Composition comprising cancer cells administered by syringe

Abstract

It has been found that cellular immunity is efficiently activated by administering a composition containing cancer cells expressing damage-related molecular patterns (DAMPs) to a subject through a needleless syringe, the cancer cells in the composition being damaged by shear force while being administered, and the DAMPs being released from the cancer cells to the outside of the cells.

Description

Technical Field

[0001] The present disclosure relates to a composition comprising cancer cells that is administered by syringe. Background Art

[0002] The following method for cancer treatment is under development (Non-Patent Document 1): by inactivating cancer cells, administering the inactivated cancer cells to a subject as an antigen, and activating the subject's cellular immunity against cancer. As a product using this method, for example, Vaccinogen's OncoVax (registered trademark), which is promoting clinical trials as a cancer vaccine, can be cited. OncoVax (registered trademark) is a product obtained by inactivating cancer cells in cancer tissue removed from a patient by radiation treatment, and formulating the inactivated cancer cells in a manner that is administered together with a BCG (Bacillus Calmette-Guérin) adjuvant. It can be expected that the preparation can be administered to a patient via a needle syringe, thereby obtaining immunity against the original cancer possessed by the cancer patient.

[0003] However, when such a method is used, there are technical problems such as the following.

[0004] (1) When only a syringe with a needle is used to administer radiation-treated cancer cells, the cell contents of the cancer cells cannot be effectively released outside the cells, so it is difficult to stimulate immune cells and activate cellular immunity. Therefore, in order to facilitate the activation of cellular immunity, it is necessary to add an adjuvant such as BCG to the inactivated cancer cells for the purpose of activating cellular immunity.

[0005] (2) In order to completely inactivate cancer cells by radiation treatment, high doses of radiation treatment are required depending on the type of cancer cells.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: Japanese Patent Application Publication No. 2012-061269

[0009] Non-patent literature

[0010] Non-patent literature 1: Cancer research 1979; 39: 1365-1360 Summary of the invention

[0011] Problems to be solved by the invention

[0012] The technical problem of the present disclosure is to provide a composition containing at least cancer cells that can be administered by a needle-free injector.

[0013] Solutions for solving problems

[0014] The present inventors have found that by administering a composition containing cancer cells expressing damage-associated molecular patterns (DAMPs) to a subject via a needle-free syringe, the cancer cells in the composition are damaged by shear force during administration, and DAMPs are released from the cancer cells to the outside of the cells, thereby activating cellular immunity efficiently. Furthermore, it has been found that a method of efficiently delivering a substance (antigen) into cells by administration via a needle-free syringe can be combined to more efficiently activate cellular immunity.

[0015] That is, the present disclosure is as follows.

[0016] [1] A composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs), wherein the composition is administered to a subject via a needle-free injector.

[0017] [2] The composition according to [1], wherein the DAMPs contain at least HMGB1.

[0018] [3] A composition comprising inactivated cancer cells, wherein the composition is administered to a subject via a needle-free injector.

[0019] [4] The composition according to [3], wherein the cancer cells are inactivated by radiation irradiation, drug administration, or heat treatment.

[0020] [5] The composition according to [4], wherein the cancer cells are inactivated by radiation irradiation and heat treatment.

[0021] [6] The composition according to any one of [3] to [5], wherein the cancer cells are cancer cells that have been inactivated and then cultured for a certain period of time.

[0022] [7] The composition according to any one of [1] to [6], wherein the cancer cells contained in the composition are cancer cells derived from solid cancer.

[0023] [8] The composition according to [7], wherein the solid cancer is pancreatic cancer, brain tumor, pharyngeal cancer, colorectal cancer, cervical cancer, prostate cancer, breast cancer, ovarian cancer, malignant melanoma, lung adenocarcinoma, liver cancer or kidney cancer.

[0024] [9] The composition according to any one of [1] to [8], wherein the cancer cells are cancer cells collected from the subject.

[0025]

[10] The composition according to any one of [1] to [9], wherein the composition is used to treat or prevent cancer.

[0026]

[11] The composition according to

[10] , wherein the cancer to be treated or prevented is a cancer derived from cancer cells contained in the composition.

[0027]

[12] The composition according to any one of [1] to [9], wherein the composition is used to activate cellular immunity against cancer.

[0028]

[13] A kit for treating or preventing cancer, comprising a needle-free syringe and the composition described in any one of [1] to

[12] .

[0029]

[14] A composition comprising cancer cells inactivated by radiation irradiation and heat treatment, wherein the composition is administered to a subject via a syringe with a needle.

[0030]

[15] A kit for treating or preventing cancer, comprising a needle syringe and the composition as described in

[14] .

[0031]

[16] A method for treating or preventing cancer, the method comprising: administering a composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) to a subject via a needle-free injector.

[0032]

[17] A method for treating or preventing cancer, comprising administering a composition comprising inactivated cancer cells to a subject via a needle-free injector.

[0033]

[18] A method for activating cellular immunity against cancer, the method comprising: administering a composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) to a subject via a needle-free injector.

[0034]

[19] A method for activating cellular immunity against cancer, the method comprising: administering a composition comprising inactivated cancer cells to a subject via a needle-free injector.

[0035]

[20] A composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs), for use in the treatment or prevention of cancer, is administered to a subject via a needle-free injector.

[0036]

[21] A composition comprising inactivated cancer cells for use in the treatment or prevention of cancer, which is administered to a subject via a needle-free injector.

[0037]

[22] A method for treating or preventing cancer, comprising administering a composition comprising cancer cells inactivated by radiation irradiation and heat treatment to a subject via a syringe with a needle.

[0038]

[23] A composition comprising cancer cells inactivated by radiation irradiation and heat treatment, which is used for the treatment or prevention of cancer and is administered to a subject via a syringe with a needle.

[0039]

[24] Use of a composition comprising cancer cells inactivated by radiation irradiation and heat treatment for manufacturing a medicine for treating or preventing cancer, wherein the composition is administered to a subject via a syringe with a needle.

[0040]

[25] A composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs), which is used to activate cellular immunity against cancer, is administered to a subject via a needle-free injector.

[0041]

[26] A composition comprising inactivated cancer cells for activating cellular immunity against cancer, which is administered to a subject via a needle-free injector.

[0042]

[27] Use of a composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) for the manufacture of a medicament for treating or preventing cancer, wherein the composition is administered to a subject via a needle-free injector.

[0043]

[28] Use of a composition comprising inactivated cancer cells for manufacturing a medicine for treating or preventing cancer, wherein the composition is administered to a subject via a needle-free injector.

[0044]

[29] Use of a composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) for the manufacture of a medicament for activating cellular immunity against cancer, wherein the composition is administered to a subject via a needle-free injector.

[0045]

[30] Use of a composition comprising inactivated cancer cells for manufacturing a medicine for activating cellular immunity against cancer, wherein the composition is administered to a subject via a needle-free injector.

[0046]

[31] A method for activating cellular immunity against cancer, the method comprising administering a composition containing cancer cells inactivated by radiation irradiation and heat treatment to a subject via a syringe with a needle.

[0047]

[32] A composition comprising cancer cells inactivated by radiation irradiation and heat treatment, which is used to activate cellular immunity against cancer and is administered to a subject via a syringe with a needle.

[0048]

[33] Use of a composition comprising cancer cells inactivated by radiation irradiation and heat treatment for the manufacture of a medicine for activating cellular immunity against cancer, wherein the composition is administered to a subject via a syringe with a needle.

[0049] Effects of the Invention

[0050] According to the present disclosure, a composition comprising at least cancer cells that can be administered via a needle-free injector can be provided.

[0051] Furthermore, by using the composition of the present disclosure, for example, the following effects are achieved.

[0052] First, when a composition containing cancer cells is administered to a subject using a needle-free syringe, cellular immunity can be activated more efficiently than when a composition containing cancer cells in the same state is administered to a subject using a needle-containing syringe.

[0053] In addition, by administering the composition of the present disclosure with a needle-free syringe, cellular immunity against cancer can be activated without using an adjuvant (such as BCG, etc.) for the purpose of activating cellular immunity. That is, the adjuvant commonly used in the development process of vaccine preparations can be omitted, or the amount of adjuvant used can be reduced. Moreover, compared with administering a composition containing cancer cells with a needle syringe, administering a composition containing cancer cells with a needle-free syringe can superiorly activate cellular immunity, so adjuvants other than adjuvants for the purpose of activating cellular immunity such as BCG can also be used.

