Method for producing dendritic cell preparation comprising dendritic cells stimulating NKT cells

Abstract

[Problem] To provide a method for producing a dendritic cell preparation for stimulating NKT cells from mononuclear cells collected from a beneficiary (mononuclear cells obtained by component blood collection or peripheral blood including mononuclear cells), said dendritic cell preparation exhibiting a faster effect than in the prior art. [Solution] A method for producing a dendritic cell preparation that returns to the body of a beneficiary who has been sampled, the method comprising: an adhesion step for adding the collected monocytes to a culture vessel using a liquid medium, leaving the vessel to stand, and causing some of the monocytes to adhere to the inner surface of the vessel; a non-adherent cell removal step for removing non-adherent cells including mononuclear cells other than the cells adhering to the inner surface of the culture vessel; a differentiation step in which a predetermined factor is added to the culture vessel and the monocytes adhering to the inner surface of the culture vessel are differentiated into immature dendritic cells; a pulsing step for pulsing alpha-galactosylceramide into the culture vessel in which the immature dendritic cells are present in a non-adherent state; and an NKT-stimulated dendritic cell induction step in which NKT-stimulated dendritic cells, which are dendritic cells that stimulate NKT cells, are induced from the immature dendritic cells.

Description

Technical Field

[0001] The present invention relates to a method for producing a dendritic cell preparation containing dendritic cells that stimulate NKT cells (where "T" refers to Thymus, i.e., thymus) for any one or more of cancer treatment, cancer prevention, and enhancement of the immunity of healthy individuals, and having fewer side effects. In particular, it relates to a method for producing a dendritic cell preparation based on cells collected from the beneficiary himself / herself, and blood added with the dendritic cell preparation involved in this production method. Background Art

[0002] In recent years, cancer immunotherapies or methods for enhancing the immunity of healthy individuals targeting NKT cells have been developed. This therapy does not target cancer cells themselves, etc., but activates NKT cells, which are the command towers of the immune system. Through the adjuvant action of activated NKT cells (the action of promoting the activation of other immune system cells by activating a part of the immune system cells, thereby comprehensively activating the immune system), a strong anti-tumor effect is exhibited. Furthermore, since there is no such limitation as targeting specific antigens, effects can also be expected against new mutant cancer cells, pathogens other than cancer, viruses, parasites, etc.

[0003] Patent Document 1 describes the following method: Mononuclear cells are added to a first culture container, left standing to fix a part of the cells in the aforementioned mononuclear cells to the bottom surface of the container, and non-adherent cells other than the cells adhered to the bottom surface of the container are recovered and stored. Monocytes among the remaining cells adhered to the bottom surface are subjected to prescribed treatments to differentiate them into immature dendritic cells, and further subjected to prescribed treatments to mature them. Then, α-Galactosylceramide (hereinafter referred to as α-galactosylceramide) is added to the mature dendritic cells, dendritic cells that stimulate NKT cells are induced from the mature dendritic cells, and are cultured together with the stored non-adherent cells in a second culture container, and NKT cells are induced from a part of the aforementioned non-adherent cells.

[0004] Prior Art Documents

[0005] Patent Documents

[0006] Patent Document 1: Japanese Patent No. 6854290 Summary of the Invention

[0007] Problems to be Solved by the Invention

[0008] Patent Document 1 discloses that a large amount of IFN-γ (interferon-γ) is produced when NKT cells are activated, and when the CD-1d molecule on the surface of antigen-presenting cells (dendritic cells) is presented, an activation signal is transmitted through the T cell receptor on the surface of NKT cells, and the NKT cells are activated.

[0009] Regarding the cell composition containing dendritic cells and NKT cells that stimulate NKT cells disclosed in Patent Document 1, although the proportion of NKT cells is shown, the production amount of IFN-γ that has various effects on cancer cells is not disclosed, so its effects cannot be said to be clear. In addition, induction for stimulating NKT cells is carried out after the dendritic cells are matured. However, there is no record of research on the time correlation between the timing of induction and the timing of maturation.

[0010] To solve such problems, the inventors of the present application found, during the research and development process, a preparation of the present invention that produces a larger amount of IFN-γ and the production of IFN-γ starts faster compared to the method of Patent Document 1. The present invention studied the timing of performing induction for stimulating NKT cells on dendritic cells, and as a result, compared with the prior art (Patent Document 1), the amount of IFN-γ produced by NKT cells can be made larger. The produced IFN-γ stimulates and activates both the innate immunity system and the acquired immune system in the human body. Therefore, in cancer treatment and the like, greater effects brought about by these two pathways stimulated by a large amount of produced IFN-γ can be expected, and a manufacturing method of a dendritic cell preparation containing dendritic cells that stimulate NKT cells, which can expect higher therapeutic effects, is provided.

[0011] Means for Solving the Problems

[0012] First, to summarize the means for solving the problems, regarding the pulse timing of the ligand on dendritic cells, performing it at the stage when the dendritic cells are immature will increase the effect. In addition, regarding the timing of pulsing the ligand on immature dendritic cells, it is not the case that any timing is acceptable as long as it is in the immature stage. Even in the immature stage, a large effect can be obtained by pulsing at a specified timing. The following describes the specific means for solving the problems.

[0013] To solve the related problems of the dendritic cell preparation as described above, in the present application, as a first invention, there is provided a manufacturing method of a dendritic cell preparation, wherein the NKT cell-stimulating dendritic cell preparation is returned to the body of the beneficiary from whom monocytes have been collected to stimulate NKT cells. The manufacturing method is a manufacturing method of a dendritic cell preparation containing dendritic cells that stimulate natural killer T (NKT) cells, for any one or more of cancer treatment, cancer prevention, and immunity enhancement, and is characterized by including:

[0014] (1) Adhesion step: Monocytes collected from a beneficiary (monocytes obtained by component blood collection or blood collection, or peripheral blood containing monocytes) are added to a culture container using a liquid medium, and left standing to allow some of the monocytes to adhere to the inner surface of the container.

[0015] (2) Non - adherent cell removal step: Non - adherent cells containing monocytes other than the cells adhering to the inner surface of the culture container are removed.

[0016] (3) Differentiation step: A predetermined factor is added to the culture container to differentiate the monocytes adhering to the inner surface of the culture container into immature dendritic cells.

[0017] (4) Pulsing step: α - galactosylceramide is pulsed into the culture container in which the immature dendritic cells exist in a non - adherent state; and

[0018] (5) NKT - stimulated dendritic cell induction step: Dendritic cells that stimulate NKT cells, namely NKT - stimulated dendritic cells, are induced from the immature dendritic cells.

[0019] In addition, as a second invention, there is provided a method for manufacturing the following dendritic cell preparation: Based on the first invention, taking the day when the predetermined factor is added in the differentiation step of (3) as day 0, the pulsing step of (4) of the first invention is carried out on the 3rd day to the 4th day.

[0020] In addition, as a third invention, there is provided a method for manufacturing the following dendritic cell preparation: Based on the first invention, the pulsing step of (4) of the first invention is carried out 60 hours to 108 hours after the addition of the predetermined factor in the differentiation step of (3).

[0021] In addition, as a fourth invention, there is provided a method for manufacturing the dendritic cell preparation recited in claim 1 below: Based on the first invention, taking the day when the monocytes collected from the beneficiary are added to the culture container in the adhesion step of (1) as day 0, the pulsing step of (4) is carried out until the 4th day to the 5th day.

[0022] In addition, as a fifth invention, there is provided a method for manufacturing the following dendritic cell preparation: Based on the first invention, the pulsing step of (4) is carried out 84 hours to 132 hours after the monocytes collected from the beneficiary are added to the culture container in the adhesion step of (1).

