JP6868862B2 - Mesoporous silica particles - Google Patents

Description

The present invention relates to mesoporous silica particles that alone serve as an immunoadjudicant for immunotherapy of tumors (including carcinomas and sarcomas). More specifically, the present invention relates to spherical mesoporous silica particles that serve as an immune adjuvant capable of stimulating tumor cell-specific adaptive acquired immunity alone without the coexistence of other immunostimulatory substances. The spherical mesoporous silica particles of the present invention can be used for tumor immunotherapy including treatment and / or prevention of tumors.

Treatment methods for tumors include surgical treatment, radiotherapy, chemotherapy, and immunotherapy. Immunotherapy is a treatment method for fighting tumors by utilizing the immunity inherent in the living body. Tumor immunotherapy has recently been shown to be highly effective against some tumors that have failed conventional surgery, radiotherapy, and chemotherapy, and tumor treatment with immunotherapy as the main treatment. Tumor immunotherapy is developing rapidly, including the development of methods.

By the way, pathogenic microorganisms and viruses usually have two elements, an antigen and an immunostimulator, integrated, and the immune system in the body is activated by the immunostimulator coexisting with the antigen, which is innate immunity or antigen-specific. Acquired immunity is induced. Typical immunostimulatory substances include Toll-like receptor (TLR) agonists such as pathogen-associated molecular patterns (PAMPs) commonly found in pathogens. On the other hand, although tumors have antigens, they have low antigenicity due to the fact that they do not contain immunostimulatory substances and the immune escape mechanism works, and it is difficult to induce antigen-specific acquired immunity. Therefore, in order to induce tumor antigen-specific acquired immunity, in addition to the tumor antigen, it is essential to use an immune adjuvant mainly composed of PAMP and other Toll-like receptor agonists (Non-Patent Documents 1 and 2). ..

In general, immune adjuvants can be roughly classified into two types according to their mechanism of action (Non-Patent Documents 3 and 4). One is the action of affecting the temporal, spatial or concentration change and attenuation of the vaccine antigen to facilitate or increase the chances of immune cells utilizing the antigen (S1). The other is an action of giving an auxiliary stimulus to the antigen-specific immune cells when the antigen-specific immune cells recognize the antigen (S2). In other words, the former S1 is an antigen delivery and antigen protection action. Immune adjuvants in which S1 is the main action include W / O emulsions, liposomes, calcium phosphate and the like. The latter S2 is an action that directly activates the immune cells themselves. Pathogen-associated molecular patterns (PAMP), agonists of pattern recognition receptors such as Toll-like receptors, heat shock proteins, damage-associated molecular patterns (DAMP), and various cytokines are classified as immunoadjudicants whose main action is S2. Some immune adjuvants have both S1 and S2 effects.
By mixing an immunoadjugant and an antigen, an immunogenic composition such as a vaccine is obtained. As the immune adjuvant, a ceramic compound, that is, an inorganic one, an aluminum compound (alum), aluminum phosphate, calcium phosphate and the like have been conventionally used and applied to medical treatment. Dead bacteria and attenuated pathogens are themselves vaccines because they are an integral part of the antigen and PAMP, which is an immunoadjuct itself.

From another point of view, when classified according to the subjects to be immunostimulated by the immune adjuvant, humoral innate immunity (A), humoral acquired immunity or antibody production (B), cellular innate immunity (C), and cellular innate immunity (C). It is divided into four subjects of acquired immunity (D) (Non-Patent Document 5). For example, the target to be immunostimulated by an immunostimulating aluminum compound (Alam) such as aluminum hydroxide or aluminum phosphate is mainly humoral adaptive immunity (B). In addition, many PAMPs effectively stimulate cellular adaptive immunity (D).
As an adjuvant for tumor immunotherapy, natural killer cells responsible for cellular innate immunity (C), natural killer T cells, those that stimulate γδ T cells, cellular innate immunity (C) and cellular acquired immunity ( It is preferable that it stimulates dendritic cells straddling D), type I helper T cells responsible for cellular adaptive immunity (D), and killer T cells.

Amorphous calcined silica (SiO ₂ ) is an inorganic porous substance with pores, has adsorptive properties, and is not biotoxic, so it has been used for a long time as an additive to health foods and pharmaceuticals. In recent years, "mesoporous silica" having a large number of micropores (mesopores) of 2 nm to 50 nm has been attracting attention because of its high specific surface area and excellent adsorption performance. In particular, it has been proposed that mesoporous silica SBA-15 having a hexagonal uniform and regular two-dimensional structure manufactured by using an amphipathic block copolymer is formed into a rod shape or the like and used as an immune adjuvant. (Patent Document 1, Non-Patent Documents 6 to 7). It is described that an immunogenic complex in which antigens such as proteins, bioactive peptides, toxins, and viruses are encapsulated in the pores of SBA-15 is used as a vaccine preparation. In this complex, SBA-15 is used. It coexists with an immunostimulatory substance derived from an antigen (Patent Document 1). Neither document describes its use for tumor-targeted immunotherapy (Patent Document 1, Non-Patent Documents 6 to 7).
Silica-based immune adjuvants have been listed as examples of substances that are expected to have immunoadjugant (S2 + D) activity that induces a cell-mediated immune response even in tumor immunotherapy (Patent Document 2). There has never been a case where it was actually used alone as an immune adjuvant targeting a tumor. When "mesoporous silica" particles are used as an immunoadjugant that can be used for tumor immunotherapy, PAMP or Toll-like receptor agonists such as tuberculin and BCG vaccine preparations, granulocyte macrophage colony stimulating factor (GM-CSF), etc. are pored. The composition carried therein is used as an immunoadjugant (Patent Documents 3 and 4).
Although the effects of these immunoadjudicant compositions were sufficiently satisfactory, there were many problems in commercializing them to meet the pharmaceutical standards. That is, when a bio-derived substance such as PAMP or a Toll-like receptor agonist or a physiologically active protein is supported on mesoporous silica particles, which are inorganic substances having no problem in storage stability and stability, the process becomes complicated. In addition to this, not only is it inconvenient to handle and distribute, such as requiring refrigerated storage, but it is also extremely difficult to maintain uniform and stable quality for a long period of time.

Patent No. 5091863 Special Table 2009-500544 Gazette Special Table 2015-516398 Japanese Unexamined Patent Publication No. 2013-129655

Joseph N.Blattman et al., Science, 9 July 2004, Vol.305,200-205 Achal Pashine et al., Nature Medicine Supplement, Vol.11, No.4, April 2005, S63-S68 Riberio C.M.et al., Methods in Mol. Biol. 2010; 626: 1-14. Schijns, V.E.J.C, Crit Rev Immunol. 2001; 21 (1-3): 75-85 Delves, P.J .; Martin, S.J .; Burton D.R .; Roitt I.M .; Translated by Masayuki Miyasaka; Roat, Color Basic Immunology, Nishimura Bookstore, 2011 Mercuri, L.P. et al., Small. 2006 Feb; 2 (2): 254-6. Kupferschmidt N. et al., Nanomedicine (Lond). 2014; 9 (12): 1835-1846

An object of the present invention is an immunoadjugant function capable of inducing a high cell-mediated immune response by itself without using an immunostimulatory substance such as PAMP or a Toll-like receptor agonist even in immunotherapy targeting a tumor. It is an object of the present invention to provide mesoporous silica particles having a specific structure and a specific particle size.

The present inventors produced various different silica fine particles in which the presence / absence, shape, and particle size of mesopores were variously changed by appropriately adjusting the silicon raw material and the firing method, and diligently studied the action of each of them. Specifically, silica fine particles having different shapes, particle sizes, and structures are suspended in physiological saline together with a model antigen (albumin derived from chicken egg white (hereinafter referred to as OVA)) and administered intradermally to mice. The production amounts of IFNγ, IL-2, IL-4, and IL-10 produced by the collected lymphocytes on the 8th day after administration were compared. As a result, it was clarified that spherical mesoporous silica particles with a roundness of 20% or less and a particle size of 50 to 5,000 nm can strongly stimulate humoral acquired immunity and cell-mediated acquired immunity by themselves. I made it.
Specifically, the metal-containing or metal-free spherical mesoporous silica particles obtained in the present invention alone are IFNγ, IL-2, IL-4 and / or IL-10 of lymphocytes of model tumor antigen OVA-administered mice. It was confirmed that the production amount can be enhanced or the growth of a tumor formed subcutaneously in a mouse by using a mouse lung cancer-derived LLC cell disrupted product as a tumor antigen or as an OVA model tumor antigen can be effectively suppressed. Next, the tumor recurrence prevention effect in the recurrence prevention model mice obtained by removing the formed LLC tumor tissue and using it as an autologous tumor antigen was confirmed, and a significant increase in the number of effector memory T cells that control immunological memory was confirmed. In addition, a model in which a tumor formed by seeding tumor cells GL261-mKO under the skin of a mouse thigh was denatured by X-ray irradiation, and then spherical mesoporous silica particles were administered 6 times, and GL261-mKO was seeded in the brain 24 hours after irradiation. Follow-up of recurrent mice revealed that the survival time was significantly extended.
The present invention has been completed by obtaining the above findings.

