing vaccine of the present invention in immunoradiotherapy enables cancer treatment with low-dose external radiation. Furthermore, by inducing the production of personalized in situ cancer vaccines in the body, it is possible to achieve not only the treatment of primary cancers but also the prevention and treatment of recurrent and distant metastatic cancers.

Claims

1. A radiosensitizing vaccine for use in cancer immunoradiotherapy, comprising: the radiosensitizing vaccine comprises mesoporous silica-radiosensitizer particles in which mesoporous silica and a radiosensitizer are integrated; the immunoradiotherapy comprises administering the radiosensitizing vaccine to a subject having cancer and external radiation therapy to the cancer; A radiosensitizing vaccine, wherein the radiosensitizing vaccine induces cancer radiosensitization and immune activation in a subject.

2. The radiosensitizing vaccine of claim 1, which further induces the abscopal effect.

3. The radiosensitizer is copper oxide (CuO) or hafnium oxide (HfO ２ 2. The radiosensitizing vaccine of claim 1, wherein the

4. when the radiosensitizer is copper oxide (CuO), the molar ratio of copper (Cu) to silicon (Si) (Cu / Si) in the mesoporous silica-radiosensitizer particles is 0.1% to 40%, The radiation sensitizer is hafnium oxide (HfO ２ ) when the molar ratio of hafnium (Hf) to silicon (Si) in the mesoporous silica-radiation sensitizer particles (Hf / Si) is 1% to 40%; The radiosensitizing vaccine of claim 3.

5. 2. The radiosensitizing vaccine according to claim 1, wherein the particle diameter of the mesoporous silica-radiosensitizer particles is 10 nm to 5000 nm.

6. 2. The radiosensitizing vaccine of claim 1, wherein the dose of the external radiation is 0.5 to 20 Gy.

7. The radiosensitizing vaccine of claim 1, further comprising an immune checkpoint inhibitor.

8. The radiosensitizing vaccine of claim 1, wherein the immunoradiotherapy further comprises administration of an immune checkpoint inhibitor.

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