[0023] In addition, as the sixth invention, there is provided a method for producing the following dendritic cell preparation: Based on the first invention, taking the day when non-adherent cells other than the cells adhering to the inner surface of the culture vessel, which include monocytes, are removed in the non-adherent cell removal step of (2) as day 0, the pulse step of (4) is carried out on the 4th to 5th day.

[0024] In addition, as the seventh invention, there is provided a method for producing the following dendritic cell preparation: Based on the first invention, the pulse step of (4) is carried out 84 to 132 hours after the non-adherent cells including monocytes other than the cells adhering to the inner surface of the culture vessel are removed in the non-adherent cell removal step of (2).

[0025] In addition, as the eighth invention, there is provided a method for producing the following dendritic cell preparation: Based on any one of the first to seventh inventions, the concentration of α-galactosylceramide pulsed in the pulse step of (4) is 300 to 1000 ng / mL.

[0026] In addition, as the ninth invention, there is provided the blood of a beneficiary, to which a dendritic cell preparation produced by the production method described in any one of the first to seventh inventions is added.

[0027] In addition, as the tenth invention, there is provided the blood of a beneficiary, to which a dendritic cell preparation produced by the production method described in the eighth invention is added.

[0028] Advantages of the Invention

[0029] According to the method for producing a dendritic cell preparation that stimulates NKT cells in the body of a beneficiary from whom monocytes have been collected and returned according to the present invention, compared with the prior art (Patent Document 1), the amount of IFN-γ produced by NKT cells can be made larger. The produced IFN-γ stimulates and activates both the innate immune system and the acquired immune system in the human body. Therefore, in cancer treatment and the like, greater effects brought about by these two pathways stimulated by a large amount of produced IFN-γ can be expected, and a method for producing a dendritic cell preparation containing dendritic cells that stimulate NKT cells, which can expect higher treatment effects, is provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A graph showing the relationship between the pulse timing of α-galactosylceramide during the production of the dendritic cell preparation in Embodiment 2 and the ratio of the number of NKT cells after co-culture

[0031] Figure 2a ​​Graph 1 showing the relationship between the pulse timing of α-galactosylceramide during the production of dendritic cell preparations in Embodiment 2 and the maximum ratio of the number of NKT cells after co-culture under each condition (when the day of addition of the differentiation-inducing factor is set as day 0)

[0032] Figure 2b Graph 2 showing the relationship between the pulse timing of α-galactosylceramide during the production of dendritic cell preparations in Embodiment 2 and the maximum ratio of the number of NKT cells after co-culture under each condition (when the start of culture is taken as the starting point)

[0033] Figure 3 Graph showing the relationship between the pulse timing of α-galactosylceramide during the production of dendritic cell preparations in Embodiment 3 and the ratio of the number of NKT cells after co-culture

[0034] Figure 4a Graph 1 showing the relationship between the pulse timing of α-galactosylceramide during the production of dendritic cell preparations in Embodiment 3 and the maximum ratio of the number of NKT cells after co-culture under each condition (when the day of addition of the differentiation-inducing factor is set as day 0)

[0035] Figure 4b Graph 2 showing the relationship between the pulse timing of α-galactosylceramide during the production of dendritic cell preparations in Embodiment 3 and the maximum ratio of the number of NKT cells after co-culture under each condition (when the start of culture is taken as the starting point)

[0036] Figure 5 Graph showing the relationship between the concentration of α-galactosylceramide pulsed during the production of dendritic cell preparations in Embodiment 4 and the amount of IFN-γ produced after co-culture

[0037] Figure 6 NK cell activity value and IFN-γ production amount in the case of using the dendritic cell preparation of the present invention for humans

[0038] Figure 7 Comparison graph of the prior art (Patent Document 1) and the inventive effect of the present invention (amount of IFN-γ produced after co-culture) Detailed Description of the Invention

[0039] First, before describing the embodiments of the present invention, the immunity of the body will be described.

[0040] Hereinafter, in the present specification, the effect of the preparation of the present invention on cancer cells will be taken as an example for description, but the same effect can also be obtained for other pathogens and the like. Immunity is divided into innate immunity that an organism originally has and acquired immunity that is acquired later.

[0041] ​​​​​​​Innate immunity is the immunity that humans are born with and is centered around phagocytes that engulf and remove foreign substances such as bacteria and viruses. Phagocytes have receptors that recognize molecules and structures and are roughly divided into types that process and eliminate foreign substances and types that play a role in signal transmission between cells. As innate immune cells, there are eosinophils, neutrophils, basophils, macrophages, dendritic cells, and NK (natural killer) cells.

[0042] On the other hand, acquired immunity is the immunity that memorizes pathogens in the body and can effectively eliminate them when encountered again. Compared with innate immunity, it takes time to respond, but it has diversity in the response to various pathogens. As acquired immune cells, there are B cells and T cells, and T cells further include helper T cells, cytotoxic T cells, and regulatory T cells. B cells change into plasma cells and memory B cells according to their state.

[0043] In addition, there are NKT cells that stimulate both innate immunity and acquired immunity.

[0044] Among the aforementioned immune cells, the cells directly acted on by this dendritic cell preparation are NKT cells, but through the adjuvant effect of IFN-γ produced by the activation of NKT cells, NK cells, dendritic cells, T cells (helper T cells, cytotoxic T cells), B cells, etc. are also activated together.

[0045] Hereinafter, a brief description will be given of the immune cells and substances related to immune functions involved in the action of the dendritic cell preparation generated by the manufacturing method of this dendritic cell preparation.

[0046] <Explanation of terms for immune cells and substances related to immune functions>

[0047] <Term explanation: Antigen>

[0048] Foreign substances that enter the body are targets for acquired immune cells to recognize as objects of attack. In the case of cancer antigens, the structure of proteins that do not exist in normal cells is targeted, and immune cells recognize them as objects of attack.

[0049] <Term explanation: Antibody>

[0050] It is the general term for proteins called immunoglobulins. Cancer antibodies bind to the cancer antigens of cancer cells and attack cancer cells through the following three functions.

[0051] Neutralization function: The function of hindering the proliferation of cancer cells

[0052] ADCC activity: The function of becoming the target of cytotoxic T cells and NK cells and assisting in the attack on cancer

[0053] CDC activity: Complement (synthesized in vivo, one of the proteins present in serum, a substance involved in immune responses such as infection defense, with 9 types from C1 to C9) binds to an antibody that has already bound to a cancer antigen of a cancer cell, activating the complement from C1 to C9 in a chain reaction, creating pores in the cell membrane of the cancer cell to kill it and cause its death

[0054] <Term Explanation: Cytokine>

[0055] A protein secreted by immune cells, a general term for physiologically active substances involved in cell - to - cell interactions. It transmits signals to target cells, causing various cellular responses such as cell activation, cell proliferation, differentiation, cell death, and functional expression. Interleukins, interferons, tumor necrosis factors, etc. are types of cytokines

[0056] <Term Explanation: Cytokine: Interleukin>

[0057] Currently, more than 30 types of interleukins, which are a type of cytokine, have been confirmed. In particular, they are secreted in large amounts by phagocytic cells such as lymphocytes, macrophages, and neutrophils, which are a type of white blood cell that fights foreign substances in the body. Interleukins are named with numbers like interleukin - 2 (hereinafter, interleukins are abbreviated as IL. For example, interleukin - 4 is IL - 4). For example, regarding interleukins, several sources of production and functions are described below