That is, the present invention is as follows.
[1] An adjuvant for tumor immunotherapy containing spherical mesoporous silica, which does not contain immunostimulatory substances other than endotoxin of 0.1 EU / mg or less that are unintentionally mixed, and has an immunostimulatory effect by itself, tumor immunity. Therapeutic adjuvant.
Here, typical immunostimulatory substances include toll-like receptor agonists, cytokines, C-type lectin receptor agonists, NOD-like receptor agonists, RIG-like receptor agonists, and exogenous substances that bind to cyclic GMP-AMP synthase. DNA, exogenous allamines.
[2] The adjuvant for tumor immunotherapy according to the above [1], wherein the spherical mesoporous silica contains 65% or more of particles having a roundness of 20% or less and has a particle size of 50 to 5,000 nm.
[3] The adjuvant for tumor immunotherapy according to any one of [1] to [2] above, wherein the spherical mesoporous silica is hollow.
[4] The hollow spherical mesoporous silica has an outer shell having a thickness of 10 to 200 nm, and has a mesopore having a diameter of 2 to 50 nm in the outer shell. The adjuvant for tumor immunotherapy described in.
[5] Spherical mesoporous silica contains at least one metal selected from calcium, magnesium, zinc, sodium, potassium, phosphorus, boron and aluminum in a metal / silicon molar ratio of 0 to 20 mol%. The adjuvant for tumor immunotherapy according to any one of the above [1] to [4].
[6] The adjuvant for tumor immunotherapy according to any one of [1] to [5] above, which is administered together with a tumor antigen into the body of a mammal including a human to induce a systemic immune response against the tumor antigen. ..
[7] The tumor immunity according to the above [6], wherein the tumor antigen is selected from the group consisting of tumor tissue, tumor cells, tumor cell lysates or denatured products thereof, tumor cell components, tumor antigen proteins, and tumor antigen peptides. Therapeutic adjuvant.
[8] The tumor according to any one of claims 1 to 5, for inducing an anti-tumor immune response by degenerating the tumor tissue of a mammalian animal including human by physical means and then administering it into the tumor tissue. An immunotherapeutic adjuvant whose physical means are microwave irradiation, radiofrequency coagulation method, freeze coagulation method, electric scalpel heating, hot water injection, alcohol injection, embolization method, radiation irradiation, laser light irradiation, and ultrasonic destruction. An adjuvant for tumor immunotherapy, which is one or more means selected from the group consisting of.
[9] A vaccine containing the adjuvant for tumor immunotherapy according to any one of [1] to [5] above and a tumor antigen.
[10] (1) It is characterized by containing (2) an immune checkpoint inhibitor together with the adjuvant for tumor immunotherapy according to any one of the above [1] to [8] or the vaccine according to the above [9]. A composition for treating and / or preventing a tumor.
[11] The immune checkpoint inhibitor is anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-CD40 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-TGF-β. The tumor according to the above [10], which is one or more antibodies selected from an antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-TIM3 antibody, an anti-CD96 antibody, and an anti-TIGIT antibody. A therapeutic and / or prophylactic composition.
[12] (1) The adjuvant for tumor immunotherapy according to any one of the above [1] to [8] or the vaccine according to the above [9] is combined with (2) an immune checkpoint inhibitor. Tumor treatment and / or preventive medication.
Here, the combined tumor therapeutic and / or preventive drug mixes (1) and (2) separately or simultaneously with a pharmacologically acceptable carrier, excipient, additive or the like. Can be formulated and administered as a pharmaceutical composition. When they are formulated separately, both may be mixed and administered with a diluent or the like at the time of use, but both may be administered separately, simultaneously or at different times.
[13] Immune checkpoint inhibitors are anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-CD40 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-TGF-β. The tumor according to the above [12], which is one or more antibodies selected from an antibody, an anti-LAG3 antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-TIM3 antibody, an anti-CD96 antibody, and an anti-TIGIT antibody. A therapeutic and / or prophylactic drug.

The spherical mesoporous silica particles provided by the present invention having a particle size of 50 nm to 5000 nm and a roundness of 20% or less and 65% or more of particles are cellular properties that are effective against tumor cells even without the addition of an immunostimulatory substance. Since it can stimulate acquired immunity, it can be used alone as an immune adjuvant for tumor immunotherapy. Since it is not necessary to add an immunostimulatory substance with low stability, it is used as an immunoadjudicant preparation with excellent ease of manufacture, storage stability, and quality stability for tumor immunotherapy including tumor treatment and / or prevention. can do.

The figures showing the quantification results of cytokines in the culture medium of the lymphocytes collected from the mice in which the model antigen OVA was intradermally administered at the same time as each fine particle, which was carried out in Example 1, were cultured by OVA stimulation in vitro. is there. (A): IFNγ, (B): IL-2, (C): IL-4, (D): IL-10. The sample names (a) to (k) correspond to Table 1. It is a figure which showed the transmission electron microscope image of each sample prepared in Example 2. The sample names (l) (m) (h) (n) (o) (p) correspond to Table 1. It is a figure which showed the pore distribution and nitrogen adsorption isotherm of each sample prepared in Example 2. The sample names (l) (m) (h) correspond to Table 1. It is a figure which showed the result of the animal experiment which examined the antitumor immunostimulatory action of the spherical mesoporous silica carried out in Example 3. (A): Kaplan-Meier curve whose endpoint is tumor development with a diameter of 15 mm or more (hemispherical approximation) in sample (h), (B): time course of tumor volume calculated from tumor diameter in sample (h). .. * In the figure indicates that the p-value between the two groups is less than 0.05. (C): Kaplan-Meier curve whose endpoint is tumor development with a diameter of 15 mm or more (hemispherical approximation) in sample (g) (n) (o) (p), (D): sample (g) (n) (o) ) (P) Shows the time course of tumor volume calculated from the diameter of the tumor in It is a figure which showed the result of the animal experiment of the antitumor immunostimulatory action of spherical mesoporous silica (h) in the recurrence prevention model carried out in Example 4. It is a figure which showed the abundance ratio of the effector memory T cell in the immune cell collected from the bone marrow of the mouse after the animal experiment of Example 4 carried out in Example 5. * In the figure indicates that the p-value between the two groups determined by the two-sided t-test is less than 0.05. (A): Results for CD4 positive cells, (B): Results for CD8 positive cells. Survivorship curve of mice in which the thigh tumor tissue was denatured by physical means (X-ray irradiation), spherical mesoporous silica (h) was administered to the degenerated tumor tissue, and then tumor cells were seeded in the brain, which was carried out in Example 6. The proportion of mice formed by E.G7-OVA cells with an average tumor diameter of 15 mm or less was treated with anti-PD-1 antibody to the local administration of a mixture of spherical mesoporous silica (h) and OVA in the negative control group with OVA topical administration only. Kaplan-Meier curves compared between the combined drug group combined with systemic administration and the systemic administration group with anti-PD-1 antibody alone. The proportion of mice with an average tumor diameter of 15 mm or less formed by E.G7-OVA cells was divided into a negative control group with only OVA topical administration, and a composition group in which spherical mesoporous silica (h), OVA, and anti-PD-1 antibody were mixed. And Kaplan-Meier curves compared in the systemic administration group of anti-PD-1 antibody alone. ^{The proportion of mice with a total tumor volume of 1690 mm 3} (equivalent to an average diameter of 15 mm) or less formed by E.G7-OVA cells was measured in a negative control group with OVA topical administration only, a mixture of spherical mesoporous silica (h) and OVA. Kaplan-Meier curves compared between a combination drug group in which local administration is combined with systemic administration of anti-PD-L1 antibody and a systemic administration group in which anti-PD-L1 antibody alone is administered. ^{The proportion of mice with a total tumor volume of 1690 mm 3} (equivalent to an average diameter of 15 mm) or less formed by E.G7-OVA cells was measured in the negative control group with OVA topical administration only, spherical mesoporous silica (h), OVA, and anti-PD. Kaplan-Meier curves compared between the composition group mixed with -L1 antibody and the systemic administration group with anti-PD-L1 antibody alone. The proportion of mice formed by E.G7-OVA cells with an average tumor diameter of 15 mm or less was treated with anti-PD-1 antibody to the local administration of a mixture of spherical mesoporous silica (q) and OVA in the negative control group with OVA topical administration only. Kaplan-Meier curves compared between the combined drug group combined with systemic administration and the systemic administration group with anti-PD-1 antibody alone. The proportion of mice with an average tumor diameter of 15 mm or less formed by E.G7-OVA cells was divided into a negative control group with only local OVA administration, and a composition group in which spherical mesoporous silica (q), OVA, and anti-PD-1 antibody were mixed. , And Kaplan-Meier curves compared in the systemic administration group of anti-PD-1 antibody alone.

1. 1. About spherical mesoporous silica particles of the present invention (1) What is mesoporous silica?
IUPAC defines pores with a diameter of 2 to 50 nm as mesopores. In the present invention, "mesoporous silica" is silicon dioxide having mesopores, and the silicon dioxide contains metal / silicon = 0 to 20 mol%, preferably 0 to 15 mol%, and more preferably 0 to 10 mol%. It may contain calcium, magnesium, zinc, sodium, potassium, phosphorus, boron and aluminum. As the contained metal, calcium, magnesium or zinc is preferable. If a large number of mesopores are regularly arranged, it can be determined that there are mesopores by whether or not a peak appears at a position corresponding to a lattice spacing of 2 to 50 nm by powder X-ray diffraction method. Such a peak appears in the range of 2θ of 0.5 to 3.0o by the X-ray diffraction method using CuKα. When the mesopores are arranged irregularly, the mesopores can be confirmed by a transmission electron microscope. Alternatively, the pore diameter can be measured using an automatic specific surface area / pore distribution measuring device. Mesoporous silica uses tetraethoxysilane, tetramethoxysilane, tetrapropoxysilane, sodium silicate, etc. as silicon source raw materials, block copolymers such as poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide), and hexadecyltrimethylammonium. Surfactants typified by long-chain alkylammoniums such as bromide are used for mesoporous templates to make the liquid acidic or basic under the conditions of 20 to 150 ° C, preferably pH 1.0 or less or pH 12. It can be prepared by various methods such as adjusting to 0 or more, hydrolyzing the raw material of the silicon source, and then removing the template by extraction method or burning. In this production step, if a metal nitrate or a metal alkoxide is mixed with the raw material of the silicon source, mesoporous silica to which a metal is added can be synthesized, but the method of adding the metal is not limited to this.