[0054] In addition, in the past, when cancer cells were administered to a subject, in order to prevent cancer from occurring, the cancer cells needed to be completely inactivated. On the other hand, if a composition containing cancer cells is administered to a subject using a needle-free syringe, when the cancer cells in the composition are administered to a living body, the cancer cells are damaged and die, so there is no need to completely inactivate the cancer cells. Thus, methods and conditions for inactivating cancer cells before administering a composition containing cancer cells to a subject are added. It should be noted that according to the methods and conditions for inactivating cancer cells, cellular immunity can be efficiently activated even when a needle syringe is used.

[0055] Furthermore, in order to induce cell death of immunogenic cells, the following operation has been performed: irradiating cancer cells in vivo with an appropriate type of radiation at an intensity that induces cell death of cancer cells. On the other hand, the composition disclosed herein is, for example, a composition containing cancer cells expressing DAMPs, which is administered to a subject via a needle-free syringe, that is, the cancer cells are obtained by optimal inactivation treatment such as radiation irradiation, drug administration, or heat treatment in vitro, and therefore, cellular immunity can be efficiently activated even without radiation irradiation in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 This is a diagram showing a schematic configuration of a needle-free syringe according to an embodiment of the present disclosure.

[0057] Figure 2The photographs show the results of tumorigenesis when irradiated breast cancer cells according to one embodiment of the present disclosure are administered to mice using a needleless syringe or a needle-based syringe.

[0058] Figure 3 The photograph (A) and the graph (B) show the results of ELISpot (enzyme-linked immunospot) assay of interferon-γ using splenocytes excised from mice to which irradiated breast cancer cells were administered by needleless or needle-based syringes according to one embodiment of the present disclosure.

[0059] Figure 4 The photographs show the results of tumor development when irradiated colon cancer cells or non-irradiated colon cancer cells were administered to mice using a needleless syringe or a needle syringe, respectively, according to one embodiment of the present disclosure.

[0060] Figure 5 This is a graph showing the results of ELISpot assay of interferon-γ using spleen cells excised from mice to which irradiated colon cancer cells or non-irradiated colon cancer cells were administered by needleless or needle-based syringes, respectively, according to one embodiment of the present disclosure.

[0061] Figure 6 These are photographs showing the HMGB1 expression levels of various cancer cells after radiation irradiation according to one embodiment of the present disclosure.

[0062] Figure 7 This is a graph showing the results of ELISpot assay of interferon-γ using spleen cells excised from mice to which colorectal cancer cells treated with radiation, heat, or radiation and heat were administered by needle syringe according to one embodiment of the present disclosure. In the graph, * indicates p<0.05.

[0063] Figure 8 This is a graph showing the results of ELISpot assay of interferon-γ using spleen cells excised from mice to which irradiated colon cancer cells were administered by needleless or needle-based syringes according to one embodiment of the present disclosure. In the graph, * indicates p<0.05. DETAILED DESCRIPTION

[0064] Each configuration and combination thereof in each embodiment is an example, and addition, omission, substitution, and other changes of the configuration may be appropriately made within the scope not departing from the gist of the present invention. The present disclosure is not limited to the embodiments but only to the claims.

[0065] Each solution disclosed in this specification may also be combined with any other feature disclosed in this specification.

[0066] <Compositions containing cancer cells expressing damage-associated molecular patterns (DAMPs)>

[0067] One embodiment of the present disclosure is a composition comprising cancer cells expressing damage associated molecular patterns (DAMPs), wherein the composition is administered to a subject via a needle-free injector.

[0068] Damage-associated molecular patterns (DAMPs) are molecules expressed by damaged cells and are rarely expressed in undamaged cells.

[0069] The cancer cells contained in the composition of this embodiment are damaged cells, for example, cells damaged by inactivation treatment such as radiation irradiation treatment. On the other hand, the undamaged cells are typically normal cells that have not been damaged by the inactivation treatment, but may also be cancer cells that have not been damaged by the treatment.

[0070] The cancer cells contained in the composition of the present embodiment are cells that express DAMPs more than at least undamaged cells.

[0071] The DAMPs expressed by cancer cells contained in the composition of the present embodiment are not particularly limited as long as they do not hinder the effect of the composition of the present embodiment, and examples thereof include: high mobility group protein B1 (HMGB1), heat shock protein, S100A8, S100A9, cyclophilin A, cytochrome c, enolase-1, uric acid, matrilin-2, histone, fibrinogen, IL-33, hyaluronan, ferritin, profilin-1, fibronectin, ATP (Adenosine triphosphate), LL-37, mtDNA, calreticulin, etc.

[0072] The DAMPs expressed by the cancer cells contained in the composition of the present embodiment are preferably DAMPs containing at least HMGB1, and may also contain one or more DAMPs other than HMGB1. Here, a plurality of means that there is no particular limitation as long as the effect of the composition of the present embodiment is not hindered, and for example, it may be 2 or more, 3 or more, 5 or more, 10 or more, and it may be 100 or less, 50 or less, 30 or less, or 15 or less. In addition, these lower limits and upper limits may be arbitrarily combined, for example, it may be 2 or more and 100 or less, 3 or more and 50 or less, 5 or more and 30 or less, or 10 or more and 15 or less.

[0073] The expression level of DAMPs in cancer cells contained in the composition of the present embodiment is not particularly limited as long as it does not hinder the effect of the composition of the present embodiment. The expression level of DAMPs can be greater than that of control cells. For example, the expression level of DAMPs relative to the expression level of control cells can be greater than 1.00 times, 1.04 times or more, 1.10 times or more, 1.20 times or more, 1.22 times or more, 1.30 times or more, 1.40 times or more, 1.41 times or more, 1.50 times or more, 1.70 times or more, 1.71 times or more, or 2.00 times or more.

[0074] The needle-free syringe of this embodiment is one embodiment of an injector that can administer the composition to a subject without an injection needle. That is, in this embodiment, the composition can be administered to a subject by an injector that can administer the composition to a subject without an injection needle.

[0075] By administering the composition of the present embodiment to a subject using a needle-free syringe, there is an effect of more efficiently activating cellular immunity compared to the case of using a needle syringe.

[0076] That is, first, by using a needleless syringe, unlike the case of using a needle syringe, the cancer cells contained in the composition are damaged by shear force while being administered, so the DAMPs in the cancer cells are efficiently released outside the cancer cells. In addition, local inflammation is caused by the DAMPs released from the cancer cells, thereby causing an immune response. As a result, it is speculated that the phagocytic action of dendritic cells is enhanced, and the administered composition activates efficient cellular immunity. Then, not only the local effect of the administration site of the composition is activated, but also the cellular immunity against cancer cells present away from the administration site is activated.

[0077] That is, by administering a composition containing cancer cells expressing DAMPs to a subject via a needle-free syringe, an effect such as an abscopal effect can be produced with good reproducibility, and an effect such as an abscopal effect can be enhanced.

[0078] Therefore, administering the composition of the present embodiment to a subject via a needle-free syringe is useful for cancer treatment, cancer prevention, etc. Since effects such as abscopal effects can be expected even in cases where surgery is not possible or colorectal cancer cases where radiotherapy is not possible, administering the composition of the present embodiment to a subject via a needle-free syringe is useful.

[0079] The cancer cells contained in the composition of the present embodiment may be cancer cells derived from solid cancer. As solid cancers, for example, pancreatic cancer, brain tumors, pharyngeal cancer, colorectal cancer, cervical cancer, prostate cancer, breast cancer, ovarian cancer, malignant melanoma, lung adenocarcinoma, liver cancer or renal cancer, etc., and the types of cancer cells contained in the composition may be one or more of them. Solid cancer is preferably colorectal cancer or breast cancer. Breast cancer may be triple-negative breast cancer in which the markers of estrogen, prolactin and Her2 (human epidermal growth factor receptor 2) are all negative.

[0080] The cancer cells may be cancer cells collected from the subject (i.e., cancer cells collected from the subject to which the composition of the present embodiment is to be administered), or cancer cells collected from a subject other than the subject to which the composition of the present embodiment is to be administered, but the former is preferred. Regardless of the subject from which the cancer cells are collected, the cancer cells are preferably collected in the form of cancer tissue, and then the tissue is decomposed by, for example, enzyme treatment to make them into single-cell cancer cells.

[0081] Cancer cells may be subjected to pre-treatment such as purification of cancer cells as necessary.

[0082] The object is a mammalian individual (organism). There is no particular limitation on the mammal, and humans can be listed. In addition, mammals other than humans can be listed. Mammals other than humans include mice, rats, guinea pigs, hamsters, cattle, goats, sheep, pigs, monkeys, dogs, cats, etc. There are no particular limitations on the site of administration, and for example, the following can be listed: intradermal, subcutaneous, muscular, lymph node, etc., and the site of administration can be one or more of them. In addition, as the site of administration, for example, organs or tissues such as the pancreas, brain, pharynx, large intestine, cervix, prostate, breast, ovary, skin, lung, liver, and kidney can also be listed, and it can be one or more of these organs or tissues.