[0058] <Term Explanation: Cytokine: Interleukin: IL - 4>

[0059] IL - 4 is one of the Th2 cytokines, produced by activated helper T cell type 2 (Th2 cells), NKT cells, etc. It promotes the proliferation and differentiation of antigen - presenting cells. It has an impact on B cells, T cells, macrophages, monocytes, etc. Naive T cells differentiate into Th2 cells when stimulated by cytokines such as IL - 4 and IL - 13. When IL - 4 acts on activated B cells, the immunoglobulin (antibody) produced undergoes a class switch from IgM to IgE and IgG1. Thus, it is involved in the humoral immune system

[0060] NKT cells are activated when they recognize α - galactosylceramide presented by antigen - presenting cells (dendritic cells) and produce IL - 4 and IFN - γ

[0061] <Term Explanation: Cytokine: Interleukin: IL - 12>

[0062] IL-12 is produced by phagocytes (which migrate between tissues in animals and are phagocytic cells responsible for cellular immunity: macrophages, neutrophils, etc.) and dendritic cells. It is an NK cell stimulatory factor and, together with IFN-γ, acts on naive T cells to differentiate them into Th1 cells, which are a type of helper T cell. Th1 cells produce Th1 cytokines such as IFN-γ and are involved in cellular immunity, activating macrophages, cytotoxic T cells, etc.

[0063] α-Galactosylceramide causes dendritic cells (antigen-presenting cells) to produce IL-12. IL-12 produced by antigen-presenting cells acts on NKT cells to cause them to produce IFN-γ, or shows cytotoxic activity against target cells expressing Fas antigen (Fas receptor) via the Fas ligand on the surface of NKT cells.

[0064] <Term Explanation: Cytokine: Interferon>

[0065] As a type of cytokine, interferon has the effects of activating NK cells, macrophages that attack cancer cells or virus-infected cells, etc., inhibiting the proliferation of viruses and tumor cells, and promoting the phagocytosis of cancer cells (adjuvant effect). It is also recognized by the state as an antiviral drug and an anticancer agent and is used in the treatment of multiple myeloma, brain tumors, and renal cell carcinoma.

[0066] <Term Explanation: Cytokine: Interferon: IFN-γ>

[0067] IFN-γ is an important cytokine in both the innate and adaptive immune systems. As part of the innate immune response, it is mainly produced by NK cells and NKT cells, and after the induction of antigen-specific immunity, it is produced by Th1 cells, cytotoxic T cells, macrophages, etc. It accelerates the processing of a large number of invading non-self antigens, hyperactivates the cellular immune response. It is an important activator of macrophages, stimulating them to phagocytose and kill bacteria. It promotes NK cell activity and, in addition, differentiates naive T cells into Th1 cells.

[0068] <Term Explanation: Cytokine: Tumor Necrosis Factor>

[0069] Tumor necrosis factor (TNF) is a substance secreted by cells such as lymphocytes that causes tumor cells to necrose. In addition to acting on almost all malignant cells, it also promotes immune function. Narrowly defined, it includes three types: TNF-α, TNF-β (lymphotoxin (LT)-α), and LT-β.

[0070] <Term Explanation: Cytokine: Tumor Necrosis Factor: TNF-α>

[0071] TNF-α is mainly produced by macrophages and was discovered as a cytokine that causes hemorrhagic necrosis in solid cancers. Generally, tumor necrosis factor refers to TNF-α. It is involved in the expression of cell adhesion molecules and the induction of apoptosis. Apoptosis refers to the suicide of regulated cells, that is, programmed cell death.

[0072] <Term Explanation: NK cells>

[0073] NK cells have various receptors for capturing abnormal cells and have the function of recognizing cells infected with viruses, cancer cells, etc., which are different from the cells that make up the normal human body (remember the cells that make up the human body), as abnormal and killing the abnormal cells when they are found.

[0074] <Term Explanation: Dendritic cells>

[0075] They are antigen-presenting cells. They obtain the antigen-presenting ability unique to the foreign substance by ingesting (phagocytosing) foreign substances (cells infected with pathogens, viruses, cancer cells, etc.), or obtain the antigen-presenting ability by antigen presentation by macrophages, etc. And they have the function of presenting the antigen to T cells and being recognized as the target of attack. Dendritic cells have the phagocytic function of phagocytosing viruses and cancer cells, and thus obtain the ability to acquire antigens and present antigens to other immune cells. Early dendritic cells (immature dendritic cells) have the ability to ingest antigens (phagocytosis), but cannot activate naive T cells (it can be said that they have no antigen-presenting ability). Mature dendritic cells obtain the antigen-presenting ability to activate T cells due to maturation, but lose the ability to ingest antigens (phagocytosis).

[0076] <Term Explanation: Helper T cells>

[0077] They recognize the antigen as the target of attack by antigen presentation by dendritic cells, etc., and instruct cytotoxic T cells to attack. In addition, they generate cytokines to activate B cells. Furthermore, they judge whether the antigen is a dangerous foreign substance by antigen presentation by B cells and transmit the result to B cells. In addition, they have the function of assisting antibody production.

[0078] <Term Explanation: Cytotoxic T cells>

[0079] Cytotoxic T cells recognize antigens presented by MHC class I molecules (MHC class I molecules mainly bind to peptides generated by the degradation of cytoplasmic proteins based on proteasomes (protein-degrading enzyme complexes) (as an indication of the target of a human, i.e., oneself). Then, the MHC I peptide complex is inserted into the outer side of the cell membrane through the endoplasmic reticulum membrane. The peptide serving as an epitope binds to the extracellular portion of the MHC I peptide molecule. In this way, the MHC I peptide molecule presents intracellular proteins to cytotoxic T cells. However, it is known that MHC I peptide molecules sometimes also present peptides formed from foreign proteins, and this process is cross-presentation), recognize self-nonself, and kill nonself cells. Among them, the ability to recognize self-nonself is conferred by thymic competence education (negative selection), and this ability is used by MHC I peptide molecules. In this way, the response of T cells as acquired immunity starts from the antigen presentation of antigen peptides based on MHC molecules.

[0080] Under the instruction of helper T cells, cytotoxic T cells kill and remove cells infected with viruses, cancer cells, etc., which are dangerous or unnecessary for the organism.

[0081] <Term Explanation: B cells>

[0082] Antibodies are generated by the stimulation of cytokines produced by helper T cells. They have the function of becoming memory B cells and remembering antigens even after the antigens are eliminated, preparing for the next invasion.

[0083] <Term Explanation: NKT cells>

[0084] Cells that have the characteristics of both T cells and NK cells. When activated, they produce cytokines (IFN-γ) and also have the function of inducing responses of immune stimulation and inhibition therapies. In addition, the most expected effect of the preparation based on the present invention is the adjuvant effect. While activating various immune cells in the body, it is important that it also has the function of proliferating them.

[0085] <Function of dendritic cell preparation that stimulates NKT cells>

[0086] The main effects expected by activating NKT cells with this preparation include the promotion of dendritic cell maturation, adjuvant effects (activation and proliferation of various immune cells), induction of apoptosis in cancer cells, inhibition of angiogenesis, etc., which are the effects of both nonspecific immunity and specific immunity. Apoptosis refers to the self-suicide of regulated cells, i.e., programmed cell death.

[0087] As described above, by using a dendritic cell preparation that stimulates NKT cells to activate the innate immune system, various immune functions in the body can be activated to attack cancer cells.

[0088] Hereinafter, the embodiments will be described in detail. Regarding the substances used in the manufacturing method, they are common to all embodiments. The manufacturer, etc. will be described only when they first appear.