(2) Spherical Mesoporous Silica Particles The present invention provides spherical mesoporous silica having an immunostimulatory effect by itself. The present invention targets "spherical mesoporous silica particles", but the mesoporous silica fine particles are "spherical". The presence or absence is determined based on the roundness of the particles and their content when observed on electron microscopic images. In the present invention, the roundness of the particles is "the circumscribed circle of the diameter difference between the two circles when the contour of the particle is sandwiched between the circumscribed circle and the inscribed circle that have the same center. Ratio to diameter ". In the present invention, the spherical mesoporous silica is a mesoporous silica containing 65% or more, preferably 75% or more, more preferably 80% or more of mesoporous silica particles having a roundness of 20% or less when observed with an electron microscope. is there. As long as the mesoporous silica particles can be observed, the electron microscope may be a scanning type or a transmission type.
Further, it is preferable that the spherical mesoporous silica of the present invention not only has mesopores but also has a hollow inside (hollow type). Since the inside is hollow, not only the immunostimulatory action is enhanced, but also the internal volume for adsorbing the antigen inside is increased, so that the antigen delivery and the antigen protective action effect (S1 action) are also excellent.

(3) Hollow Mesoporous Silica Particles In the present invention, the "hollow" mesoporous silica (hollow type) is a particle whose bright area has high electron beam transmittance when observed with a transmission electron microscope. Silica with more than 5% of cross-sectional area and mesoporous outer shell (Teng J. et al., Chem Mater. 2013; 25: 98-105, etc.). The pores of the outer shell may be composed of only mesopores (5 to 50 nm), or may be composed of mesopores and pores larger than mesopores. If the pores are larger than the mesopores, a hollow mesoporous silica having an excellent S1 action that easily supports an antigen or the like inside the hollow is provided.
Whether or not the mesoporous silica is hollow can be confirmed with a transmission electron microscope. When there is no hollow space inside, there is no big difference in the transmittance of the electron beam between the center of the particle and the edge, so the transmission electron microscope image has no difference in brightness between the center and the edge, or the edge. The part is brighter. If there is a hollow space inside, the hollow space in the center is bright because the electron beam transmittance is high, and the edge portion is dark because the electron beam transmittance is low. The portion of this peripheral portion where the electron beam transmittance is low is the outer shell.
The thickness of the outer shell of the hollow mesoporous silica can be appropriately changed depending on the particle size, the convenience of supporting the antigen and the like, and the purpose of use, but is preferably 10 to 200 nm, more preferably 10 to 80 nm. The reason why the lower limit is 10 nm is that the mesopore (2 to 50 nm) needs to be present in the outer shell.

(4) Particle size of spherical mesoporous silica particles The particles must be of a size that can be phagocytosed by cells in order to exert an immunostimulatory effect. Also in the experimental results of the present invention, it was confirmed that the immunostimulatory action depends on the particle size. According to the preferred embodiment of the present invention, the particle size is 50 nm to 5000 nm, preferably 50 nm to 900 nm, more preferably 100 to 500 nm, still more preferably 170 to 420 nm.

(5) Method for Producing Spherical Mesoporous Silica Particles A typical method for producing spherical mesoporous silica particles of the present invention is shown below, but the method is not limited to this method.
For the spherical mesoporous silica particles of the present invention, refer to the known methods of Radu et al. (J Am Chem Soc. 2004; 126: 13216-7) and Teng et al. (Chem Mater. 2013; 25: 98-105). Can be manufactured. Specifically, as a template agent, for example, hexadecyltrimethylammonium bromide (CTAB), polyoxyethylene (42) polyoxypropylene (67) glycol (Pluronic P-123) and the like can be used. Dissolve the template in a basic or acidic solution, add tetraethyl orthosilicate (TEOS) with vigorous stirring at 20-150 ° C, and continue stirring for 2-48 hours. Instead of tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate, tetrapropyl orthosilicate, sodium silicate and the like may be used.
In this example, TEOS: CTAB: ammonia: ultrapure water: ethanol (99.5%) was set to a molar ratio of 1.00: 0.0922: 2.96: 621: 115. This setting value can be appropriately set in the range of 0.10 to 2.00 for TEOS, 0.03 to 0.20 for CTAB, 1.00 to 5.00 for ammonia, 60 to 2000 for ultrapure water, and 20 to 2000 for ethanol (99.5%).
The white precipitate obtained by this operation is recovered by centrifugation, washed multiple times with ethanol (99.5%), dispersed in a TEOS-containing solution (1 mL / 480 mL of ultrapure water), and stirred at 20 to 100 ° C. Do. Spherical fine particles are formed by spontaneous shape change during that period, and the thickness of the outer shell can be adjusted by changing the treatment conditions at that time as follows. The obtained fine particles are collected by centrifugation, washed with ultrapure water and ethanol (99.5%), dried (80 ° C), and calcined (550 ° C, 5 hours) to remove CTAB.
As for the specific treatment conditions for adjusting the thickness of the outer shell, the thickness of the outer shell can be adjusted by appropriately changing the temperature condition and the treatment time. Specifically, the outer shell is not formed when treated at room temperature (20 to 30 ° C) for 1 to 3 hours (dense type), but the outer shell is formed when treated at high temperature (50 to 100 ° C) for 10 minutes to 3 hours. Formed at 100 to 10 nm (hollow type).

(6) Method for Producing Metal-Containing Spherical Mesoporous Silica Particles From another viewpoint, spherical mesoporous silica in which the above-mentioned spherical mesoporous silica contains a metal that controls immune response, biocompatibility and bioabsorption is provided. These metals are metals selected from calcium, magnesium, zinc, sodium, potassium, phosphorus, boron and aluminum, and the ratio of metal / silicon = 0 to 20 mol%, more preferably metal / silicon = 0 to 12 mol% is added. Will be done. The upper limit is 20 mol% from the viewpoint that the metal is dissolved in silicon dioxide and not precipitated as metal oxide crystals.

A typical method for producing the metal-containing spherical mesoporous silica particles of the present invention is shown below, but the method is not limited to this method.
The metal-containing spherical mesoporous silica particles of the present invention can be produced by referring to the known method of Li et al. (Micropor Mesopor Mater 2007; 102: 151-158) and appropriately changing the metal to be contained. Specifically, the template agent (CTAB) is dissolved in a sodium hydroxide solution, and TEOS and a metal nitrate solution (for example, calcium nitrate solution, magnesium nitrate solution, zinc nitrate solution, etc.) are vigorously stirred while maintaining 70 ° C. Is added dropwise at 0.01 to 20 mol%. After dropping, stirring is continued until a white precipitate is obtained by hydrolysis (5 hours), the precipitate is centrifuged, thoroughly washed with pure water and ethanol, and dried (80 ° C.). The dried product is calcined (550 ° C, 5 hours) to remove CTAB.

2. Immunoadjuvant preparation for tumor immunotherapy of the present invention (1) Anti-tumor immunostimulatory action that the spherical mesoporous silica particles of the present invention can exert alone In the present invention, the immunostimulatory action is intended for antigen-specific immune cells. It is the above-mentioned S2 action that gives an auxiliary stimulus to the antigen-specific immune cells when recognizing a tumor antigen, and includes humoral acquired immunity, cellular acquired immunity, and antitumor immunity.
If it has an S2 action, it affects the temporal, spatial or concentration change and attenuation of the vaccine antigen, and makes it easier for immune cells to utilize the antigen, or increases the chance of utilizing the antigen. May have.

When the spherical mesoporous silica particles of the present invention are said to have an immunostimulatory effect even when they are used alone, they have an immunostimulatory effect even when they do not contain an S2 agonist other than endotoxin of 0.1 EU / mg or less that is unintentionally mixed. It means that there is S2. By the way, the S2 agonist is specifically a Toll-like receptor agonist such as CpG-ODN or Poly (I: C), cytokines such as GM-CSF or interferon β, and C type lectin receptor such as β glucan. Agonists, NOD-like receptor agonists such as muramildipeptides, RIG-like receptor agonists such as viral RNA, exogenous DNA that binds to cyclic GMP-AMP synthase, exogenous allamines such as heat shock protein and HMGB1 and S100 protein (Alarmin), but not limited to these.

(2) Measurement method and regulation method of endotoxin The amount of endotoxin mixed in the prepared mesoporous silica is based on the endotoxin test method described in the Japanese Pharmacopoeia, the United States Pharmacopeia, and the European Pharmacopoeia, that is, the blood cell extract component of horseshoe crab. Quantify using the prepared lysate reagent. As a quantification method, the gel formation of the lysate reagent by the action of endotoxin is measured by a gelation method, a turbidimetry method or a colorimetric method. The turbidimetry method and the colorimetric method may be endpoint measurement or kinetic measurement. Endotoxin standard products are used for all measurements, but the titers of endotoxin standard products are standardized based on the World Health Organization endotoxin international standard products. Endotoxin units are shown in the EU, where 1 EU is equal to 1 endotoxin international unit (IU). In this way, the amount of endotoxin mixed in mesoporous silica is defined as the amount of endotoxin (EU) per 1 mg of mesoporous silica. In the present invention, the amount of endotoxin mixed in the spherical mesoporous silica is 0.1 EU / mg or less, preferably 0.06 EU / mg or less.