[0083] The content of cancer cells in the composition is not particularly limited as long as the effect of the composition of the present embodiment is not impaired.

[0084] For example, the lower limit can be set to 1.0×10 5 pcs / mL or more, 5.0×10 5 / mL or more, 1.0×10 6 pcs / mL or more, 5.0×10 6 / mL or more, 1.0×10 7 pcs / mL or more, 5.0×10 7 / mL or more, 1.0×10 8 pcs / mL or more, 5.0×10 8 / mL or more, 1.0×10 9 pcs / mL or more, 5.0×10 9 / mL or more, 1.0×10 10 pcs / mL or more, 5.0×10 10 / mL or more, 1.0×10 11 The upper limit can be set to 1.0×10 12 pc / mL or less, 5.0×10 11 pc / mL or less, 1.0×10 11 pc / mL or less, 5.0×10 10 pc / mL or less, 1.0×10 10 pc / mL or less, 5.0×10 9 pc / mL or less, 1.0×10 9 pc / mL or less, 5.0×10 8 pc / mL or less, 1.0×10 8 pc / mL or less, 5.0×10 7 pc / mL or less, 1.0×10 7 pc / mL or less, 5.0×10 6 pc / mL or less, 1.0×10 6 In addition, these lower and upper limits can be arbitrarily combined to set a certain range, for example, 1.0×10 5 / mL or more and 1.0×10 12 pc / mL or less, 5.0×10 5 / mL and above 5.0×10 11 pc / mL or less, 1.0×10 6 / mL or more and 1.0×10 11 pc / mL or less, 5.0×10 6 / mL and above 5.0×10 10 pc / mL or less, 1.0×10 7 / mL or more and 1.0×10 10 pc / mL or less, 5.0×107 / mL and above 5.0×10 9 pc / mL or less, 1.0×10 8 / mL or more and 1.0×10 9 When expressed as weight per 1 mL of the composition, it may be 1 ng / mL to 1000 mg / mL, 1 μg / mL to 1000 mg / mL, or 1 mg / mL to 1000 mg / mL.

[0085] The dosage of the composition of the present embodiment is not particularly limited as long as it does not hinder the effect of the composition, and can be appropriately adjusted according to the age, sex, weight, type, stage, and route of administration of the subject. For example, in the case of non-oral administration to an adult human (weight 60 kg), the composition of the above content can be administered at 1 ng to 1000 mg per day, 1 μg to 1000 mg, or 1 mg to 1000 mg. It can be administered once or multiple times.

[0086] The composition of the present embodiment is administered parenterally using a needle-free syringe, for example, intradermal administration, subcutaneous administration, and intramuscular administration, and may be one or more of these administration routes.

[0087] The composition of the present embodiment may be in a liquid form, and examples of the dosage form include injections.

[0088] The composition of the present embodiment may contain cancer cells expressing DAMPs alone, or may be mixed with a carrier or an additive. Here, as a carrier, pharmacologically permitted solvents, diluents, etc. may be listed. In the preparation of a liquid composition, for example, water, physiological saline, buffer, etc. may be used. As additives, stabilizers, pH adjusters, thickeners, antioxidants, isotonic agents, buffers, solubilizers, suspending agents, preservatives, antifreeze agents, cryoprotectants, freeze-drying protectants, antibacterial agents, etc. may be listed. The composition of the present embodiment may be manufactured by methods known to those skilled in the art.

[0089] The method for causing cancer cells to express DAMPs is not particularly limited, and for example, it can be carried out by the method for inactivating cancer cells described in the section <Composition containing inactivated cancer cells> described later.

[0090] <Needle-free syringe>

[0091] Another embodiment of the present disclosure is a needle-free syringe, which administers the composition to the object, and the needle-free syringe comprises: a container, which contains the composition; a nozzle, which is connected to the container and has an ejection port for ejecting the composition toward the object; and a pressurizing portion, which pressurizes the composition contained in the container during operation, thereby ejecting the composition from the ejection port toward the object.

[0092] The needle-free injector of the present embodiment is also one aspect of the needle-free injector in the item <Composition containing cancer cells expressing damage-associated molecular patterns (DAMPs)>.

[0093] In the needle-free syringe of this embodiment, the "distal end side" refers to the side where the ejection port for ejecting the composition from the needle-free syringe is arranged, and the "base end side" refers to the side opposite to the distal end side in the needle-free syringe, and these expressions do not limit to specific parts or positions.

[0094] In the needle-free injector of the present embodiment, the drive unit imparts injection energy in order to eject the composition toward the object. The "injection" achieved by the needle-free injector of the present embodiment is achieved by pressurizing the composition contained in the containing part by the pressurizing part using the injection energy imparted by the drive unit, thereby causing the composition to flow through the flow path of the containing part. The injection energy may be the injection energy used for conventional needle-free injectors, for example, the combustion energy of gunpowder, the generation energy of a gas generator, the electrical energy of a piezoelectric element, the mechanical energy of a spring, etc., and energy appropriately combined with these forms of energy may be used.

[0095] When the combustion energy of the gunpowder is used as the injection energy, the gunpowder includes, for example, a gunpowder containing zirconium and potassium perchlorate (ZPP), a gunpowder containing titanium hydride and potassium perchlorate (THPP), a gunpowder containing titanium and potassium perchlorate (TiPP), a gunpowder containing aluminum and potassium perchlorate (APP), a gunpowder containing aluminum and bismuth oxide (ABO), a gunpowder containing aluminum and molybdenum oxide (AMO), a gunpowder containing aluminum and copper oxide (ACO), a gunpowder containing aluminum and iron oxide (AFO), or a gunpowder containing a combination of multiple thereof. As a feature of these gunpowders, even if the combustion product thereof is gaseous at high temperature, it does not contain gaseous components at room temperature, so the combustion product condenses immediately after ignition. Thus, in the pressurization process for injecting the composition, the temperature of the combustion product during pressurization can be shifted to near room temperature in a short time after the pressure applied to the composition reaches the first peak injection force by the combustion of the ignition powder.

[0096] In addition, when the generation energy of the gas generating agent is used as the injection energy, a single-base smokeless gunpowder (for example, a single-base smokeless gunpowder containing 98 mass % of nitrocellulose, 0.8 mass % of diphenylamine, and 1.2 mass % of potassium sulfate) or various gas generating agents used in gas generators for air bags and gas generators for seat belt pretensioners can also be used as the gas generating agent.

[0097] In the needle-free syringe of the present embodiment, the composition contained in the containing portion is pressurized by the pressurizing portion during operation, thereby causing the composition to be ejected from the ejection port toward the object.

[0098] There is no particular limitation on the pressurization by the pressurizing unit as long as the system, for example, the housing portion, is not damaged, and the pressurization conditions of a normal needle-free syringe can be adopted.

[0099] The pressure refers to the pressure inside the receiving part. There is no particular limitation on the measuring method, and for example, the measurement can be performed as described below. That is, as in the measuring method described in Japanese Patent Gazette No. 2005-21640, the measurement is performed by the following method: the force of the injection is dispersedly applied to the diaphragm of the force sensor disposed downstream of the nozzle, and the output from the force sensor is collected by a data acquisition device via a detection amplifier and stored as the injection force (N) according to time. The injection pressure is calculated by dividing the injection pressure thus measured by the area of ​​the injection port 31a of the needleless syringe. The measured value of the internal pressure of the receiving part is the same as the injection pressure, and the injection pressure can be used as the pressure inside the receiving part.

[0100] The injection energy obtained by the driving unit is transmitted to the plunger via the piston, causing the plunger to slide in the container, whereby the composition contained in the container is extruded along the flow path formed in the nozzle unit and finally injected toward the object from the injection port.

[0101] The containing part may contain the composition from the beginning, or may not contain the composition. When not contained, the composition is contained by sucking the composition into the containing part through a nozzle having an ejection port. In this way, by adopting a structure that requires a containing operation of the containing part, any desired composition can be administered to the subject. Therefore, in the needle-free syringe of this embodiment, the syringe part and the syringe body can be configured to be detachable.

[0102] The administration to a subject by a needle-free injector in this embodiment may be administration to a subject by jet injection.

[0103] Here, in the present disclosure, "jet injection" is an injection without an injection needle, and the injection is characterized in that the composition is ejected from the ejection port toward the object, thereby forming a through hole that penetrates the inner and outer boundaries of the object, and generating a high-pressure ultrafine liquid flow that can pass through the through hole and deliver the composition into the object. For example, it refers to an injection characterized by generating a high-pressure ultrafine liquid flow that can penetrate the skin of the object. Or, it refers to an injection characterized by generating a high-pressure ultrafine liquid flow that can penetrate the organ when it is directly administered to the organ during laparotomy of the object.