[0089] <Embodiment 1: Outline> Mainly for claim 1

[0090] A manufacturing method for preparing a dendritic cell preparation by changing monocytes collected from a beneficiary (monocytes obtained by apheresis or blood collection, or peripheral blood containing monocytes) into dendritic cells that stimulate NKT cells.

[0091] Hereinafter, in this specification, a "beneficiary" refers to a person who hopes to obtain beneficial effects through the administration of a dendritic cell preparation for any one or more of cancer treatment, cancer prevention, and immune enhancement, and who provides their own monocytes as the raw material for the aforementioned preparation.

[0092] <Embodiment 1: Manufacturing Method>

[0093] First, an outline of the manufacturing method of the NKT cell-stimulating dendritic cell preparation of the present invention will be described. Next, the action of this preparation will be described as follows.

[0094] <Embodiment 1: (1) Adhesion step: Preparation of monocytes>

[0095] Prepare monocytes collected from a beneficiary (monocytes obtained by apheresis or blood collection, or peripheral blood containing monocytes). "Prepare" means transporting the monocytes collected from a beneficiary (monocytes obtained by apheresis or blood collection, or peripheral blood containing monocytes) to the manufacturing site, and does not include blood collection itself. In this adhesion step, preparing the culture containers and liquid media to be used subsequently in a manner that can be transported to the manufacturing site for use may also be included in the preparation. It should be noted that in this specification, unless otherwise specified, a "culture container" refers to a container for culture purposes that houses a culture medium such as a liquid medium inside.

[0096] "Apheresis" means taking blood out of the body and using a dedicated device to separate the target blood components / factors from the blood and then returning them to the body. In the present invention, as the blood component, peripheral blood mononuclear cells containing monocytes are separated, and about 100 cc of blood components are collected. In the blood components after separating the aforementioned peripheral blood mononuclear cells containing monocytes, in addition to the aforementioned target blood components, plasma and platelets are also included. Regarding apheresis, in order to prevent blood coagulation, an anticoagulant is mixed into the blood before separating the peripheral blood mononuclear cells containing monocytes, and then the anticoagulant returns to the body. Or when the apheresis for separating the peripheral blood mononuclear cells containing monocytes takes a long time, the burden on the body is large.

[0097] It should be noted that it is also possible to collect 100 to 400 mL of peripheral blood instead of collecting by apheresis. Although the proportion of monocytes contained is higher in apheresis, since anticoagulants can be avoided and blood can be collected in a short time, it is also possible to collect based on peripheral blood.

[0098] <Embodiment 1: (1) Adhesion step: Static incubation>

[0099] Next, mononuclear cells are collected from the collected peripheral blood mononuclear cells containing monocytes by density centrifugation using Ficoll (registered trademark). (Ficoll (registered trademark) is part of Ficoll-Paque (registered trademark) and is used to separate blood into its components (red blood cells, white blood cells, etc.)) The collected mononuclear cells are added to a culture container containing a liquid medium. The mononuclear cells collected from the beneficiary are preferably added to a culture container containing a medium (preferably a liquid medium) within 72 hours from the time of collection. The time point when the mononuclear cells containing monocytes are added to a culture container containing a medium (preferably a liquid medium) is taken as the culture start time point. Hereinafter, in the present specification, unless otherwise specified, when it is described as "adding monocytes to a culture container", it means adding to a culture container containing a medium (preferably a liquid medium). Incubation is carried out by statically incubating for about 30 minutes in a sterilized constant temperature environment of 37°C. Unless otherwise specified, the steps after static incubation are carried out in a sterilized constant temperature environment of 37°C. The static incubation time can be longer than 30 minutes. If it is shorter than 30 minutes, a sufficient number of monocytes cannot completely adhere to the inner surface of the container, so it is preferably statically incubated for at least about 30 minutes.

[0100] In the present specification, "adhesion" to the inner surface of the culture container does not mean deliberately pressing the cells against the inner surface of the culture container or using some substance for the purpose of the action like an adhesive to stick the cells in a state where they are not easily detached from the inner surface of the culture container. "Adhesion" means that even without applying an external force other than gravity, the cells can directly maintain the state of contacting the inner surface after contacting the inner surface.

[0101] <Embodiment 1: (2) Non-adherent cell removal step: Removal of non-adherent cells>

[0102] After the above-mentioned static incubation (e.g., static incubation for 30 minutes), the non-adherent cells containing monocytes that are not adhered to the inner surface of the culture container and suspended in the liquid medium are removed (transferred to another container). The cells (monocyte fraction) adhered to the inner wall surface of the container remain in the culture container containing the liquid medium.

[0103] The non-adherent cells containing monocytes to be removed are preferably cultured separately or cryopreserved. The removed non-adherent cells (including NKT cells) can be used for verifying the efficacy of the preparation by co-culturing with the prepared preparation. It is also possible to provide a stripping step in which, after removing the non-adherent cells, the cells (monocyte fraction) adhered to the inner wall surface of the container are stripped and suspended in a liquid medium. During the aforementioned static state, monocytes mainly adhere to the inner surface of the container, so mainly monocytes are suspended in the liquid medium after stripping.

[0104] <Embodiment 1: (3) Differentiation step: Differentiation from monocytes to immature dendritic cells>

[0105] The following substances are added to the culture container of the above-mentioned existing liquid medium after removing non-adherent cells as the specified factors for differentiating monocytes into immature dendritic cells: granulocyte-macrophage colony-stimulating factor (hereinafter referred to as GM-CSF) at 50 ng / mL (about 30 ng / mL to 70 ng / mL is sufficient. If less, the stimulation is too weak and the differentiation is slow. If more, the differentiation rate has reached the peak and it is unreasonable in terms of cost), and interleukin 4 (hereinafter referred to as IL-4) at 50 ng / mL (about 30 ng / mL to 70 ng / mL is sufficient. If less, the stimulation is too weak and the differentiation is slow. If more, the differentiation rate has reached the peak and it is unreasonable in terms of cost). After adding, continue the culture. By adding GM-CSF and IL-4 to stimulate monocytes, the monocytes in the culture differentiate into immature dendritic cells. It should be noted that the substances used for the stimulation of differentiation are preferably the above-mentioned GM-CSF and IL-4, but are not limited thereto. By adding the stimulating substances, the state in the culture container becomes a state where there are very many (predominantly present) immature dendritic cells compared to undifferentiated monocytes and mature dendritic cells. The differentiated immature dendritic cells account for more than half of the cells in the culture container, preferably more than 80%, more preferably more than 90%.

[0106] <Embodiment 1: (4) Pulsing step: Pulsing of α-galactosylceramide>

[0107] As described above, the liquid medium in the culture vessel containing more than half of the immature dendritic cells was pulsed with α-galactosylceramide. The α-galactosylceramide used was the product number KRN7000 of Kyowa Kirin Co., Ltd. By pulsing with α-galactosylceramide, the immature dendritic cells became immature dendritic cells that could stimulate NKT cells after maturation. α-Galactosylceramide is a glycolipid antigen necessary for activating NKT cells. The feature of the present invention is that this α-galactosylceramide is directly imparted to immature dendritic cells. Generally, mature dendritic cells acquire the antigen-presenting ability of α-galactosylceramide by phagocytosing various foreign substances, but the feature of the present invention is that the antigen-presenting ability is imparted to the dendritic cells after maturation by imparting (pulsing) α-galactosylceramide to the dendritic cells at the immature stage. In Patent Document 1 as the prior art, it is described that the antigen-presenting ability is imparted by pulsing α-galactosylceramide to mature dendritic cells, but the inventors of the present application confirmed that according to the present invention, at least when pulsing immature dendritic cells, the effect of antigen-presenting and activating NKT cells is higher.