3. 3. Immunotherapy of tumors using the spherical mesoporous silica particles of the present invention as an immune adjuvant (1) Immunoadjudicant preparation consisting of the spherical mesoporous silica particles of the present invention The spherical mesoporous silica particles of the present invention can be used together with tumor antigens in mammals including humans. It is useful as an immune adjuvant for inducing a systemic immune response against the antigen when administered into the body. At that time, the spherical mesoporous silica particles of the present invention can be used alone as an immunoadjugant preparation for tumor immunotherapy without using a substance having another immunostimulatory action (S2 action) in combination. Since it is not necessary to add an immunostimulatory substance having low stability by using a single preparation, the manufacturing process is not complicated, and the obtained preparation has advantages of excellent storage stability and quality stability. Microwave irradiation, radio frequency coagulation method, freeze coagulation method, electric scalpel heating, hot water injection, alcohol injection, embolization method, irradiation, laser light irradiation, ultrasonic destruction, anticancer drug treatment, surgical treatment, micro When administered alone or in combination with a tumor antigen during or after wave treatment, the therapeutic effect can be enhanced and the recurrence prevention effect of prognosis can be enhanced. It can also be used in combination with an immune checkpoint inhibitor to enhance the therapeutic effect.

The administration method is microwave irradiation, radio frequency coagulation method, freeze coagulation method, electric scalpel heating, hot water injection, alcohol injection, embolization method, irradiation, laser light irradiation, ultrasonic destruction, anticancer drug treatment, etc. If degenerated tumor tissue remains in the body after treatment, it is directly administered to the degenerated tumor tissue by a method such as injection or endoscopy. The immunostimulatory action (S2 action) of the administered spherical mesoporous silica induces antitumor immunity using degenerated tumor tissue as a tumor antigen. In addition, as a different administration method, tumor tissue or tumor cells denatured after being removed by surgery or the like, or tumor antigen proteins or peptides and spherical mesoporous silica are mixed and homogenized to provide intradermal, subcutaneous, submucosal, etc. It can also be administered by injection or the like to a tissue in which a large number of antigen-presenting cells are present.
The timing and frequency of administration are appropriately determined depending on the condition of the tumor, the type and condition of the tumor antigen, the severity of the symptoms, the condition of the patient, and the like. For example, it may be administered all at once or in several divided doses at the same time as the treatment of other tumors such as alcohol injection, or within the range of 1 hour to 10 weeks thereafter, but the present invention is limited to this. It's not a thing.

The dose is also appropriately determined from the viewpoint of safety and efficacy depending on the condition of the tumor, the type and condition of the tumor antigen, the severity of the symptom, the condition of the patient, and the like. For example, it can be administered at a dose of 0.4 to 9.4 mg / kg body weight, but it is not limited to this range, and when standardized by body surface area instead of body weight, even if the dose is different. Good.
When a tumor antigen peptide or other synthetic tumor antigen is used in combination with an immunoassistant consisting of spherical mesoporous silica particles alone, it can be combined with these synthetic tumor antigens to form a kit.
The type of tumor targeted in the present invention is not particularly limited, and may be any of solid cancer, cell cancer, and hematological cancer, and may be high-grade or low-grade, or benign sarcoma. .. Further, although the present invention is mainly intended for humans, it is not limited to humans, and is not limited to humans, but widely targets mammalian tumors such as primates such as monkeys and rodents such as dogs, cats, cows, horses, and mice. ..

(2) Antitumor vaccine Tumor-derived antigens (tumor antigens) include tumor tissues, tumor cells, tumor cell lysates or denatured products thereof, tumor cell components, tumor antigen proteins, tumor antigen peptides, or purified products thereof. Examples include processed preparations. The immunoadjudicul of the present invention is formulated with one or more of these tumor antigens and known pharmacologically acceptable carriers, excipients, additives and the like to form an antitumor vaccine composition. You can also. In that case, the antigen delivery function and the protective function (S1 action) of the immune adjuvant of the present invention can be utilized.
The tumor antigen used in the present invention differs depending on the type of tumor to be targeted. For known tumor antigens, epitopes and peptides (eg MAGE-A4, Survivin, WT1, MUC1, LMP2, HER-2 / neu, MAGE-A3, NY-ESO-1, Glypican-3, SVN-2B, IMP -3 etc. antigens, epitopes, peptides), such purified ones can be used as antitumor vaccine preparations in advance. In addition, a patient-specific antitumor vaccine can be provided by degenerating the tumor tissue or tumor cells of the patient himself / herself collected by biopsy or surgery and then using it as a tumor antigen.
In addition, after degenerating the tumor tissue in the patient's body by the physical means described in (3) below, the tumor cells remaining in the patient's body are treated by administering the immunoadjudicant or vaccine of the present invention. It can be used as an immunotherapeutic agent for tumors that induces an antitumor immune response.

(3) Method for Administering ImmunoAdulgent to Tumor Tissue After Degeneration After denatured tumor tissue of mammalian animals including humans by physical means, an immunoadjugant preparation containing spherical mesoporous silica particles of the present invention is placed in the tumor tissue. By administration, an antitumor immune response can be induced. Physical means for degenerating tumor tissue include microwave irradiation, radiofrequency coagulation, cryocoagulation, electrosurgical heating, hot water injection, alcohol injection, embolization, irradiation, laser light irradiation, and ultrasound. Means such as destruction can be adopted. However, the physical degeneration means are not limited to these, and any means that can induce cell death of tumor cells in the tumor tissue may be used. Two or more physical means may be combined as appropriate.

(4) Concomitant use of immune checkpoint inhibitors A method for inducing antitumor immunity in combination with an immune checkpoint inhibitor for releasing an immunosuppressive state, and a composition for treating and / or preventing tumors, or treating tumors. And / or a combination drug for prevention is provided. When separately formulated with a combination drug, the separately formulated ones can be mixed and administered at the time of use, but the separately formulated ones are administered separately, simultaneously or at different times to the same subject. You may. According to a preferred embodiment of the invention, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-CD40 antibody, an anti-CD137 antibody, an anti-OX40 antibody, One or more checkpoint inhibitors selected from anti-TGF-β antibody, anti-LAG3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-TIM3 antibody, anti-CD96 antibody, anti-TIGIT antibody, and immune check. Methods of treating and / or preventing tumors that enhance the effectiveness of point inhibitors or reduce the amount of immune checkpoint inhibitors used, as well as compositions for treating and / or preventing tumors or for treating and / or preventing tumors. Combination medicines are provided.

(5) Concomitant use with other immunostimulatory substances The spherical mesoporous silica particles of the present invention are effective as an immunostimulant alone against tumor cells without adding other immunostimulatory substances (S2 action). It exerts immunostimulatory activity (S2 action), but does not prevent its combined use with other immunostimulatory substances. When used in combination with other immunostimulatory substances, higher immunostimulatory activity (S2 action) can be exhibited.

4. Measurement method and evaluation method of antigen-specific immunostimulatory action (1) Evaluation of immunostimulatory action of the present invention The immunostimulatory action of the present invention is carried out by the method specifically described in the specification of the present invention and the method described below. It can be easily measured by those skilled in the art. As an example, albumin derived from chicken egg white (hereinafter referred to as OVA) is used as an antigen, and OVA and mesoporous silica as a test substance are subcutaneously administered to mice. A few days to a few weeks after administration, lymphocytes are collected from lymphoid tissues such as lymph nodes and bone marrow and cultured, and at that time, OVA is added to stimulate them. From the type and amount of cytokines produced by lymphocytes by OVA stimulation, it is possible to confirm the activation of antigen-specific cellular acquired immunity and humoral immunity. For example, IL-2, IFNγ, and TNFβ are cytokines that characterize cellular acquired immunity, and IL-4, IL-5, and IL-10 are cytokines that characterize humoral immunity.

(2) Evaluation method for anti-tumor immune response-inducing effect-1
Whether or not to induce an antitumor immune response among antigen-specific cellular acquired immunity can be confirmed, for example, as follows.
^{A Lewis lung cancer cell (LLC) strain derived from 2.5 × 10 5} mouse lung cancers as a tumor antigen is crushed, mixed with the spherical mesoporous silica of the present invention, and then administered subcutaneously to the left mouse on the back. After another 7 to 11 days, the spherical mesoporous silica of the present invention is administered to the same site. After 15 days, 1 × 10 ⁴ live cell LLCs will be seeded subcutaneously in the right lower extremity and followed for 30 days to see if tumors form. In the control group, 2.5 × 10 ⁵ Lewis lung cancer cell (LLC) strains ground as tumor antigens were administered subcutaneously to the left left mouse on the back, and 15 days later, 1 × 10 ⁴ live cell LLCs were subcutaneously administered to the right lower extremity. Disseminate and follow up for tumor formation over the next 30 days. If the spherical mesoporous silica of the present invention has the activity of inducing a tumor antigen-specific immune response, tumor growth is inhibited or suppressed as compared with the control group. However, the administration timing and observation period of the spherical mesoporous silica, the tumor antigen, and the spherical mesoporous silica can be appropriately set. Further, a tumor antigen or a model tumor antigen and a tumor cell expressing the antigen may be used. For example, the antigen to be administered first may be OVA, and the live cells seeded thereafter may be OVA antigen-expressing mouse lymphoma cells (E.G7-OVA).