[0104] The needle-free syringe of this embodiment can be obtained by containing the composition in the containing part in the previous needle-free syringe. As such previous needle-free syringes, for example, there can be listed: the needle-free syringe described in International Publication No. 2019 / 156238, the needle-free syringe described in International Publication No. 2019 / 156239, the needle-free syringe described in International Publication No. 2019 / 156237, and the needle-free syringe described in the specification of Japanese Patent No. 5989039. They are needle-free syringes that can perform the jet injection. In addition, there are many commercially available needle-free syringes that can perform the jet injection. For example, Straits (Pharmajet), Tropis (Pharmajet), Vitajet (Bioject Medical Technologies Inc.), Biojector 2000 (Bioject Medical Technologies Inc.), Bioject Zetajet (Bioject Medical Technologies Inc.), Glide (Glide Pharma), MediJector Vision (Antares), Sumaval DosePro (Zogenix), SQ Pen (Bespak), Injex (Equidyne), etc. can be mentioned.

[0105] Hereinafter, with reference to the accompanying drawings, a syringe 1 (needle-free syringe) will be described as an example of a needle-free syringe of the present embodiment. It should be noted that the configuration of the following embodiment is an example and is not limited to the configuration of the present embodiment. It should be noted that "top end side" and "base end side" are used as terms to indicate the relative positional relationship of the syringe 1 in the longitudinal direction. The "top end side" indicates a position close to the top end of the syringe 1 described later, that is, close to the ejection port 31a, and the "base end side" indicates the direction on the opposite side of the "top end side" in the longitudinal direction of the syringe 1, that is, the direction on the driving unit 7 side. In addition, this example is an example in which the combustion energy of the gunpowder ignited by the ignition device is used as the ejection energy for pressurization, but the present embodiment is not limited to this.

[0106] (Structure of the syringe 1)

[0107] Figure 1 1 is a diagram showing a schematic structure of the syringe 1, and is also a cross-sectional view of the syringe 1 along its longitudinal direction. The syringe 1 is constructed as follows: a syringe assembly 10 is mounted on a housing (syringe housing) 2, and the syringe assembly 10 is formed by integrally assembling a subassembly consisting of a syringe barrel 3 and a plunger 4 and a subassembly consisting of a syringe body 6, a piston 5, and a drive unit 7.

[0108] As described above, the syringe assembly 10 is configured to be detachable relative to the housing 2. The accommodating portion 32 formed between the syringe barrel 3 and the plunger 4 included in the syringe assembly 10 is filled with the composition, and the syringe assembly 10 is a unit that is used up and thrown away each time the composition is ejected. On the other hand, the housing 2 side includes a battery 9 that supplies power to the igniter 71 included in the drive unit 7 of the syringe assembly 10. The power from the battery 9 is supplied between the electrode on the housing 2 side and the electrode on the drive unit 7 side of the syringe assembly 10 through wiring by the user pressing the button 8 provided on the housing 2. It should be noted that the shape and position of the electrode on the housing 2 side and the electrode on the drive unit 7 side of the syringe assembly 10 are designed to automatically contact when the syringe assembly 10 is mounted on the housing 2. In addition, the housing 2 is a unit that can be used repeatedly as long as there is power remaining in the battery 9 that can be supplied to the drive unit 7. It should be noted that, in the casing 2 , when the power of the battery 9 is exhausted, only the battery 9 may be replaced and the casing 2 may continue to be used.

[0109] Next, the details of the syringe assembly 10 will be described. First, when describing the subassembly including the syringe barrel 3 and the plunger 4, the syringe barrel 3 has a housing 32 formed therein as a space capable of housing the composition. More specifically, Figure 1As shown, the plunger 4 is configured to slide freely along the inner wall surface extending in the axial direction of the injection barrel portion 3, and the inner wall surface of the injection barrel portion 3 and the plunger 4 define a housing portion 32. In addition, the injection barrel portion 3 has a nozzle portion 31 connected to the housing portion 32, and an ejection port 31a is formed on the top side of the nozzle portion 31. The nozzle portion 31 is a flow path whose cross-sectional area gradually decreases from the housing portion 32 side to the ejection port 31a side, and is used to guide the composition filled in the housing portion 32 to the ejection port 31a. Figure 1 In the example shown, the shape of the tip end side of the plunger 4 is substantially identical to the shape of the nozzle portion 31 .

[0110] Next, a subassembly including the syringe body 6, the piston 5 and the drive unit 7 will be described. The piston 5 is made of metal, for example, and is constructed to slide in a through hole formed inside the syringe body 6 by pressurizing the combustion product (combustion gas) generated by the igniter 71 of the drive unit 7. The syringe body 6 is a roughly cylindrical member that accommodates the piston 5, and the piston 5 is able to slide freely along the inner wall surface extending in the axial direction of the syringe body 6. It should be noted that the piston 5 can also be made of resin. In this case, metal can also be used in parts that require heat resistance and pressure resistance. In addition, if Figure 1 As shown, the piston 5 is integrally connected to the plunger 4 .

[0111] Next, the driving unit 7 will be described. Figure 1 As shown, the driving unit 7 is fixed to the base end side with the through hole in the syringe body 6 as a reference. The driving unit 7 has an igniter 71 as an electric igniter. The igniter 71 is arranged in a manner facing the inside of the through hole in the syringe body 6, and ignition powder is contained therein. As the ignition powder, various gunpowders can be used as described above. In addition, the ignition powder can be contained in a gunpowder cup formed of an appropriate thin-walled metal, for example.

[0112] Next, the operation of the syringe 1 constructed as described above will be described. Figure 1As shown, in a state where the syringe assembly 10 is mounted on the housing 2, the composition is sucked from the ejection port 31a of the nozzle portion 31. Thus, the composition can be filled into the housing portion 32. From this state, for example, when the ejection port 31a of the syringe 1 is brought into contact with the object, when the user presses the button 8 provided on the housing 2, this is used as a trigger to supply operating power from the battery 9 to the igniter 71 of the drive portion 7, and the igniter 71 operates. When the igniter 71 operates, the ignition powder is ignited and burns, generating combustion products (flame, combustion gas, etc.). As a result, for example, the gunpowder cup of the igniter 71 is cracked, and the combustion gas of the ignition powder is released into the through hole of the syringe body 6. As a result, the pressure in the through hole of the syringe body 6 rises sharply, and the piston 5 is pressed toward the top side of the syringe body 6, and as a result, the piston 5 slides toward the top side along the inner wall surface of the through hole of the syringe body 6. As described above, since the plunger 4 is integrally connected to the piston 5, the plunger 4 also slides along the inner wall surface of the injection barrel 3 in a manner linked with the piston 5. That is, by pressing the plunger 4 into the nozzle portion 31 located at the top end side of the injection barrel 3, the volume of the container 32 containing the composition decreases, and the pressure is increased rapidly. As a result, the composition filled in the container 32 is pressed into the nozzle portion 31 and ejected from the ejection port 31a at high pressure. Thus, the composition can be administered to the subject.

[0113] In addition, Figure 1 Although no additional explosive component is particularly arranged in the syringe body 6 shown, in order to adjust the pressure shift applied to the composition via the piston 5, a gas generating agent that generates gas by burning the combustion product generated by the combustion of the explosive in the igniter 71 can also be arranged in the igniter 71 and in the through hole of the syringe body 6. The configuration of arranging the gas generating agent in the igniter 71 is a well-known technology as disclosed in International Publication No. 01-031282 and Japanese Patent Publication No. 2003-25950. In addition, as an example of the gas generating agent, a single-base smokeless powder containing 98% by mass of nitrocellulose, 0.8% by mass of diphenylamine, and 1.2% by mass of potassium sulfate can be cited. In addition, various gas generating agents used for gas generators for air bags and gas generators for seat belt pretensioners can also be used. By adjusting the size, shape, and especially the surface shape of the gas generating agent when it is arranged in the through hole, the combustion completion time of the gas generating agent can be changed, thereby, the pressure shift applied to the composition can be set to the desired shift, that is, the shift that can make the composition reach the object appropriately. In this embodiment, the driving unit 7 also includes a gas generating agent used as needed. In this embodiment, the "pressurizing unit" includes a plunger 4 and a piston 5.

[0114] <Composition containing inactivated cancer cells>

[0115] Another embodiment of the present disclosure is a composition comprising inactivated cancer cells, wherein the composition is administered to a subject via a needle-free injector.