[0108] <Embodiment 1: (5) NKT-stimulating dendritic cell induction step: Maturation of dendritic cells>

[0109] As described above, after pulsing with α-galactosylceramide, GM-CSF (5 ng / mL: about 3 ng / mL to 7 ng / mL is sufficient. If less, the stimulation is too weak and the maturation is slow. If more, the maturation speed has reached the peak and it is unreasonable in terms of cost), IL-4 (5 ng / mL: about 3 ng / mL to 7 ng / mL is sufficient. If less, the stimulation is too weak and the maturation is slow. If more, the maturation speed has reached the peak and it is unreasonable in terms of cost), OK-432 (10 ng / mL: about 7 ng / mL to 13 ng / mL is sufficient. If less, the stimulation is too weak and the maturation is slow. If more, the maturation speed has reached the peak and it is unreasonable in terms of cost), and PGE2 (50 ng / mL: about 30 ng / mL to 70 ng / mL is sufficient. If less, the stimulation is too weak and the maturation is slow. If more, the maturation speed has reached the peak and it is unreasonable in terms of cost) were added to the liquid medium in the aforementioned culture vessel. These four substances can be added from the start of the culture until the 6th day. The immature dendritic cells are stimulated by the aforementioned added components and mature into mature dendritic cells. It should be noted that OK-432 is the trade name PICIBANIL manufactured by Chugai Pharmaceutical Co., Ltd., and PGE2 is prostaglandin E2 (English: Prostaglandin E2, hereinafter referred to as PGE2). It should be noted that the substances used for the maturation stimulation are preferably the aforementioned GM-CSF, IL-4, OK-432, and PGE2, but are not limited thereto.

[0110] <Embodiment 1: (5) NKT-stimulating dendritic cell induction step: final inspection>

[0111] After producing mature dendritic cells, perform sterility tests, endotoxin tests, and mycoplasma testing to confirm the safety of injection administration to humans. In addition, perform surface marker assays to evaluate the quality of dendritic cells. These quality confirmation tests can also be supplemented with other tests as appropriate. In addition, when the administration to the beneficiary is not immediate or is planned to be administered in several doses, etc., as needed, the manufactured dendritic cells are recovered and formulated with a cryopreservation solution to be 0.5×10 7 cells or more / ml / vial (0.5×10 6 cells or more / ml / vial in the case of using whole blood as a raw material), and cryopreserved under liquid nitrogen. A vial is a type of container for storing injections and is classified as a sealed container in the Japanese Pharmacopoeia. To obtain the desired effect, it is preferably set to the concentration or higher described above. So far is the manufacturing method of the dendritic cell preparation for stimulating NKT cells of the present invention.

[0112] <Embodiment 1: Effects of dendritic cell preparation for stimulating NKT cells>

[0113] When the dendritic cell preparation for stimulating NKT cells of the present invention is used for the beneficiary after blood collection, in the beneficiary's body, the preparation of the present invention stimulates and activates Vα24-NKT cells, causing them to produce IFN-γ, which is one type of cytokine, thereby producing an adjuvant effect. NK cells are strongly activated by this adjuvant effect and attack cancer cells or cells other than normal cells through cytotoxic responses. Activated NKT cells activate the acquired immune system by promoting the maturation of immature dendritic cells in the body. By activating both the innate immune system and the acquired immune system, it has effects not only on cancer treatment but also on cancer prevention and disease prevention based on the improvement of the immunity of healthy individuals.

[0114] Next, the effects in the case where the dendritic cell preparation for stimulating NKT cells of the present invention is returned to the body of the beneficiary from whom monocytes were collected for manufacturing the aforementioned preparation will be described.

[0115] <Embodiment 1: Action of this preparation: return to the body by intravenous drip>

[0116] This preparation containing dendritic cells matured after pulsing α-galactosylceramide in immature dendritic cells is returned to the beneficiary's body by methods such as intravenous drip.

[0117] <Embodiment 1: Action of this preparation: Activation of Vα24-NKT cells>

[0118] Through the blood vessels in the body, the dendritic cells of the preparation of the present invention bind the ingested glycolipid antigen, i.e., α-galactosylceramide, to a ligand, CD1d, and present it to Vα24-NKT cells in the body. In the case of Vα24-NKT cells in the body, the receptor binds to α-galactosylceramide (receives antigen presentation) and is activated.

[0119] <Embodiment 1: Action of this preparation: Activation of NKT cells: TNF-α production, angiogenesis inhibition>

[0120] The activated Vα24-NKT cells produce TNF-α (tumor necrosis factor α: one of the tumor necrosis factors), which stimulates immature dendritic cells (immature DCs). At the same time, TNF-α exerts an angiogenesis-inhibiting effect on cancer cells.

[0121] <Embodiment 1: Action of this preparation: Maturation of dendritic cells and activation of the acquired immune system>

[0122] Immature dendritic cells phagocytose neoantigens, which are cancer antigens released from cancer cells or dead cancer cells. Alternatively, immature dendritic cells stimulated by TNF-α from activated Vα24-NKT cells phagocytose neoantigens. The dendritic cells that have phagocytosed neoantigens are also matured by the effect of TNF-α stimulation. As a stimulus, cytotoxic T cells and helper T cells receive antigen presentation from the matured dendritic cells. The helper T cells that have received antigen presentation stimulate B cells through cytokines. The stimulated B cells produce and release cancer cell-specific antibodies.

[0123] <Embodiment 1: Action of this preparation: Apoptosis, cell killing activity>

[0124] NK cell activation is stimulated by IFN-γ produced by activated Vα24-NKT cells. Then, the activated NK cells and activated Vα24-NKT cells carry out the following attacks (innate immune system): killing cancer cells (or pathogens) that are not the beneficiary's own cells (cytotoxic activity), or promoting apoptosis. On the other hand, the antibodies released by the aforementioned B cells bind to cancer cells. Cytotoxic T cells carry out the following attacks (acquired immune system): creating pores in the cell membrane of cancer cells (which can also be pathogens) bound with antibodies to cause cancer cell necrosis (cytotoxic activity), or producing substances such as TNF-β (tumor necrosis factor, a type of cytokine) to promote apoptosis. The attacks from activated NK cells and activated Vα24-NKT cells (innate immune system) occur 1 to 2 days after intravenous infusion of this preparation. The attacks from cytotoxic T cells (acquired immune system) occur after antigen presentation by dendritic cells, so it takes time, about 7 to 14 days later.

[0125] Cytotoxic T cells and helper T cells that attack cancer cells cannot survive for a long time, but a part of the aforementioned T cells exist as memory cytotoxic T cells and memory helper T cells in lymph nodes, etc. (it is considered that they are maintained by replicating and exchanging T cells with memory). Even after cancer cells temporarily disappear, in the case of cancer cells that reappear and display the same antigen, the aforementioned memory helper T cells can immediately give instructions to immune cells to attack due to having the previous memory, and cytotoxic T cells also respond quickly. In addition to T cells, B cells that have released antibodies also exist as memory B cells in the same way and can quickly release antibodies during recurrence.

[0126] <Effect>

[0127] By manufacturing dendritic cells that stimulate NKT cells, when returning to the beneficiary's body, NK cells are activated from the innate immune system pathway and dendritic cells are stimulated, thereby indirectly activating helper T cells, cytotoxic T cells, and B cells can be expected. Since it does not specifically target only cancer but improves overall immunity, it is expected to prevent the beneficiary, who is weakened by cancer onset, from suffering from diseases other than cancer. Even if suffering from diseases other than cancer, it will end in mild symptoms or recover quickly. In addition to cancer treatment, it is also effective for cancer prevention and prevention of other diseases based on improved immunity. Furthermore, this dendritic cell preparation is a preparation obtained by culturing based on the beneficiary's own cells and is dedicated to the beneficiary, so it is a preparation with no rejection reaction and very high safety.