(3) Evaluation method for anti-tumor immune response-inducing effect-2
Furthermore, whether or not to induce an anti-tumor immune response as another method can be confirmed, for example, as follows.
^{C57BL / 6J mice are seeded with 5 × 10 5} Lewis lung cancer cells (LLC) subcutaneously on the left back of the mouse and bred for 7 to 10 days to form tumor tissue, and then the tumor tissue is removed and frozen in liquid nitrogen. The cells are immobilized by coagulation, pulverized, mixed with the spherical mesoporous silica of the present invention, and then administered subcutaneously to mice 3 days after tumor resection. Further, 7 to 14 days after the tumor resection, the spherical mesoporous silica of the present invention is administered to the same site. Four weeks after tumor resection, 5 × 10 ⁵ LLCs are seeded again on the right back back and followed for 30 days to see if tumors form. In the control group, only the excised tumor crushed 3 days after the tumor excision or a mixture of the excised tumor and alum was administered, and after 7 to 14 days, physiological saline was applied to the excised tumor group, and a mixture group of the excised tumor and alum was administered. Administers Alam and follows for 30 days to see if tumors form. If the spherical mesoporous silica of the present invention has the activity of inducing an antitumor immune response, tumor growth is inhibited or suppressed as compared with the control group. However, the tumor cells used are not limited to LLC, and the initial tumor immobilization treatment is not limited to freeze coagulation, and the administration time and observation period of spherical mesoporous silica and excised tumor, and spherical mesoporous silica can be appropriately set.

Hereinafter, the present invention will be described in more detail 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 art, and the techniques used to carry out the present invention are not particularly those for which the source is specified. Can be easily and surely carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, the instruction manuals for the kits, catalogs, and the like mutatis mutandis.
The technical documents, patent gazettes, and patent application specifications cited in the present specification shall be referred to as the description contents of the present invention.

(Example 1) Immunostimulatory action of silica fine particles having different shapes, particle sizes and structures Of the various shapes, particle sizes and structures of silica fine particles (Tables 1 (c) to (k)) shown in the following (Table 1). The immunostimulatory effect (cell-mediated immunity and / or humoral immunity) was examined. Mice (C57BL / 6J, 6-) in which 2 mg of aluminum hydroxide powder (Alam) (Table 1 (b)) and silica fine particles ((c)-(k)) in Table 1 were suspended in 100 μL of physiological saline. It was injected into the base of the tail (50 μL) and both sole bulbs (25 μL each) of a 10-week-old female). Two groups were prepared: an OVA-stimulated group in which OVA as an antigen was added at a concentration of 0.5 mg / mL to physiological saline in which each fine particle was suspended, and a non-stimulated group in which OVA was not added. As a negative control, a group to which physiological saline (Table 1 (a2)) to which fine particles were not added was also examined. On the 8th day after administration, the excised inflow region lymph nodes were crushed to obtain a single suspension of lymphocytes. The collected lymphocytes were cultured in RPMI1640 culture medium containing 10% FBS for 72 hours while stimulating with OVA. The immune activity of lymphocytes after a series of operations was compared by the amount of cytokines produced by lymphocytes, specifically IFNγ, IL-2, IL-4 and IL-10. It was evaluated that cell-mediated immunity was activated when the production of IL-2 and IFNγ increased, and humoral immunity was activated when the production of IL-4 and IL-10 increased.

FIG. 1 shows the amount of each cytokine produced after OVA stimulation of lymphocytes collected from the inflow region lymph node. FIG. 1A shows the production amount of IFNγ, FIG. 1B shows the production amount of IL-2, FIG. 1C shows the production amount of IL-4, and FIG. 1D shows the production amount of IL-10. Silica particles without mesopores ((c) to (f)) show cytokine production equal to or less than that of alum ((b)), whereas mesoporous silica tends to produce more cytokines and immunize alone. It was found to have a stimulating effect. Spherical particles (g) (h) (i) alone have a high immunostimulatory effect, especially (g) has a high IL-2 and IL-10 production, and (i) has a high IL-2 production. , (H) show that the production of all IFNγ, IL-2, IL-4 and IL-10 is high, and that they have a strong enhancing effect on both cell-mediated immune activity and humoral immune activity. It was.

(Example 2) Method for synthesizing spherical mesoporous silica fine particles (2-1) Synthesis of hollow and dense mesoporous silica spherical particles Hollow and dense mesoporous silica spherical particles (Table 1 (l) (m) (h)) are synthesized. However, the hollow structure, outer shell thickness, and mesopore diameter were controlled. _{Hexadecyltrimethylammonium bromide (C 19} H ₄₂ BrN, also known as cetyltrimethylammonium bromide, hereinafter abbreviated as "CTAB"), which is a template agent, is dissolved in ethanol (99.5%) supplemented with 28% aqueous ammonia, and the solution is 35 It was heated to ° C. After vigorously stirring the solution while maintaining the temperature, tetraethyl orthosilicate (Si (OC ₂ H ₅ ) ₄ , also known as Tetraethyl orthosilicate, hereinafter abbreviated as TEOS) was quickly added, and the mixture was continuously stirred for 24 hours. The mixing ratio of each chemical component in this solution was set to 1.00: 0.0922: 2.96: 621: 115 for TEOS: CTAB: ammonia: ultrapure water: ethanol (99.5%) in terms of molar ratio. The white sediment obtained by this operation was recovered by centrifugation (4400 rpm, 10 minutes), and further washed with ethanol (99.5%) three times. This white sink is stirred in a solution of 480 mL of ultrapure water plus 1 mL of TEOS, and the thickness of the outer shell of the fine particles is increased by the spontaneous shape change caused by changing the treatment conditions thereafter. Adjusted to 3 levels. The obtained three types of fine particles were recovered by centrifugation (4400 rpm, 10 minutes), and further washed with ultrapure water and ethanol (99.5%). After that, it was dried at 80 ° C. and further calcined at 550 ° C. for 5 hours to remove CTAB. Specific treatment conditions for adjusting the thickness of the outer shell are shown in (Table 2: Conditions for adjusting the outer shell thickness of hollow mesoporous silica fine particles).

(2-2) Synthesis of metal-containing and dense mesoporous silica spherical particles The metal-containing and dense mesoporous silica spherical particles (Table 1 (n) (o) (p)) are obtained by the method of Li et al. (Micropor Mesopor Mater 2007). It was synthesized as follows with reference to 102: 151-158).
0.28 g of sodium hydroxide and 1 g of template agent CTAB were dissolved in 480 mL of water. After visually confirming that CTAB was completely dissolved, the solution was heated to 70 ° C. After vigorously stirring the CTAB solution while maintaining the temperature, any combination of TEOS and calcium nitrate solution, TEOS and magnesium nitrate solution, or TEOS and zinc nitrate solution was added dropwise. At that time, the metal / silicon molar ratio was set to 10 mol%. After the dropping, stirring was continued for 5 hours to obtain a white precipitate by hydrolysis. The precipitate was centrifuged, washed thoroughly with pure water and ethanol, and dried at 80 ° C. The dried product was calcined at 550 ° C. for 5 hours to remove CTAB. When the product was dissolved in acid and chemically analyzed by induced binding plasma emission spectroscopic analysis, the Ca / Si molar ratio of (n) was 7.4 ± 0.3 mol%, and the Mg / Si molar ratio of (o) was 9.6 ± 0.3 mol. %, The Zn / Si molar ratio in (p) was 10.7 ± 0.1 mol%.

(2-3) Shape and characteristics of each mesoporous silica spherical particle Fig. 2 shows a transmission electron microscope image of mesoporous silica (l) (m) (h) (n) (o) (p). All of the fine particles are spherical with a diameter of about 100 to 200 nm, and (l) (n) (o) (p) have no high electron beam transmittance in the particles and are dense to the inside without an outer shell. Mesoporous particles (also called dense type). (m) is bright because the electron beam transmittance is high in the center of the particle, and dark because the electron beam transmittance is low at the edge. This dark part is the outer shell, which is about 70 nm thick. That is, (m) is a spherical hollow mesoporous silica particle (also referred to as a hollow type) having an outer shell having a thickness of 70 nm. Similarly, (h) was a spherical hollow mesoporous silica (hollow type) having an outer shell of about 30 nm to 40 nm. By observing at a higher magnification, fine changes in shading with a period of about several nm were observed, indicating that the outer shell is a mesoporous body. Furthermore, from the measurement of the roundness of the electron microscope image, 80% or more of spherical mesoporous silica with a roundness of 20% or less is contained in each of (l) (m) (h) (n) (o) (p). It turns out. For fine particles other than (l) (m) (h) (n) (o) (p), the diameter and roundness were measured from the electron microscope image in the same manner. The results are shown in the above (Table 1).

3A, B and C show the measurement results of the pore distribution and nitrogen adsorption isotherm of (l), (m) and (h), respectively. To support the observation with the transmission electron microscope image, the pore distribution measurement results show that all the fine particles have peaks with a diameter of 2 to 6 nm, and the nitrogen adsorption isotherms of all the fine particles are the adsorption and desorption isotherms by IUPAC. Similarity to type IV of line classification indicates that (l), (m) and (h) are mesoporous forms. (Table 3) shows the "specific surface area of hollow mesoporous silica". In particular, the hollow type (m) and (h) have a higher BET specific surface area than the dense type (l) and have a large hysteresis loop in terms of the amount of nitrogen adsorbed. It was demonstrated that adsorption occurs. This indicates that it favors the adsorption, delivery and protection of antigens (FIGS. 3A-C).

(Example 3) Evaluation of antigen-specific anti-tumor immunostimulatory effect of spherical mesoporous silica (3-1) Evaluation of anti-tumor immunostimulatory effect of spherical mesoporous silica particles Spherical mesoporous silica alone has immunostimulatory effect, particularly cellularity acquisition In order to confirm that it exerts an immunostimulatory effect, the antitumor immunostimulatory effect was examined using mice as a model animal. As the tumor antigen, a crushed product of LLC cells (cell number RCB0558: RIKEN BioResource Center) was used. Back of mice (C57BL / 6J, 6-10 weeks old, female, n = 5) with 0.1 mL saline containing 0.8 mg spherical mesoporous silica (h) and ^{5 crushed LLC 2.5 × 10} It was injected subcutaneously on the left side. After 7 and 11 days, 0.1 mL of physiological saline containing 0.8 mg (h) was subcutaneously injected into the same site. The same procedure was performed on the negative control group (n = 5) infused with only saline containing ⁵ LLC 2.5 × 10 crushed products. After 15 days, LLC (1 × 10 ⁴ cells) obtained as live cells was injected subcutaneously into the right lower limb of the mouse, and subsequent tumorigenesis was followed up for 1 month. If LLC did not engraft, it was evaluated that anti-tumor immunity was established.