[0116] In this embodiment, inactivating cancer cells means inducing immunogenic cell death by any method in cells that accumulate DAMPs represented by HMGB1, etc. Cancer cells inactivated in this way typically express the DAMPs described in the above-mentioned <Compositions containing cancer cells expressing damage-associated molecular patterns (DAMPs)>.

[0117] In this embodiment, the degree of inactivation of cancer cells can be more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98% or more than 99% of all cancer cells contained in the composition, and the degree of inactivation can also be 100%.

[0118] The degree of inactivation of cancer cells can be determined, for example, by performing MTS colorimetry (MTS assay), which is a method known to those skilled in the art, 24, 48, or 72 hours after the inactivation treatment of the cells, to confirm the state of cell death or growth. Specifically, for example, if living cells can be confirmed 24 hours after the inactivation treatment of the cells, but no living cells can be confirmed after 72 hours, it can be determined that the cells are completely inactivated, that is, it can be determined that 100% of all cancer cells contained in the composition have been inactivated. On the other hand, if living cells can be confirmed 72 hours after the inactivation treatment of the cells, it can be determined that the cells are incompletely inactivated, for example, if 50% of all cancer cells contained in the composition are confirmed to be alive after 72 hours after the inactivation treatment of the cells, it can be determined that the degree of inactivation is 50%.

[0119] The method for inactivating cancer cells is not particularly limited as long as it can inactivate cancer cells, and examples thereof include radiation treatment, drug treatment, heat treatment, etc., and one or more of these treatments may be used. Preferably, a combined treatment combining radiation treatment and heat treatment is performed.

[0120] When radiation treatment is used as a method for inactivating cancer cells, the following methods can be cited: treatment with X-rays, treatment with heavy particle rays such as carbon ion rays, treatment with α rays, treatment with β rays, or treatment with γ rays. In the case of treatment with X-rays, as long as the cancer cells can be inactivated, the dose of X-rays is not particularly limited. For example, the lower limit can be set to 12 Gy or more, 25 Gy or more, 50 Gy or more, or 100 Gy or more. In addition, the upper limit can be set to 100 Gy or less, 50 Gy or less, or 25 Gy or less. These upper and lower limits can be arbitrarily combined, for example, 12 Gy or more and 100 Gy or less, 25 Gy or more and 100 Gy or less, 50 Gy or more and 100 Gy or less, 12 Gy or more and 50 Gy or less, 25 Gy or more and 50 Gy or less, 12 Gy or more and 25 Gy or less, etc.

[0121] It is preferred to adjust the cell number before radiation treatment. For example, it can be adjusted to 1×10 5 / ml~1×10 7 / ml, 5×10 5 / ml~5×10 6 / ml, etc., can also be adjusted to 1×10 6 Pieces / ml.

[0122] In the case of using drug treatment as a method for inactivating cancer cells, examples include treatment with drugs that cause apoptosis or necroptosis, such as mitomycin c, doxycycline, and B / B homodimerizer. Specifically, in the case of using mitomycin c, for example, mitomycin c can be added to a liquid containing cancer cells to a final concentration of 1.5 μg / ml to 150 μg / ml (e.g., 15 μg / ml), and treated in a water bath at 37° C. for 2 hours. In addition, in the case of using doxycycline, for example, doxycycline can be added to a liquid containing cancer cells to a final concentration of 0.1 mg / ml to 10 mg / ml (e.g., 1 mg / ml), and cultured for 24 hours for treatment. In addition, in the case of using B / B homodimers, for example, B / B homodimers can be added to a liquid containing cancer cells to a final concentration of 1 nM to 100 nM (e.g., 10 nM), and cultured for 24 hours for treatment. When using each chemical, the chemical concentration, reaction temperature, reaction time, etc. can be changed appropriately.

[0123] When heat treatment is used as a method for inactivating cancer cells, for example, the cells may be heated to 1×10 7The suspension was suspended in PBS at a concentration of 100 μg / ml or less and placed in a microcentrifuge tube (1.5 ml) and treated in a dry bath incubator at 42°C to 55°C (eg 45°C) for 30 minutes. The reaction temperature, reaction time, etc. can be appropriately changed.

[0124] In the present embodiment, cancer cells can be cultured for a certain period after inactivation. By culturing for a certain period, DAMPs can be accumulated in the inactivated cancer cells. Here, culturing for a certain period is not particularly limited, for example, as a lower limit, more than 12 hours, more than 24 hours, more than 48 hours, more than 72 hours can be cultivated, in addition, as an upper limit, less than 24 hours, less than 48 hours, less than 72 hours can be cultivated. These upper and lower limits can be combined arbitrarily, for example, more than 12 hours and less than 24 hours, more than 12 hours and less than 48 hours, more than 12 hours and less than 72 hours, more than 24 hours and less than 48 hours, more than 24 hours and less than 72 hours, more than 48 hours and less than 72 hours, more than 48 hours and less than 72 hours, etc.

[0125] Furthermore, the culture conditions for the inactivated cancer cells are not particularly limited as long as the cancer cells can be cultured, and the conditions such as the culture medium, temperature, and humidity used are not limited.

[0126] In this embodiment, the cancer tissue collected from the subject is preferably decomposed into a single-cell state and then inactivated by radiation treatment or the like and cultured. Alternatively, the cancer tissue may be inactivated and cultured by radiation treatment or the like in a state of only subdividing and slicing or in the original state of the cancer tissue without being decomposed into a single-cell state.

[0127] The descriptions about DAMPs, needle-free syringes, effects of administration, cancer cells, subjects, the content of cancer cells in the composition, the dosage of the composition, administration routes, forms, dosage forms, etc. can refer to the descriptions in the above-mentioned <Compositions comprising cancer cells expressing damage-associated molecular patterns (DAMPs)>.

[0128] <Composition containing cancer cells inactivated by radiation irradiation and heat treatment>

[0129] Another embodiment of the present disclosure is a composition comprising cancer cells inactivated by radiation irradiation and heat treatment, wherein the composition is administered to a subject via a syringe with a needle.

[0130] As a method for inactivating cancer cells, in the case of a combined treatment combining radiation treatment and heat treatment, not only when a needleless syringe is used to administer to an object, but also when a needle syringe is used to administer to an object, DAMPs in cancer cells are also efficiently released outside the cancer cells. In addition, local inflammation is caused by the DAMPs released from cancer cells, thereby causing an immune response. As a result, it is speculated that the phagocytosis of dendritic cells is enhanced, and the administered composition activates efficient cellular immunity. Then, not only the local effect of the administration site of the composition is activated, but also the cellular immunity against cancer cells present away from the administration site is activated.

[0131] That is, by administering a composition containing cancer cells subjected to a composite treatment combining radiation treatment and heat treatment to a subject via a syringe with a needle, an effect such as an abscopal effect can be produced with good reproducibility and an effect such as an abscopal effect can be enhanced.

[0132] Therefore, administering the composition of the present embodiment to a subject via a syringe with a needle is useful for cancer treatment, cancer prevention, etc. Since effects such as abscopal effects can be expected even in cases where surgery is not possible or colorectal cancer cases where radiotherapy is not possible, administering the composition of the present embodiment to a subject via a syringe with a needle is useful.

[0133] When radiation treatment is used as a method for inactivating cancer cells, the following methods can be cited: treatment with X-rays, treatment with heavy particle rays such as carbon ion rays, treatment with α rays, treatment with β rays, or treatment with γ rays. In the case of treatment with X-rays, as long as the cancer cells can be inactivated, the dose of X-rays is not particularly limited. For example, the lower limit can be set to 12 Gy or more, 25 Gy or more, 50 Gy or more, or 100 Gy or more. In addition, the upper limit can be set to 100 Gy or less, 50 Gy or less, or 25 Gy or less. These upper and lower limits can be arbitrarily combined, for example, 12 Gy or more and 100 Gy or less, 25 Gy or more and 100 Gy or less, 50 Gy or more and 100 Gy or less, 12 Gy or more and 50 Gy or less, 25 Gy or more and 50 Gy or less, 12 Gy or more and 25 Gy or less, etc.

[0134] It is preferred to adjust the cell number before radiation treatment. For example, it can be adjusted to 1×10 5 / ml~1×10 7 / ml, 5×10 5 / ml~5×10 6 / ml, etc., can also be adjusted to 1×10 6 Pieces / ml.

[0135] When heat treatment is used as a method for inactivating cancer cells, for example, the cells may be heated to 1×10 7 The suspension was suspended in PBS at a concentration of 100 μg / ml or less and placed in a microcentrifuge tube (1.5 ml) and treated in a dry bath incubator at 42°C to 55°C (eg 45°C) for 30 minutes. The reaction temperature, reaction time, etc. can be appropriately changed.