[0128] <Embodiment 2: Outline> Mainly Claim 2

[0129] In the second embodiment, based on the method for manufacturing the dendritic cell preparation of the first embodiment, the day when a specified factor is added in the differentiation step (3) is taken as day 0, and the pulsing step (4) is carried out on the 3rd to 4th day.

[0130] <Embodiment 2: (4) Pulsing step: Pulsing with α-galactosylceramide>

[0131] Compared with the timing of pulsing with α-galactosylceramide in the first embodiment, in the second embodiment, α-galactosylceramide is pulsed into the liquid medium in the culture vessel dominated by immature dendritic cells on any day of the 3rd or 4th day when the day when GM-CSF and IL-4 are added as specified factors in the differentiation step is taken as day 0. α-galactosylceramide is the same as that in the first embodiment and uses the product number KRN7000 of Kyowa Kirin Co., Ltd.

[0132] <Embodiment 2: (4) Pulsing step: Optimal pulsing timing>

[0133] It is expected that the dendritic cell preparation produced by the production method of the present invention can stimulate and activate Vα24-NKT cells, thereby enabling them to produce cytokines such as IFN-γ and activate various participants in the immune system. As a judgment of the efficacy of the preparation, for the preparation of the present invention, non-adherent cells (lymphocytes (T cells, B cells, NK cells, NKT cells), etc.) removed and separately cultured and preserved in the non-adherent cell removal step (2) added in the production method after the preparation is completed are co-cultured, and the ratio of the number of NKT cells producing IFN-γ to the total number of lymphocytes is investigated. The relationship between the pulsing timing and the ratio of the number of NKT cells after the co-culture is investigated, and the optimal pulsing timing is obtained. The above-mentioned pulsing timing is determined in this way. It should be noted that the use of non-adherent cells is to avoid imposing a physical burden on the beneficiary and has no other purpose. Therefore, it is also possible to collect peripheral blood from the beneficiary again, add the dendritic cell preparation produced by the production method of the present invention to the collected peripheral blood, and similarly compare and confirm the effects. In addition, the difference in the increase in the proportion of NKT cells relative to all lymphocytes was observed during co-culture, and it was found from this observation that the proliferation rate of NKT cells (which can be considered self-dividing proliferation) is affected by the dendritic cell preparation of the present invention. Therefore, the step (5) described in claim 1 of the present application is simply described as "NKT-stimulating dendritic cell induction step" in the claim, in other words, it can also be said to be "NKT proliferation-stimulating dendritic cell induction step".

[0134] In this embodiment, the day when a specified factor (e.g., GM-CSF and IL-4) is added for the differentiation of monocytes into immature dendritic cells is defined as day 0, and the time is recorded in days hereinafter. After the preparation is completed, non-adherent cells are added and co-cultured to confirm the effect of the preparation. To verify the most suitable pulsing timing, the collected monocytes are divided into three parts, and three samples of dendritic cell preparations with different pulsing timings of α-galactosylceramide are prepared. Samples are respectively prepared by pulsing α-galactosylceramide (concentration 100 ng / mL) on the 2nd day (48 hours later) after the addition of the specified factor (Condition 1), by pulsing on the 4th day (96 hours later) (Condition 2), and by pulsing on the 5th day (120 hours later) (Condition 3).

[0135] To confirm the effect of this preparation, the day when the specified factor for directing monocytes to immature dendritic cells is added is defined as day 0, and non-adherent cells are added on the 6th day to start co-culture. At three times, namely, on the day of the start of co-culture (day 0 since the start of co-culture), on the 7th day since the start of co-culture, and on the 14th day since the start of co-culture, the ratio of the number of NKT cells to the number of lymphocytes is investigated.

[0136] Figure 1 It is a graph showing the investigation results of the pulsing timing conditions (Conditions 1 to 3) of the above-mentioned α-galactosylceramide and the ratio of NKT cells producing IFN-γ (NKT cells contained in non-adherent cells) to the total number of lymphocytes at each timing after co-culture. This is the result of investigation using a flow cytometer. Under the three conditions, on the 7th day after the start of co-culture, the ratio of the number of NKT cells to the number of lymphocytes reaches the maximum. In addition, among the three conditions, when the day when the specified factor is added for the differentiation of monocytes into immature dendritic cells is defined as day 0 and pulsing is performed on the 4th day, the ratio (%) of the number of NKT cells after co-culture reaches the maximum. The relationship between the ratio (%) of the number of NKT cells on the 7th day after co-culture, which has reached the maximum, and the pulsing timing (days) with the day when the specified factor is added as day 0 is shown in Figure 2a the scatter plot. Figure 2aThe approximation curve when approximated by a quadratic equation is described by extrapolating forward and backward. The coefficient of determination of this approximation curve is 1, and it can be considered to have sufficient reference value. The peak of the curve is between the 3rd and 4th days of the pulse timing (calculated by the approximation formula as 3.7 days). The preferred range is up to -10% of the peak value, approximately from the 2.5th day to the 5th day. Since the lifespan of dendritic cells is several days to about 1 week, if the manufacturing using this manufacturing method (which refers to the period from collecting monocytes or adding a specified factor for differentiating monocytes into immature dendritic cells until they mature into mature dendritic cells, that is, until adding GM-CSF, IL-4, OK-432, and PGE2 for inducing into mature dendritic cells (it should be noted that the time length from then until complete maturity is about 10 hours to 20 hours, so the process of this time length part can also be included)) takes too long, the time available for use as a preparation becomes shorter. Therefore, considering that the preparation should be completed within 1 week from the start of cultivation, the range up to -5% of the peak value, that is, between the 3rd day and the 4.5th day, is more preferred, and the pulse can be carried out on the 3rd day or the 4th day. Most preferably, the pulse is carried out on the 4th day.

[0137] In the case of pulsing α-galactosylceramide to dendritic cells before maturation, in order to differentiate from monocytes into immature dendritic cells, after adding a specified factor (e.g., GM-CSF and IL-4), the pulse is carried out on either the 4th day or the 3rd day, and the preparation thus manufactured can exert the maximum effect in the beneficiary's body.

[0138] <Embodiment 3: Summary> Mainly Claim 3

[0139] In this Embodiment 3, based on the manufacturing method of the dendritic cell preparation of Embodiment 1, (4) the pulsing step is carried out 60 hours to 108 hours after adding the specified factor in the (3) differentiation step.

[0140] <Embodiment 3: (4) Pulsing step: Pulse of α-galactosylceramide>

[0141] Compared with the pulse timing of α-galactosylceramide in Embodiment 1, in this Embodiment 3, α-galactosylceramide is pulsed 60 hours to 108 hours after the time point when GM-CSF and IL-4 are added as specified factors in the differentiation step earlier. The α-galactosylceramide used the same product number KRN7000 of Kyowa Kirin Co., Ltd. as in Embodiment 1.