4A and 4B show the evaluation results of the antitumor immunostimulatory effect of spherical mesoporous silica (h). When the presence of the tumor was confirmed by palpation, the Kaplan-Meier curve (Fig. 4A, Fig. 4C) was created using this as an endpoint. In group (h), the tumor inhibition rate continued to be maintained at 100% during the observation period, whereas in the saline group, tumor development was observed after 12 days, and the log-rank test performed after the elapsed period of 30 days also showed. The detection of a significant difference also showed (h) a clear antigen-specific antitumor immunostimulatory effect alone (FIGS. 4A and 4B).
It was proved that administration of a mixture of antigen and spherical mesoporous silica induced antigen-specific antitumor immunity (cell-mediated acquired immunity) and effectively suppressed tumor growth.

(3-2) Evaluation of anti-tumor immunostimulatory effect of metal-containing dense spherical mesoporous silica particles It was confirmed that metal-containing dense spherical mesoporous silica alone exerts an immunostimulatory effect, particularly a cellular adaptive immune stimulatory effect. Therefore, the antitumor immunostimulatory effect was examined using mice as a model animal. OVA was used as a model tumor antigen. Mice (C57BL / 6J) in 0.1 mL physiological saline containing 2 mg of metal-containing dense spherical mesoporous silica (n) (o) (p) or metal-free dense spherical mesoporous silica (g) and 150 μg of OVA. , 6-10 weeks old, female, n = 5) injected subcutaneously on the left side of the back. After 3 and 10 days, 0.1 mL of physiological saline containing 2 mg (n) (o) (p) (g) was subcutaneously injected into the same site. The same procedure was performed on the negative control group (n = 5) infused with saline (not including OVA) only. ^{After 14 days, 5 × 10 5} E.G7-OVA (American Type Culture Collection (ATCC)) obtained as living cells were injected subcutaneously into the right back of the mouse, and subsequent tumorigenesis was followed up for 1 month. .. If E.G7-OVA did not engraft or the tumor remained less than 15 mm in diameter (diameter by hemisphere approximation), it was evaluated that antitumor immunity was established.

4C and 4D show the evaluation results of the antitumor immunostimulatory effect of metal-containing spherical mesoporous silica (g) (n) (o) (p). A Kaplan-Meier curve was created with tumor growth up to a diameter of 15 mm or more as the endpoint. As a result, in all cases of (g) (n) (o) (p), an antigen-specific anti-tumor immunostimulatory effect alone was shown (FIGS. 4C and D).
It was demonstrated that administration of a mixture of antigen and metal-containing spherical mesoporous silica alone induced antigen-specific antitumor immunity (cell-mediated acquired immunity) and effectively suppressed tumor growth.

(Example 4) Evaluation of antitumor immunostimulatory effect of spherical mesoporous silica in recurrence prevention model Furthermore, the antitumor immunostimulatory effect of spherical mesoporous silica (h) alone was examined in the recurrence prevention model. ^{That is, 5 × 10 5} LLCs suspended in 0.1 mL of saline were injected subcutaneously into the left side of the back of a mouse (C57BL / 6J, 6-10 weeks old, female, n = 12) and 7-10. The tumor tissue formed after a day was surgically removed. The excised tumor tissue was put into liquid nitrogen for 30 minutes, then pulverized and used as an autologous tumor antigen. Three days after tumor tissue removal, 0.1 mL of physiological saline containing 0.8 mg of spherical mesoporous silica (h) and autologous tumor antigen was injected subcutaneously into the left side of the back of the surgically treated mouse (tumor removal site). After 7 and 14 days, 0.1 mL of physiological saline containing 0.8 mg (h) was subcutaneously injected into the same site. The same procedure was performed for two control groups infused with autologous tumor antigen only (n = 11) and saline suspension of autologous tumor antigen with alum (n = 12). After 4 weeks, LLC (5 x 10 ⁵ cells / 0.1 mL physiological saline) obtained as a live cell suspension was injected subcutaneously on the right side of the back of the mouse (model recurrence), simulating a tumor recurrence lesion, and thereafter. Tumor formation was followed up for 1 month. If no tumor was formed, it was evaluated that the tumor recurrence prevention effect was shown.

FIG. 5 shows the evaluation results of the tumor recurrence prevention effect of spherical mesoporous silica (h) alone. The Kaplan-Meier curve was created by regarding the time when tumor development on the right side of the back was confirmed by palpation as the endpoint. The tumor recurrence prevention rate in group (h) tended to be the highest among the three groups. This result shows that simply mixing spherical mesoporous silica alone with a tumor tissue pulverized product, which is a tumor antigen, can induce anti-tumor immunity, which is cellular acquired immunity, and reduce the tumor recurrence rate. There is.

(Example 5) Evaluation of immunological memory effect of spherical mesoporous silica In order to examine how spherical mesoporous silica is involved in immune induction, spherical mesoporous silica (h) was evaluated from the viewpoint of immunological memory.

Since the spherical mesoporous silica (h) can suppress the recurrence of cancer, it is expected that (h) will increase the number of effector memory T cells that control immunological memory and contribute to the extension of the cancer recurrence suppression period. Therefore, the abundance ratio was confirmed based on the characteristics of effector memory T cells, which are said to highly express CD44 and hardly express CD62L. Bone marrow was collected after euthanasia from mice (n = 3) that survived until the end of the observation period after the second administration of LLC in (Example 4), and 0.5% bovine was prepared after preparing a single cell suspension. Washed with serum albumin / PBS (-). The recovered bone marrow cells were previously blocked with an anti-CD16 / CD32 antibody (BD bioscience) from the Fc receptor, and then the presence or absence of expression of CD4, CD8, CD44 and CD62L was counted by flow cytometry.

FIG. 6A shows the ratio of CD4 positive (corresponding to helper T cells) and FIG. 6B to the total number of CD8 positive (corresponding to killer T cells) effector memory T cells collected from bone marrow.
(h) In mice administered alone and a mixture of autologous tumor antigens, the number of effector memory T cells was higher than in mice administered a mixture of alum and autologous tumor antigens or physiological saline containing only autologous tumor antigens. The number increased, and a significant difference was observed especially in the case of CD4 positive cells. This result indicates that (h) alone is involved in immunological memory and has the effect of sustaining the established cellular adaptive immunity against tumor antigens for a long period of time.

(Example 6) Anti-tumor immunostimulatory action of administering spherical mesoporous silica after denatured tumor tissue by physical means
^{1 × 10 7} tumor cells expressing wedge orange were seeded under the skin of one side of the C57BL / 6 albino mouse. Tumor size was monitored with IVIS, and 10 days after dissemination, the tumor was degenerated by irradiating the tumor with 20 Gy X-rays as a physical means. Next, 0.1 mg of spherical mesoporous silica (h) was administered to the degenerated tumor part of the thigh irradiated with X-rays 4,7,11,14,18,21 days after irradiation, and 24 days after irradiation, the brain was treated as a model recurrence 6 × 10 ^Five GL261-mKO cells were seeded (6 dose group; 10 animals). In the control non-administration group (16 animals), tumor formation under the skin of the thigh, degeneration of the thigh tumor by X-ray irradiation, and cell dissemination to the brain were performed with the same number of cells and schedule. After dissemination of tumor cells into the brain, follow-up was performed while monitoring the tumor size of the thigh and brain with IVIS.

FIG. 7 shows the survival curve of a mouse in which spherical mesoporous silica (h) was independently administered to a tumor tissue denatured by a physical means called X-ray, and tumor cells were seeded in the brain as a model recurrence. In the control group without spherical mesoporous silica, the mean overall survival was 59.8 days. In contrast, in the 6-dose group of spherical mesoporous silica (h), the average overall survival was 76.3 days (p = 0.038), and (h) alone was administered to the tumor site degenerated by physical means. A significant difference was observed in the tendency to prolong overall survival.

(Example 7) Measurement of endotoxin content of mesoporous silica 11.4 mg of spherical mesoporous silica (h) was weighed and placed in a 15 mL tube. 11.4 mL of distilled water for injection was added thereto to prepare a uniform suspension. Using 100 μL of this suspension as a sample, quantification was performed by the Kinetic colorimetric method using horseshoe crab blood cell extract components. As the endotoxin standard product, the endotoxin standard product (JP-RSE) of the Japanese Pharmacopoeia was used, and a standard stock solution of 10000 EU / mL was prepared based on the labeling titer of the standard product and used for measurement. In two measurements, the endotoxin concentration of the sample was measured to be less than 0.05 EU / mL. Therefore, it was found that the amount of endotoxin mixed in spherical mesoporous silica (h) was 0.05 EU / mg or less. That is, the amount of endotoxin unintentionally mixed in the spherical mesoporous silica (h) was 0.1 EU / mg or less.

(Example 8) Tumor therapeutic effect of a composition containing spherical mesoporous silica and anti-PD-1 antibody, and tumor therapeutic effect of a combination drug of spherical mesoporous silica and anti-PD-1 antibody Spherical mesoporous silica (h) and immune check The effects of a composition containing an anti-PD-1 antibody, which is a point inhibitor, and a combination drug of spherical mesoporous silica (h) and an anti-PD-1 antibody were examined for the treatment and / or prevention of tumors. Specifically, lymphoma cells E.G7-OVA (ATCC®) that express ovalbumin (OVA) by changing the dose and administration method of anti-PD-1 antibody and spherical mesoporous silica (h). The ^{growth inhibitory effect of CRL2113 TM} ) on tumors was observed over time. In this example, an anti-mouse PD-1 antibody was used as the anti-PD-1 antibody.