[0136] The composition of the present embodiment is administered non-orally using a syringe with a needle. For example, intradermal administration, subcutaneous administration, and intramuscular administration may be listed, and one or more of these administration routes may be used. The type of syringe with a needle is not particularly limited, and the specification, length, and shape of the needle tip of the needle may be a commonly used syringe according to the administration site.

[0137] Regarding the records of DAMPs, cancer cells, objects, the content of cancer cells in the composition, the dosage of the composition, the morphology, the dosage form, etc., the records in the above-mentioned <Composition containing cancer cells expressing damage-associated molecular patterns (DAMPs)> can be cited, and regarding the definition of inactivation of cancer cells, the degree of inactivation, and the culture after inactivation, the records in the above-mentioned <Composition containing inactivated cancer cells> can be cited.

[0138] <Composition containing cancer cells for treating or preventing cancer>

[0139] The compositions described in the above-mentioned <compositions comprising cancer cells expressing damage-associated molecular patterns (DAMPs)>, <compositions comprising inactivated cancer cells>, or <compositions comprising cancer cells inactivated by radiation irradiation and heat treatment> can all be used to treat or prevent cancer. That is, these compositions can all be used as pharmaceutical compositions.

[0140] In this embodiment, cancer treatment can be determined by the following circumstances: in the subject administered with the composition, the number of cancer cells is reduced, the cancer volume is reduced, or the cancer disappears completely, etc., compared with the control subject not administered with the composition or the subject before administration of the composition.

[0141] In this embodiment, the prevention of cancer may be, for example, the prevention of cancer in a subject who has not experienced cancer, or the prevention of recurrence or metastasis after resection of a solid cancer.

[0142] The composition of this embodiment can also be used as a vaccine for treating or preventing cancer.

[0143] The cancer to be treated or prevented is preferably a cancer from which the cancer cells contained in the composition of the present embodiment originate. Examples of cancers include pancreatic cancer, brain tumors, pharyngeal cancer, colorectal cancer, cervical cancer, prostate cancer, breast cancer, ovarian cancer, malignant melanoma, lung adenocarcinoma, liver cancer, or kidney cancer, and may be one or more of these cancers. Specifically, when the cancer to be treated or prevented is breast cancer, the cancer cells contained in the composition of the present embodiment are preferably cancer cells derived from the breast cancer to be treated or prevented.

[0144] The cancer cells may be cancer cells collected from the subject (i.e., cancer cells collected from the subject to which the composition of this embodiment is to be administered) or cancer cells collected from a subject other than the subject to which the composition of this embodiment is to be administered, but the former is preferred.

[0145] Regarding the description of DAMPs, needle-free syringes, effects of administration, cancer cells, subjects, the content of cancer cells in the composition, the dosage of the composition, the administration route, the form, the dosage form, inactivation, etc., reference can be made to the description in the above-mentioned <Composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs)>, <Composition comprising inactivated cancer cells> or <Composition comprising cancer cells inactivated by radiation irradiation and heat treatment>.

[0146] As other examples of this embodiment, the following schemes can be listed:

[0147] A method for treating or preventing cancer, the method comprising: administering a composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) to a subject via a needle-free injector;

[0148] A method for treating or preventing cancer, the method comprising: administering a composition comprising inactivated cancer cells to a subject via a needle-free injector;

[0149] A composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) for use in the treatment or prevention of cancer, which is administered to a subject via a needle-free injector;

[0150] A composition comprising inactivated cancer cells for use in the treatment or prevention of cancer, which is administered to a subject via a needle-free injector;

[0151] Use of a composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) for the manufacture of a medicament for treating or preventing cancer, wherein the composition is administered to a subject via a needle-free injector;

[0152] Use of a composition comprising inactivated cancer cells for the manufacture of a medicament for treating or preventing cancer, wherein the composition is administered to a subject via a needle-free injector;

[0153] A method for treating or preventing cancer, comprising: administering a composition containing cancer cells inactivated by radiation irradiation and heat treatment to a subject via a syringe with a needle;

[0154] A composition comprising cancer cells inactivated by radiation and heat treatment, which is used for the treatment or prevention of cancer and is administered to a subject via a syringe with a needle;

[0155] Use of a composition comprising cancer cells inactivated by radiation irradiation and heat treatment for the manufacture of a medicine for treating or preventing cancer, wherein the composition is administered to a subject via a syringe with a needle.

[0156] <Composition containing cancer cells for activating cellular immunity against cancer>

[0157] The compositions described in the above-mentioned <compositions comprising cancer cells expressing damage-associated molecular patterns (DAMPs)>, <compositions comprising inactivated cancer cells>, or <compositions comprising cancer cells inactivated by radiation irradiation and heat treatment> can all be used to activate cellular immunity against cancer. That is, these compositions can all be used as pharmaceutical compositions.

[0158] By administering the composition of the present embodiment to a subject using a needle-free syringe, cellular immunity against cancer can be activated more efficiently than when a needle-containing syringe is used.

[0159] That is, first, by using a needleless syringe, unlike the case of using a needle syringe, the cancer cells contained in the composition are damaged by shear force while being administered, so the DAMPs in the cancer cells are efficiently released outside the cancer cells. In addition, local inflammation is caused by the DAMPs released from the cancer cells, thereby causing an immune response. As a result, it is speculated that the phagocytic action of dendritic cells is enhanced, and the administered composition activates efficient cellular immunity. Then, not only the local effect of the administration site of the composition is activated, but also the cellular immunity against cancer cells present away from the administration site is activated.

[0160] Regarding the description of DAMPs, needle-free syringes, effects of administration, cancer cells, subjects, the content of cancer cells in the composition, the dosage of the composition, the administration route, the form, the dosage form, inactivation, etc., reference can be made to the description in the above-mentioned <Composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs)>, <Composition comprising inactivated cancer cells> or <Composition comprising cancer cells inactivated by radiation irradiation and heat treatment>.

[0161] As other examples of this embodiment, the following schemes can be listed:

[0162] A method for activating cellular immunity against cancer, the method comprising: administering a composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) to a subject via a needle-free injector;

[0163] A method for activating cellular immunity against cancer, the method comprising: administering a composition comprising inactivated cancer cells to a subject via a needle-free injector;

[0164] A composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) for activating cellular immunity against cancer, which is administered to a subject via a needle-free injector;

[0165] A composition comprising inactivated cancer cells for activating cellular immunity against cancer, which is administered to a subject via a needle-free injector;

[0166] Use of a composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs) for the manufacture of a medicament for activating cellular immunity against cancer, wherein the composition is administered to a subject via a needle-free injector;

[0167] Use of a composition comprising inactivated cancer cells for the manufacture of a medicament for activating cellular immunity against cancer, wherein the composition is administered to a subject via a needle-free injector;

[0168] A method for activating cellular immunity against cancer, the method comprising: administering a composition comprising cancer cells inactivated by radiation irradiation and heat treatment to a subject via a syringe with a needle;

[0169] A composition comprising cancer cells inactivated by radiation and heat treatment, which is used to activate cellular immunity against cancer and is administered to a subject via a syringe with a needle;

[0170] Use of a composition comprising cancer cells inactivated by radiation irradiation and heat treatment for the manufacture of a medicine for activating cellular immunity against cancer, wherein the composition is administered to a subject via a syringe with a needle.

[0171] <Kit for treating or preventing cancer>

[0172] Another embodiment of the present disclosure is a kit for treating or preventing cancer, the kit comprising the needle-free syringe and the composition comprising cancer cells. Or a kit for treating or preventing cancer, the kit comprising the needle-containing syringe and the composition comprising cancer cells.

[0173] These kits may optionally include a buffer, a diluent, and the like in addition to a needle-free syringe or a needle-containing syringe and a composition containing cancer cells.

[0174] Example

[0175] Hereinafter, the present disclosure will be described in more detail by way of examples, but the scope of the present disclosure is of course not limited to the examples.

[0176] <Example 1 Administration of irradiated breast cancer cells to mice>

[0177] 4T1 cells (obtained from ATCC (American Type Culture Collection)) as breast cancer cells were treated with 100 Gray doses of radiation using Gammacell (registered trademark) 40Exactor, and then cultured in RPMI 1640 complete medium for 24 hours. Cells were recovered after 24 hours and frozen with stem cell banker until use. It should be noted that although 4T1 cells were irradiated at this time, the inactivation of cancer cells was insufficient.

[0178] Before administering the above cancer cells to mice (Balb / c, 6 weeks old, male or female), cells stored in stem cell banker were thawed and washed twice with PBS. 1×10 6 Cancer cells (suspended in 30 μl PBS) were subcutaneously administered to the buttocks of mice. One week later, it was confirmed whether a tumor had formed at the site of administration of the cancer cells.