[0142] <Embodiment 3: (4) Pulse step: Optimal pulse timing>

[0143] The dendritic cell preparation produced by the production method of the present invention is expected to stimulate and activate Vα24-NKT cells, thereby enabling them to produce cytokines such as IFN-γ and activating various participants in the immune system. As a judgment of the efficacy of the preparation, for the preparation of the present invention, non-adherent cells (lymphocytes (T cells, B cells, NK cells), etc.) removed in the (2) non-adherent cell removal step added to the production method after the preparation is completed are co-cultured, and the ratio of the number of NKT cells producing IFN-γ is investigated. The relationship between the pulse timing and the ratio of the number of NKT cells after the aforementioned co-culture is investigated, and the optimal pulse timing is determined in the same manner as in the aforementioned Embodiment 2. The aforementioned pulse timing is determined in this way. It should be noted that the use of non-adherent cells is to avoid imposing a physical burden on the beneficiary and has no other purpose. Therefore, peripheral blood can also be newly collected from the beneficiary, and the dendritic cell preparation produced by the production method of the present invention is added to the collected peripheral blood, and the effects can be compared and confirmed in the same way. In addition, the difference in the increase in the proportion of NKT cells relative to all lymphocytes was observed during co-culture, and it was found from this observation that the proliferation rate of NKT cells (which can be considered self-dividing proliferation) is affected by the dendritic cell preparation of the present invention. Therefore, the step (5) described in claim 1 of the present application is simply described as "NKT-stimulating dendritic cell induction step" in the claim, or in other words, it can also be said to be "NKT proliferation-stimulating dendritic cell induction step".

[0144] In this embodiment, when a predetermined factor (e.g., GM-CSF and IL-4) is added for the differentiation of monocytes into immature dendritic cells, it is regarded as 0 hour, and the time is hereinafter recorded in hour units. After the preparation is completed, in order to confirm the effect of the preparation, non-adherent cells are added and co-cultured. In order to verify the optimal pulse timing, using the production method based on the production method of Embodiment 1, the collected monocytes are divided into three parts, and three samples of dendritic cell preparations with different pulse timings of only α-galactosylceramide are prepared. Samples (Condition 1) in which α-galactosylceramide (concentration 100 ng / mL) is pulsed 48 hours (Day 2) after the addition of the predetermined factor, samples (Condition 2) in which the pulse is performed 96 hours (Day 4) later, and samples (Condition 3) in which the pulse is performed 120 hours (Day 5) later are respectively prepared.

[0145] To confirm the effect of this preparation, non-adherent cells were added and co-culture was started 144 hours (day 6) after the addition of a specified factor for directing monocytes to immature dendritic cells. Three times, on the day of the start of co-culture (day 0 from the start of co-culture), on day 7 from the start of co-culture, and on day 14 from the start of co-culture, the ratio of the number of NKT cells to the number of lymphocytes was investigated.

[0146] Figure 1 It is a graph showing the investigation results of the pulse timing conditions (conditions 1 to 3) of the above-mentioned α-galactosylceramide and the ratio of NKT cells producing IFN-γ (NKT cells contained in non-adherent cells) to the total number of lymphocytes at each timing after co-culture. This is the result of investigation using a flow cytometer. Under the three conditions, on day 7 after the start of co-culture, the ratio of the number of NKT cells to the number of lymphocytes reached the maximum. In addition, among the three conditions, when pulsed 96 hours (day 4) after the addition of a specified factor for differentiating monocytes into immature dendritic cells, the NKT cell number ratio (%) reached the maximum. The relationship between the NKT cell number ratio (%) on day 7 after co-culture, which had reached the maximum, and the pulse timing is shown in Figure 2b a scatter plot. Figure 2b It describes extrapolating the approximate curve forward and backward when approximated by a quadratic equation. The coefficient of determination of this approximate curve is 1, and it can be considered to have sufficient reference value. The peak of the curve is between 70 hours and 96 hours after the pulse timing (calculated as 90 h by the approximate formula).

[0147] The preferred range is up to -10% of the peak value, approximately from 60 hours after to 118 hours after. Since the lifespan of dendritic cells is about several days to 1 week (up to 168 h), if the time required for the production using this production method (which refers to the period from the collection of monocytes or from the addition of a specified factor for differentiating monocytes into immature dendritic cells until they mature into mature dendritic cells, that is, until the addition of GM-CSF, IL-4, OK-432, and PGE2 for inducing maturation into mature dendritic cells (it should be noted that the time length from then until complete maturation is about 10 hours to 20 hours, so the process of this time length part can also be included)) is too long, the time available for use as a preparation becomes short. Therefore, considering that the preparation should be completed within 1 week from the start of culture, it is preferred to perform the pulse between 60 hours after and 108 hours after. More preferably, the pulse is performed within the range of -5% of the peak value, that is, between 70 hours after and 108 hours after.

[0148] In the case of pulsing dendritic cells with α-galactosylceramide before maturation, in order to differentiate monocytes into immature dendritic cells, after adding a prescribed factor (e.g., GM-CSF and IL-4), pulsing is performed 60 hours to 108 hours later. The preparation thus produced can exert the maximum effect in the body of the beneficiary.

[0149] <Embodiment 4: Summary> Mainly Claim 4

[0150] In this Embodiment 4, based on the method for producing the dendritic cell preparation of Embodiment 1, the day when monocytes collected from the beneficiary are added to the culture container in the (1) adhesion step is taken as day 0, and the (4) pulsing step is performed from day 4 to day 5.

[0151] <Embodiment 4: (4) Pulsing Step: Pulsing with α-Galactosylceramide>

[0152] Compared with the pulsing timing of α-galactosylceramide in Embodiment 1, in this Embodiment 4, α-galactosylceramide is pulsed from day 4 to day 5 when the day when monocytes collected from the beneficiary are added to the culture container is taken as day 0, earlier. The α-galactosylceramide used the product number KRN7000 of Kyowa Kirin Co., Ltd. as in Embodiment 1.

[0153] <Embodiment 4: (4) Pulsing Step: Optimal Pulsing Timing>

[0154] The dendritic cell preparation produced by the manufacturing method of the present invention is expected to stimulate and activate Vα24-NKT cells, thereby generating IFN-γ and activating various participants in the immune system. As a judgment of the efficacy of the preparation, for the preparation of the present invention, non-adherent cells (lymphocytes (T cells, B cells, NK cells), etc.) removed in the (2) non-adherent cell removal step added to the manufacturing method after the preparation is completed are co-cultured, and the ratio of the number of NKT cells producing IFN-γ is investigated. The relationship between the pulse timing and the ratio of the number of NKT cells after the co-culture is investigated, and the most suitable pulse timing is obtained. In this way, the aforementioned pulse timing is determined. It should be noted that the use of non-adherent cells is to avoid imposing a physical burden on the beneficiary, and there is no other purpose. Therefore, it is also possible to collect peripheral blood from the beneficiary again, add the dendritic cell preparation produced by the manufacturing method of the present invention to the collected peripheral blood, and similarly compare and confirm the effects. In addition, the difference in the increase in the proportion of NKT cells relative to all lymphocytes was observed during co-culture, and it was found from this observation that the proliferation rate of NKT cells (which can be considered self-dividing proliferation) is affected by the dendritic cell preparation of the present invention. Therefore, the step (5) described in claim 1 of the present application is simply recorded as "NKT-stimulating dendritic cell induction step" in the claim, in other words, it can also be said to be "NKT proliferation-stimulating dendritic cell induction step".

[0155] In the present embodiment, the time is recorded in days starting from the time when the monocytes collected from the beneficiary are added to the culture container and the culture is started. After the preparation is completed, in order to confirm the effect of the preparation, non-adherent cells are added and co-cultured. In order to verify the most suitable pulse timing, using the manufacturing method based on the manufacturing method of Embodiment 1, the collected monocytes are divided into three parts, and three samples of dendritic cell preparations with different pulse timings of only α-galactosylceramide are prepared. Samples (Condition 1) in which α-galactosylceramide (concentration 100 ng / mL) is pulsed 3 days (72 hours) after the monocytes collected from the beneficiary are added to the culture container, samples (Condition 2) in which the pulse is performed 5 days (120 hours) later, and samples (Condition 3) in which the pulse is performed 6 days (144 hours) later are respectively prepared. It should be noted that in this experiment, 24 hours after the monocytes collected from the beneficiary are added to the culture container, GM-CSF and IL-4 are added as the specified factors for differentiating monocytes into immature dendritic cells.