0.1 mL of physiological saline in which 5 × 10 ⁵ E.G7-OVAs were suspended was injected subcutaneously on the right side of the back of mice (C57BL / 6J, 7 weeks old, female, n = 5). Three days, 10 days, and 17 days after cell injection, the model tumor antigens OVA, anti-PD-1 antibody, and spherical mesoporous silica (h) were administered to mice at the single dose and administration method shown in (Table 4). It was administered. GA1 is the negative control group. GA2 is a combination drug group that combines topical administration of a mixture of spherical mesoporous silica (h) and OVA with systemic administration of anti-PD-1 antibody, GA3 and GA4 are anti-PD-1 antibody systemic administration group, and GA5 is spherical mesoporous silica. It is a composition group (composition group containing spherical mesoporous silica (h) and anti-PD-1 antibody) in which (h), OVA and anti-PD-1 antibody are mixed. OVA was administered to all groups in order to meet the conditions. Follow-up of the growth of the tumor formed on the right side of the back of the mouse was performed 2-3 times a week from 6 days to 28 days after the administration of E.G7-OVA, and the average diameter of the tumor (the sum of the minor and major lengths). It was judged that the endpoint was reached when the value (half the value of) exceeded 15 mm, and a Kaplan-Meier curve was created after the follow-up.

The effect of the combination drug of spherical mesoporous silica and anti-PD-1 antibody on the treatment and / or prevention of tumor was confirmed by comparison of GA1 to GA4. The created Kaplan-Meier curve is shown in (Fig. 8A).
In the negative control group (GA1) with topical OVA only, cases of reaching the endpoint 16 days after injection of E.G7-OVA were first confirmed, and then earlier than any group by 27 days on the back of all mice. The average diameter of the tumor formed on the right side exceeded 15 mm. In the group (GA3 and GA4) in which only anti-PD-1 antibody was systemically administered as in the existing therapy, the initial confirmation of the endpoint could be delayed until 21 days, but the average tumor diameter remained at 15 mm or less until the end of the observation period. The proportion of mice was eventually reduced to 0.0-0.2. In the case of the combination drug group (GA2) in which the same amount of anti-PD-1 antibody as GA4 is systemically administered to the topical administration of a mixture of spherical mesoporous silica (h) and OVA, the endpoint was first confirmed on the 21st day. In addition, the proportion of mice with an average tumor diameter of 15 mm or less until the end of the observation period was maintained at 0.4, which was the highest among the 4 groups compared.
This result shows that by using spherical mesoporous silica in combination as a combination drug during the existing therapy of anti-PD-1 antibody, a superior tumor treatment and / or preventive effect can be obtained as compared with treatment with anti-PD-1 antibody alone. It is shown that.

The therapeutic and / or preventive effect of the composition containing spherical mesoporous silica and anti-PD-1 antibody was confirmed by comparison of GA1, GA3 and GA5. The created Kaplan-Meier curve is shown in (Fig. 8B). In the negative control group (GA1) with only topical OVA, it was first confirmed that E.G7-OVA reached the endpoint 16 days after injection, whereas spherical mesoporous silica (h), OVA and anti-PD- The topical administration group (GA5) of the composition in which one antibody was mixed is similar to the group (GA3) in which only the anti-PD-1 antibody was systemically administered as in the existing therapy, and the initial confirmation of the endpoint is performed until the 21st. I was able to delay. Furthermore, in GA5, the proportion of mice with an average tumor diameter of 15 mm or less until the end of the observation period, which was the only of the three compared groups, did not decrease to 0.0 and remained at 0.4.
This result shows that by topically administering a composition containing both spherical mesoporous silica (h) and anti-PD-1 antibody, the tumor can be used even when the amount of anti-PD-1 antibody used is reduced compared to the existing therapy. It has been shown to be effective in treating and / or preventing. In addition, there is no need for systemic administration of anti-PD-1 antibody, the amount of anti-PD-1 antibody used is reduced, and the risk of systemic immune abnormal activation, which is a concern as a side effect, can be reduced. From the opposite point of view, according to this example, a composition containing an immune checkpoint inhibitor mixed with an immune checkpoint inhibitor, which has a tumor therapeutic and / or preventive effect that is also observed when spherical mesoporous silica is used alone. This suggests the possibility of amplification.

(Example 9) Immune checkpoints in the tumor therapeutic effect of a composition containing spherical mesoporous silica and anti-PD-L1 antibody, and the tumor therapeutic effect of a combination drug of spherical mesoporous silica and anti-PD-L1 antibody (Example 8). The effect of selecting an anti-PD-1 antibody as an inhibitor and treating and / or preventing a tumor of a combination drug of spherical mesoporous silica and anti-PD-1 antibody and a composition containing spherical mesoporous silica and anti-PD-1 antibody. However, when an equivalent combination drug or composition is composed of another type of immune checkpoint inhibitor, can a synergistic tumor treatment and / or prevention effect be obtained as in (Example 8)? Whether or not it was examined using an anti-PD-L1 antibody under the same conditions as in (Example 8). In this example, an anti-mouse PD-L1 antibody was used as the anti-PD-L1 antibody.

Inject 0.1 mL of saline containing 5 × 10 ⁵ E.G7-OVA subcutaneously on the right side of the back of mice (C57BL / 6J, 7 weeks old, female, number n = 10 at the start of the experiment). did. 3 days, 10 days, 16 days, and 24 days after cell injection, the model tumor antigens OVA, anti-PD-L1 antibody, and spherical mesoporous silica (h) were administered at a single dose and administration method shown in (Table 5). Was administered to mice. GB1 (n = 9) is the negative control group. GB2 (n = 10) is a combination drug group that combines topical administration of a mixture of spherical mesoporous silica (h) and OVA with systemic administration of anti-PD-L1 antibody, GB3 (n = 10) and GB4 (n = 10). Anti-PD-L1 antibody systemic administration group, and composition group in which GB5 (n = 9) is a mixture of spherical mesoporous silica (h), OVA and anti-PD-L1 antibody (spherical mesoporous silica (h) and anti-PD-L1 antibody) A group of compositions containing the above. OVA was administered to all groups in order to meet the conditions. Follow-up of the growth of the tumor formed on the right side of the back of the mouse was performed 2-3 times a week from 3 days to 28 days after the administration of E.G7-OVA, and the total tumor volume was 1690 mm ³ (tumor average diameter 15 mm). It was determined that the endpoint was reached when the volume of the case was exceeded, and a Kaplan-Meier curve was created after the follow-up was completed. For mice that died without waiting for the end of the follow-up period, if it could not be concluded that the direct cause of death was that the therapeutic effect on the tumor formed on the back was not obtained, it was excluded from the experimental results as a dropout. , The number of n differs depending on the group.

The effect of the combination drug of spherical mesoporous silica and anti-PD-L1 antibody on the treatment and / or prevention of tumor was confirmed by comparison of GB1 to GB4. The created Kaplan-Meier curve is shown in (Fig. 9A). In the case of the negative control group (GB1) with only topical OVA administration, the case of reaching the endpoint was first confirmed 15 days after the injection of E.G7-OVA, and the number of mice reaching the endpoint was earlier than any group thereafter. Increased. In the OVA topical administration and the medium dose (100 μg) systemic administration of anti-PD-L1 antibody (GB4), the initial confirmation of the case reaching the endpoint was slightly delayed 17 days later, but the medium dose systemic administration and spherical mesoporous In the case of the combined drug group (GB2), which is a combination of topical administration of a mixture of silica (h) and OVA, the endpoint is similar to the systemic administration group (GB3) of high-dose (200 μg) anti-PD-L1 antibody. The initial confirmation time of all mice could be delayed up to 20 days later. The progress since then was roughly the same for GB2 to GB4. When the total tumor volume was compared on the 13th day before all mice reached the endpoint, as shown in (Table 6), GB2 (spherical mesoporous silica (h) and OVA mixture was anti-PD-PD-L1). The average total tumor volume of GB3 (a group of combined drugs combined with systemic administration of L1 antibody) and GB3 (a group of systemic administration of only high-dose anti-PD-L1 antibody as in existing therapy) is GB1 (only OVA topical administration). It was about 42 to 46% of the negative control group).
This result also indicates that the growth of the tumor was delayed, and it can be seen that the effect is almost the same in GB2 and GB3. The above results show that when a combination drug of spherical mesoporous silica and anti-PD-L1 antibody is used to obtain a tumor therapeutic and / or preventive effect, the effect is remarkable at a relatively early stage after tumor formation. In addition, it has been shown that the amount of anti-PD-L1 antibody used can be halved while maintaining its effect by using a combination drug.