[0179] As a result, tumors were generated in all mice when a syringe with a needle (indicated as a normal syringe) was used. Figure 2 On the other hand, when a needle-free syringe (indicated as DD in the figure) was used, no tumors were observed in any of the mice ( Figure 2 , N=4).

[0180] In addition, before administering the above cancer cells to mice (Balb / c, 6 weeks old, male or female, n=4), cells stored in stem cell banker were thawed and washed twice with PBS. 5×10 5 Cancer cells (suspended in 30 μl PBS) were subcutaneously administered to the buttocks of mice, and 4 days later, cancer cells were administered for the second time in the same manner. Ten days after the second inoculation, cancer-specific immune responses were analyzed by the following method.

[0181] First, the spleens removed from these mice were mashed to recover spleen cells. Splenocytes were treated with AKC lysis buffer (Gibco) for 5 minutes and washed with PBS at 5 × 10 5 In addition, 4T1 cells were treated in the presence of mitomycin C (Nacalai, 15 μg / ml) in a 37°C water bath for 3 hours and then washed twice with PBS.

[0182] Splenocytes treated with AKC lysis buffer and 4T1 cells treated with mitomycin C were mixed at a cell number ratio of 10:1 and cultured for 48 hours in RPMI 1640 complete medium supplemented with 50 μM 2-mercaptoethanol (Nacalai).

[0183] After culture, spleen cells were recovered and 5×10 5 splenocytes and 2×10 3 4T1 cells were seeded together into an IFN-γ ELISpot plate and cultured for 48 hours. After the culture, the color development reaction of the ELISpot plate was performed by a method known to those skilled in the art.

[0184] As a result, in the group in which the irradiated breast cancer cells were administered by needle-free syringe (indicated as DD in the figure), a larger number of color reactions were confirmed compared with the group in which the cells were administered by needle-based syringe (indicated as ordinary syringe in the figure). Figure 3 ).

[0185] That is, first, by using a needle-free syringe, unlike when using a needle syringe, breast cancer cells are damaged by shear force during administration, and DAMPs in breast cancer cells are efficiently released outside the breast cancer cells. Furthermore, the DAMPs released from breast cancer cells cause local inflammation, thereby causing an immune response.

[0186] As a result, the phagocytic function of dendritic cells is enhanced, and the administered composition activates efficient cellular immunity, and then, not only the local effect of the administration site of the composition is activated, but also cellular immunity against cancer cells existing far from the administration site is activated.

[0187] <Example 2 Administration of irradiated colon cancer cells to mice>

[0188] MC38 cells (obtained from ATCC (American Type Culture Collection)) as colorectal cancer cells were treated with 25 Gray radiation using Gammacell (registered trademark) 40 Exactor, and then cultured in DMEM complete medium (DMEM + 10% FBS + penicillin / streptomycin) for 24 hours. After 24 hours, the cells were collected and cryopreserved using a stem cell banker until use.

[0189] Before administering the above cancer cells to mice (C57 / B6, 6 weeks old, male or female), cells stored in stem cell banker were thawed and washed twice with PBS. 1×10 6 Cancer cells (suspended in 30 μl PBS) were subcutaneously administered to the buttocks of mice. As a control, non-irradiated colorectal cancer cells were administered to the buttocks of mice using a needle syringe or a needleless syringe. One week later, it was confirmed whether a tumor had formed at the site of cancer cell administration.

[0190] As a result, in the group to which non-irradiated cells were administered by syringe with a needle (indicated as NL in the figure), tumors developed in all mice ( Figure 4 On the other hand, in the group to which irradiated cells were administered via a needle syringe (indicated as ND in the figure), the group to which non-irradiated cells were administered via a needleless syringe (indicated as DDL in the figure), and the group to which irradiated cells were administered via a needleless syringe (indicated as DDD in the figure), no tumors were observed in any of the mice ( Figure 4 , N=5 in each group).

[0191] In addition, before administering the above cancer cells to mice (C57 / B6, 6 weeks old, male or female, n=4), cells stored in stem cell banker were thawed and washed twice with PBS. 1×10 6 Cancer cells (suspended in 30 μl PBS) were subcutaneously administered to the buttocks of mice, and 4 days later, cancer cells were administered for the second time in the same manner. Ten days after the second inoculation, cancer-specific immune responses were analyzed by the following method.

[0192] First, the spleens removed from these mice were mashed to recover spleen cells. Splenocytes were treated with AKC lysis buffer (Gibco) for 5 minutes and washed with PBS at 5 × 10 5 In addition, MC38 cells were treated in the presence of mitomycin C (Nacalai, 15 μg / ml) in a 37° C. water bath for 3 hours and then washed twice with PBS.

[0193] Splenocytes treated with AKC lysis buffer and MC38 cells treated with mitomycin C were mixed at a cell number ratio of 10:1 and cultured for 48 hours in RPMI 1640 complete medium supplemented with 50 μM 2-mercaptoethanol (Nacalai).

[0194] After culture, spleen cells were recovered and 5×10 5 splenocytes and 2×10 3 MC38 cells were seeded into IFN-γ ELISpot plates and cultured for 48 hours. After culture, the color development reaction of the ELISpot plates was performed by methods known to those skilled in the art.

[0195] As a result, in the group to which the radiation-treated colorectal cancer cells were administered via a needleless syringe (indicated as DDD in the figure), a greater number of color reactions were confirmed compared to the group to which the non-radiated cells were administered via a needle syringe (indicated as NL in the figure), the group to which the radiation-treated cells were administered via a needle syringe (indicated as ND in the figure), and the group to which the non-radiated cells were administered via a needleless syringe (indicated as DDL in the figure). Figure 5 That is, first, by using a needle-free syringe, unlike when using a needle syringe, the colon cancer cells are damaged by shear force during administration, and the DAMPs in the colon cancer cells are efficiently released to the outside of the colon cancer cells. Furthermore, the DAMPs released from the colon cancer cells cause local inflammation, thereby causing an immune response.

[0196] As a result, the phagocytic function of dendritic cells is enhanced, and the administered composition activates efficient cellular immunity, and then, not only the local effect of the administration site of the composition is activated, but also cellular immunity against cancer cells existing far from the administration site is activated.

[0197] <Example 3 Study on HMGB1 expression level in various cancer cells after radiation irradiation>

[0198] 4T1 cells (obtained from ATCC (American Type Culture Collection)) as breast cancer cells were treated with 100 Gray radiation using Gammacell (registered trademark) 40Exactor, and then cultured in RPMI 1640 complete medium for 24 hours. In addition, MC38 cells (obtained from ATCC (American Type Culture Collection)) as colorectal cancer cells were treated with 25 Gray radiation using Gammacell (registered trademark) 40Exactor, and then cultured in DMEM complete medium (DMEM + 10% FBS + penicillin / streptomycin) for 24 hours.

[0199] Proteins were extracted by treating these cultured cells with RIPA buffer (obtained from Nacalai). 2.5 μg of the extracted protein was separated by 5% to 15% polyacrylamide electrophoresis (PAGE). Then, the migrated protein was transferred to a membrane by Western blot and reacted with the detection antibody. The primary antibodies used were HMGB-1 (biolegend, direct-blot, clone 3E8, cat; 651411) and β-actin (cellsignaling, clone 8H10D10, cat3700S), and the secondary antibody used was anti-mouse IgG (cytiva, cat: NA931V). After the antibody reaction, chemiluminescence was detected using immunoStar (Wako), a chemiluminescent reagent. The results are shown in Figure 6 . When 4T1 cells were used, the expression level of HMGB-1 in cancer cells irradiated with 25 Gy was 1.04 times that in the mock control group (Mock), and the expression level of HMGB-1 in cancer cells irradiated with 50 Gy was 1.22 times. In addition, when MC38 cells were used, the expression level of HMGB-1 in cancer cells irradiated with 12 Gy was 1.41 times that in the mock control group, and the expression level of HMGB-1 in cancer cells irradiated with 25 Gy was 1.7 times. <Example 4 Administration of colon cancer cells treated with a combination of radiation and heat to mice via a needle syringe>

[0200] · Acquisition of cells after irradiation

[0201] MC38 cells (obtained from ATCC (American Type Culture Collection)) as colorectal cancer cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin). The cells were cultured to 80% confluent and treated with 25Gy of radiation using Gammacell (registered trademark) 40Exactor. Then, 2 ml of trypsin EDTA solution was added and incubated at 37°C for 5 minutes to peel the cells from the culture plate. Centrifugation was performed, the supernatant was discarded, and the cells were recovered. The recovered cells were cultured for 24 hours. After 24 hours, the cells were recovered and the cell concentration was 5×10 6 The cells were added to Cellbanker as a cryopreservation solution at a concentration of 1 cell / ml and stored at -80°C.