The effect of topical administration of a composition containing spherical mesoporous silica and anti-PD-L1 antibody on tumor treatment and / or prevention was confirmed by comparing GB1, GB3 and GB5. The created Kaplan-Meier curve is shown in (Fig. 9B). In the negative control group (GB1) with only topical OVA, it was first confirmed that the endpoint reached the endpoint 15 days after the injection of E.G7-OVA, whereas spherical mesoporous silica (h) and OVA and low doses ( 25 μg) In the topical administration group (GB5) of the composition mixed with anti-PD-L1 antibody, the initial confirmation time of the endpoint can be delayed until 20 days later, and the delay effect is the systemic administration group of high-dose anti-PD-L1 antibody. It was equivalent to (GB3). In GB5, there are mice that do not reach the endpoint until the end of observation, which is also similar to GB3.
This result shows that the amount of anti-PD-L1 antibody used, which was found to have a tumor therapeutic effect by systemic administration, was 1 by topically administering a composition containing both spherical mesoporous silica (h) and anti-PD-L1 antibody. It has been shown that even if it is reduced to / 8, it exerts the same therapeutic and / or preventive effect on tumors. In addition, there is no need for systemic administration of anti-PD-L1 antibody, the amount of anti-PD-L1 antibody used is reduced, and there is a risk of inadvertent action on normal cells expressing PD-L1 at the protein level, that is, side effects. It becomes possible to reduce. From the opposite point of view, the results of this example also show that the effect of treating and / or preventing tumors, which is also observed when spherical mesoporous silica is used alone, is a composition containing an immune checkpoint inhibitor mixed with each other. This suggests the possibility of amplification.

(Example 10) Spherical mesoporous In the tumor therapeutic effect of a composition containing an anti-PD-1 antibody due to a difference in silica and a combination drug with the anti-PD-1 antibody (Example 8) and (Example 9), spherical mesoporous As the silica, the spherical mesoporous silica (h) described in (Table 1) of (Example 1) was used, but the spherical mesoporous silica (hereinafter referred to as “spheroidal mesoporous silica”) having a modified shape, structure and particle size within the scope of the patent claim. , The symbol is (q)), the composition containing the anti-PD-1 antibody, which is also an immune checkpoint inhibitor, and the treatment of tumors with a combination drug with the anti-PD-1 antibody. And / or show that a preventive effect is obtained. The experimental conditions were set with reference to (Example 8). In this example as well, an anti-mouse PD-1 antibody was used as the anti-PD-1 antibody.

The shape, structure and particle size of the spherical mesoporous silica (q) used in this example and the spherical mesoporous silica (h) used in (Example 8) and (Example 9) are compared in (Table 7). did. The grain size of spherical mesoporous silica (q) is approximately twice as large. The values of the roundness and the proportion of particles having a roundness of 20% or less were relatively close values for both mesoporous silicas.

※平均値±標準偏差

* Average ± standard deviation

0.1 mL of physiological saline in which 5 × 10 ⁵ E.G7-OVAs were suspended was injected subcutaneously on the right side of the back of mice (C57BL / 6J, 7 weeks old, female, n = 5). Three days, 10 days, and 17 days after cell injection, the model tumor antigens OVA, anti-PD-1 antibody, and spherical mesoporous silica (q) were administered to mice at the single dose and administration method shown in (Table 8). It was administered. GC1 is the negative control group. GC2 is a combination drug group that combines local administration of a mixture of spherical mesoporous silica (q) and OVA with systemic administration of anti-PD-1 antibody, GC3 and GC4 are anti-PD-1 antibody systemic administration group, and GC5 is spherical mesoporous silica. It is a composition group in which (q), OVA and an anti-PD-1 antibody are mixed (composition group containing spherical mesoporous silica (q) and an anti-PD-1 antibody). OVA was administered to all groups in order to meet the conditions. Follow-up of the growth of the tumor formed on the right side of the back of the mouse was performed 2-3 times a week from 6 days to 28 days after the administration of E.G7-OVA, and the average diameter of the tumor (the sum of the minor and major lengths). It was judged that the endpoint was reached when the value (half the value of) exceeded 15 mm, and a Kaplan-Meier curve was created after the follow-up.

The effect of the combination drug of spherical mesoporous silica (q) and anti-PD-1 antibody on the treatment and / or prevention of tumor was confirmed by comparison of GC1 to GC4. The created Kaplan-Meier curve is shown in (Fig. 10A). In the low-dose (50 μg) systemic anti-PD-1 antibody group (GC4), the endpoint was 17 days after injection of E.G7-OVA, similar to the negative control group (GC1) with topical OVA only. The average diameter of tumors formed on the right side of the back of all mice exceeded 15 mm by day 26. In the case of a combination drug group (GC2) in which the same amount of anti-PD-1 antibody as GC4 is systemically administered to the topical administration of a mixture of spherical mesoporous silica (q) and OVA, the endpoint is first confirmed at a high dose (200 μg) of anti-PD-1 antibody. It was the same as the systemic administration group of PD-1 antibody (GC3) and was delayed until the 21st day. In addition, in GC2, the proportion of mice with an average tumor diameter of 15 mm or less until the end of the observation period was maintained at 0.4, which was the only one among the four groups compared.
Since the amount of anti-PD-1 antibody administered to GC2 and GC4 is the same, it is considered that the combination of spherical mesoporous silica (q) as a combination drug has the effect of treating and / or preventing tumors. .. In addition, spherical mesoporous silica, which has the effect of treating and / or preventing tumors by using it as a combination drug during existing therapy with anti-PD-1 antibody, is a spherical mesoporous silica if the shape, structure, and particle size are appropriately adjusted. It shows that it is not limited to (h).

The effect of the composition containing spherical mesoporous silica (q) and anti-PD-1 antibody on the treatment and / or prevention of tumor was confirmed by comparison of GC1, GC3 and GC5. The created Kaplan-Meier curve is shown in (Fig. 10B). In the negative control group (GC1) with only topical OVA, it was first confirmed that E.G7-OVA reached the endpoint 16 days after injection, whereas spherical mesoporous silica (q), OVA and anti-PD- The topical administration group (GC5) of the composition mixed with one antibody delayed the initial confirmation of the endpoint until the 24th day, and compared with the group (GC3) in which only the anti-PD-1 antibody was systemically administered as in the existing therapy. Was also delayed. Furthermore, in GC5, the proportion of mice with an average tumor diameter of 15 mm or less until the end of the observation period, which was the only of the three compared groups, did not decrease to 0.0 and remained at 0.4.
From this result, even in the case of spherical mesoporous silica (q), the amount of anti-PD-1 antibody used was reduced compared to the time of existing therapy by making a composition containing both this and anti-PD-1 antibody and locally administering it. Systemic, which is a concern as a side effect, because it has the effect of treating and / or preventing tumors in the state of being left untreated, it does not require systemic administration of anti-PD-1 antibody, and the amount of anti-PD-1 antibody used can be reduced. It has been shown that it is possible to reduce the risk of abnormal immune activation. Further, if the shape, structure and particle size of the spherical mesoporous silica used as the composition containing the anti-PD-1 antibody are appropriately adjusted, the effect of treating and / or preventing the tumor is not limited to the spherical mesoporous silica (h). Is also shown to be obtained.

Claims (9)

The administered the mesoporous silica spheres of hollow or dense particle size of 50~900nm at 65% or higher circularity of 20% or less of the particles seen including, together with the tumor antigens in the body of a mammal including human An adjuvant for tumor immunotherapy to induce a systemic immune response to tumor antigens.
As an adjuvant that exerts an immunostimulatory effect when used for tumor immunotherapy, it is composed of spherical mesoporous silica alone except for endotoxin of 0.1 EU / mg or less that is unintentionally mixed;
Here, in the case of hollow spherical mesoporous silica, it has an outer shell having a thickness of 10 to 200 nm and has a mesopore having a diameter of 2 to 50 nm in the outer shell.
An adjuvant for tumor immunotherapy, characterized in that the particle size is 100 to 500 nm in the case of dense spherical mesoporous silica. Mesoporous silica spheres of the dense is calcium, magnesium, and at least one metal selected from among zinc, characterized in that it contains a metal / silicon molar ratio 0 to 20 mol%, in claim 1 The adjuvant for tumor immunotherapy described. The tumor immunotherapy according to claim 1 or 2 , wherein the tumor antigen is selected from the group consisting of tumor tissue, tumor cells, tumor cell lysates or denatured products thereof, tumor cell components, tumor antigen proteins, and tumor antigen peptides. Adjuvant. The tumor immunity according to any one of claims 1 to 3 for inducing an antitumor immune response by degenerating the tumor tissue of a mammalian animal including human by physical means and then administering the tumor tissue into the tumor tissue. Therapeutic adjuvants, the physical means of which are microwave irradiation, radiofrequency coagulation, freeze coagulation, electric scalpel heating, hot water injection, alcohol injection, embolization, irradiation, laser light irradiation, and ultrasonic destruction. An adjuvant for tumor immunotherapy, which is one or more means selected from the group. A vaccine for tumor immunotherapy containing a tumor antigen, which does not contain an immunostimulatory substance other than endotoxin of 0.1 EU / mg or less that is unintentionally contaminated, and is an adjuvant that exerts an immunostimulatory effect according to claims 1 to 1. in any one of the 4 contains only tumor immunotherapeutic adjuvants described vaccine. A tumor comprising (1) an immunotherapeutic adjuvant according to any one of claims 1 to 4 or a vaccine according to claim 5 and (2) an immune checkpoint inhibitor. Therapeutic and / or preventive composition. Immune checkpoint inhibitors are anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-CD40 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-TGF-β antibody, anti. The treatment and / or treatment of the tumor according to claim 6 , which is one or more antibodies selected from LAG3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-TIM3 antibody, anti-CD96 antibody, and anti-TIGIT antibody. Prophylactic composition. (1) Tumor immunotherapy adjuvant according to any one of claims 1 to 4 or the vaccine according to claim 5 and (2) an immune checkpoint inhibitor, which comprises a combination of tumor treatment and / Or a preventive drug. Immune checkpoint inhibitors are anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-CD40 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-TGF-β antibody, anti. The treatment and / or treatment of the tumor according to claim 8 , which is one or more antibodies selected from LAG3 antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-TIM3 antibody, anti-CD96 antibody, and anti-TIGIT antibody. Preventive medicine.

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