[0202] · Acquisition of cells after heat treatment

[0203] MC38 cells (obtained from ATCC (American Type Culture Collection)) serving as colorectal cancer cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin). The cells were cultured until 80% confluence and washed with 10 ml PBS. 2 ml of trypsin EDTA solution was added and incubated at 37°C for 5 minutes to confirm that the cells were detached from the culture plate. Centrifugation was then performed, the supernatant was discarded, and the cells were recovered. DMEM complete medium (DMEM + 10% FBS + penicillin / streptomycin) was added and stirred to prepare a cell suspension, which was then heated at 45°C for 30 minutes. The heat-treated cells were then cultured for 24 hours. The cells were recovered after 24 hours at a cell concentration of 5×10 6 The cells were added to Cellbanker at a concentration of 100 μl / ml and stored at -80°C.

[0204] ・Harvesting cells after combined treatment with radiation and heat

[0205] MC38 cells (obtained from ATCC (American Type Culture Collection)) as colorectal cancer cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin). The cells were cultured until 80% confluence and treated with 25 Gray doses of radiation using Gammacell (registered trademark) 40Exactor. Then, the culture medium was discarded and the cells were washed with 10 ml PBS. 2 ml of trypsin EDTA solution was added, and after incubation at 37°C for 5 minutes, it was confirmed that the cells were detached from the culture plate. Then, centrifugation was performed, the supernatant was discarded, and the cells were recovered. DMEM complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin) was added and stirred to prepare a cell suspension, which was then heated at 45°C for 30 minutes. Then, centrifugation was performed to recover the cells. The recovered cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin) for 24 hours. After 24 hours, the cells were recovered and the cell concentration was 5×10 6 The cells were added to Cellbanker at a concentration of 100 μl / ml and stored at -80°C.

[0206] Furthermore, before administering the cancer cells treated by the above method to mice (C57 / B6, 6-week-old, male or female, n=4), the cells were thawed and then washed twice with PBS.

[0207] Use a syringe with a needle to transfer 1 × 10 6 Cancer cells treated by the above three methods were subcutaneously administered to the buttocks of different mice (suspended in 100 μl PBS), and the second administration of cancer cells was performed in the same manner 4 days later. Ten days after the second inoculation, the cancer-specific immune response was analyzed by the following method.

[0208] First, the spleens removed from these mice were mashed to recover spleen cells. Splenocytes were treated with AKC lysis buffer (Gibco) for 5 minutes and washed with PBS at 5 × 10 5 In addition, MC38 cells were treated in the presence of mitomycin C (Nacalai, 15 μg / ml) in a 37° C. water bath for 3 hours and then washed twice with PBS.

[0209] Splenocytes treated with AKC lysis buffer and MC38 cells treated with mitomycin C were mixed at a cell number ratio of 10:1 and cultured for 48 hours in RPMI 1640 complete medium supplemented with 50 μM 2-mercaptoethanol (Nacalai).

[0210] After culture, spleen cells were recovered and 5×10 5 splenocytes and 2×10 3MC38 cells were seeded into IFN-γ ELISpot plates and cultured for 48 hours. After culture, the color development reaction of the ELISpot plates was performed by methods known to those skilled in the art.

[0211] The results showed that the colorectal cancer cells treated with radiation and the colorectal cancer cells treated with heat ( Figure 7 In the mouse groups treated with radiation and heating (indicated in the figure), spots of IFNγ-producing cells were confirmed. Figure 7 A greater number of color reactions ( Figure 7 ). That is, it was confirmed that the radiation treatment and the heat treatment had a combined effect.

[0212] <Example 5 Administration of colon cancer cells treated with radiation and heat to mice by needle-injector and needle-free syringe>

[0213] Cells treated with radiation and heat were obtained by the following method. MC38 cells (obtained from ATCC (American Type Culture Collection)) as colorectal cancer cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin). The cells were cultured to 80% confluence and treated with 25 Gray doses of radiation using Gammacell (registered trademark) 40Exactor. Then, the culture medium was discarded and the cells were washed with 10 ml PBS. 2 ml of trypsin EDTA solution was added, and after incubation at 37°C for 5 minutes, it was confirmed that the cells were detached from the culture plate. Then centrifugation was performed, the supernatant was discarded, and the cells were recovered. DMEM complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin) was added and stirred, and after making a cell suspension, it was heated at 45°C for 30 minutes. Then centrifugation was performed to recover the cells. The recovered cells were cultured in DMEM complete medium (DMEM + 10% FBS + 1% penicillin / streptomycin) for 24 hours. After 24 hours, the cells were collected and the cell concentration was 5×10 6 The cells were added to Cellbanker at a concentration of 100 μl / ml and stored at -80°C.

[0214] Furthermore, before administering the cancer cells treated by the above method to mice (C57 / B6, 6-week-old, male or female, n=4), the cells were thawed and then washed twice with PBS.

[0215] Use a syringe with a needle or a needle-free syringe to add 1×10 6The cancer cells treated with the above method were subcutaneously administered to the buttocks of different mice (suspended in 100 μl PBS in the case of a needle syringe; suspended in 30 μl PBS in the case of a needleless syringe). Four days later, the second administration of cancer cells was performed in the same manner. Ten days after the second inoculation, the cancer-specific immune response was analyzed by the following method.

[0216] First, the spleens removed from these mice were mashed to recover spleen cells. Splenocytes were treated with AKC lysis buffer (Gibco) for 5 minutes and washed with PBS at 5 × 10 5 In addition, MC38 cells were treated in the presence of mitomycin C (Nacalai, 15 μg / ml) in a 37° C. water bath for 3 hours and then washed twice with PBS.

[0217] Splenocytes treated with AKC lysis buffer and MC38 cells treated with mitomycin C were mixed at a cell number ratio of 10:1 and cultured for 48 hours in RPMI 1640 complete medium supplemented with 50 μM 2-mercaptoethanol (Nacalai).

[0218] After culture, spleen cells were recovered and 5×10 5 splenocytes and 2×10 3 MC38 cells were seeded into IFN-γ ELISpot plates and cultured for 48 hours. After culture, the color development reaction of the ELISpot plates was performed by methods known to those skilled in the art.

[0219] As a result, the mice group ( Figure 8 Compared with the conventional syringe, the Figure 8 A greater number of color reactions ( Figure 8 ). That is, it was shown that, even when cells treated with a combination of radiation and heat were used, cellular immunity could be more efficiently induced by administration using a needle-free syringe.

[0220] Description of Reference Numerals

[0221] 1: syringe; 2: housing; 3: syringe barrel; 4: plunger; 5: piston; 6: syringe body; 7: drive unit; 8: button; 9: battery; 10: syringe assembly; 31: nozzle unit; 31a: ejection port; 32: storage unit; 71: igniter.

Claims

1. A composition comprising cancer cells expressing damage-associated molecular patterns (DAMPs), wherein: The composition is administered to the subject via a needle-free injector.

2. The composition according to claim 1, wherein The DAMPs at least contain HMGB1.

3. A composition comprising inactivated cancer cells, wherein: The composition is administered to the subject via a needle-free injector.

4. The composition according to claim 3, wherein The cancer cells are inactivated by radiation, drug administration or heat treatment.

5. The composition according to claim 4, wherein The cancer cells are inactivated by radiation and heat treatment.

6. The composition according to claim 3, wherein The cancer cells are cancer cells that have been inactivated and then cultured for a certain period of time.

7. The composition according to any one of claims 1 to 6, wherein The cancer cells contained in the composition are cancer cells derived from solid cancer.

8. The composition according to claim 7, wherein The solid cancer is pancreatic cancer, brain tumor, pharyngeal cancer, colorectal cancer, cervical cancer, prostate cancer, breast cancer, ovarian cancer, malignant melanoma, lung adenocarcinoma, liver cancer or kidney cancer.

9. The composition according to any one of claims 1 to 6, wherein The cancer cells are cancer cells collected from the subject.

10. The composition according to any one of claims 1 to 6, wherein The composition is used for treating or preventing cancer.

11. The composition according to claim 10, wherein The cancer to be treated or prevented is the cancer from which the cancer cells contained in the composition originate.

12. The composition according to any one of claims 1 to 6, wherein The composition is used to activate cellular immunity against cancer.

13. A kit for treating or preventing cancer, comprising a needle-free syringe and the composition according to any one of claims 1 to 6.

14. A composition comprising cancer cells inactivated by radiation and heat treatment, wherein: The composition is administered to the subject via a syringe with a needle.

15. A kit for treating or preventing cancer, comprising a needle syringe and the composition according to claim 14.

Images