[0156] To confirm the effect of this preparation, the day when monocytes collected from the beneficiary were added to the culture vessel was taken as day 0, and non-adherent cells were added on day 7 to start co-culture. Three times, on the day of the start of co-culture (day 0 since the start of co-culture), on day 7 since the start of co-culture, and on day 14 since the start of co-culture, the ratio of the number of NKT cells to the number of lymphocytes was investigated.

[0157] Figure 3 It is a graph showing the investigation results of the pulse timing conditions (conditions 1 to 3) of the above-mentioned α-galactosylceramide and the ratio of NKT cells producing IFN-γ (NKT cells contained in non-adherent cells) to the total number of lymphocytes at each timing after co-culture. This is the result of an investigation using a flow cytometer. Under the three conditions, on day 7 after the start of co-culture, the ratio of the number of NKT cells to the number of lymphocytes reached the maximum. In addition, among the three conditions, when the pulse was applied on day 5 after the start of culture, the NKT cell number ratio (%) reached the maximum. The relationship between the NKT cell number ratio (%) on day 7 after co-culture, which had reached the maximum, and the pulse timing is shown in Figure 4a a scatter plot. Figure 4a The approximate curve when approximated by a quadratic equation is described by extrapolating forward and backward. The coefficient of determination of this approximate curve is 1, and it can be considered to have sufficient reference value. When the start of culture was taken as day 0, the peak of the curve was between day 4 and day 5 (calculated to be 4.7 days by the approximate formula). The preferred range is up to -10% of the peak, approximately from day 3.5 to day 6. Since the lifespan of dendritic cells is several days to about one week, if the manufacturing using this manufacturing method (which refers to the period from collecting monocytes or adding a specified factor for differentiating monocytes into immature dendritic cells until they mature into mature dendritic cells, that is, until adding GM-CSF, IL-4, OK-432, and PGE2 for inducing into mature dendritic cells (it should be noted that the time length from then until complete maturity is about 10 hours to 20 hours, so the process of this time length part can also be included)) takes too long, the time available for use as a preparation becomes shorter. Therefore, if it is also considered to complete the preparation within one week from the start of culture, the preferred range is -5% of the peak, that is, between day 3.9 and day 5.5. It is most preferred to apply the pulse on day 5.

[0158] When pulsing dendritic cells with α-galactosylceramide before maturation, starting from the day when mononuclear cells collected from the beneficiary are added to the culture vessel as day 0, pulsing is performed on the 4th or 5th day, and the resulting preparation can exert the maximum effect in the beneficiary's body.

[0159] <Embodiment 5: Summary> Mainly Claim 5

[0160] In this Embodiment 5, based on the method for producing the dendritic cell preparation of Embodiment 1, (4) the pulsing step is carried out 84 hours to 132 hours after adding the mononuclear cells collected from the beneficiary to the culture vessel in (1) the adhesion step.

[0161] <Embodiment 5: (4) Pulsing Step: Pulsing with α-galactosylceramide>

[0162] Compared with the timing of pulsing with α-galactosylceramide in Embodiment 1, in this Embodiment 5, α-galactosylceramide is pulsed between 84 hours and 132 hours after adding the mononuclear cells collected from the beneficiary to the culture vessel, earlier. α-galactosylceramide uses the product number KRN7000 of Kyowa Kirin Co., Ltd. as in Embodiment 1.

[0163] <Embodiment 5: (4) Pulsing Step: Optimal Pulsing Timing>

[0164] The dendritic cell preparation produced by the production method of the present invention is expected to stimulate and activate Vα24-NKT cells, produce IFN-γ, and activate various participants in the immune system. As a judgment of the efficacy of the preparation, for the preparation of the present invention, non-adherent cells (lymphocytes (T cells, B cells, NK cells), etc.) removed in the (2) non-adherent cell removal step added to the production method after the preparation is completed are co-cultured, and the ratio of the number of NKT cells producing IFN-γ is investigated. In the same manner as in the aforementioned Embodiment 4, the relationship between the pulse timing and the ratio of the number of NKT cells after the aforementioned co-culture is investigated, and the most suitable pulse timing is determined. In this way, the aforementioned pulse timing is determined. It should be noted that the use of non-adherent cells is to avoid imposing a physical burden on the beneficiary, and there is no other purpose. Therefore, it is also possible to newly collect peripheral blood from the beneficiary, add the dendritic cell preparation produced by the production method of the present invention to the collected peripheral blood, and similarly compare and confirm the effects. In addition, the co-culture was performed and the difference in the increase in the proportion of NKT cells relative to all lymphocytes was observed. From this observation, it was found that the proliferation rate of NKT cells (which can be considered self-dividing proliferation) is affected by the dendritic cell preparation of the present invention. Therefore, the step (5) described in claim 1 of the present application is simply described as "NKT-stimulating dendritic cell induction step" in the claim, or in other words, it can also be said to be "NKT proliferation-stimulating dendritic cell induction step".

[0165] In the present embodiment, the time is recorded in hours hereinafter, starting from when the monocytes collected from the beneficiary are added to the culture container and the culture is started. After the preparation is completed, in order to confirm the effect of the preparation, non-adherent cells are added and co-cultured. In order to verify the most suitable pulse timing, using the production method based on the production method of Embodiment 1, the collected monocytes are divided into three parts, and three samples of dendritic cell preparations with different pulse timings of only α-galactosylceramide are prepared. Samples (Condition 1) pulsed with α-galactosylceramide (concentration 100 ng / mL) 72 hours (3 days) after adding the monocytes collected from the beneficiary to the culture container (start of culture), samples pulsed 120 hours (5 days) later (Condition 2), and samples pulsed 144 hours (6 days) later (Condition 3) are respectively prepared. It should be noted that in this experiment, 24 hours after adding the monocytes collected from the beneficiary to the culture container, GM-CSF and IL-4 were added as specific factors for differentiating monocytes into immature dendritic cells.

[0166] To confirm the effect of this preparation, non-adherent cells were added and co-culture was started 168 hours after adding mononuclear cells collected from the beneficiary to the culture vessel. Three times, on the day of the start of co-culture (day 0 from the start of co-culture), on the 7th day from the start of co-culture, and on the 14th day from the start of co-culture, the ratio of the number of NKT cells to the number of lymphocytes was investigated.

[0167] Figure 3 It is a graph showing the results of an investigation of the pulse timing conditions (conditions 1 to 3) of the above-mentioned α-galactosylceramide and the ratio of NKT cells producing IFN-γ (NKT cells contained in non-adherent cells) to the total number of lymphocytes at each timing after co-culture. This is the result of an investigation using a flow cytometer. Under the three conditions, on the 7th day after the start of co-culture, the ratio of the number of NKT cells to the number of lymphocytes reached the maximum. In addition, among the three conditions, this is the result of an investigation using a flow cytometer. When the pulse was applied on the 5th day after the start of culture, the ratio (%) of the number of NKT cells on the 7th day after the start of co-culture reached the maximum. The relationship between the ratio (%) of the number of NKT cells on the 7th day after co-culture, which had reached the maximum, and the pulse timing is shown in Figure 4b a scatter plot. Figure 4b The approximate curve when approximated by a quadratic equation is described by extrapolating forward and backward. The coefficient of determination of this approximate curve is 1, and it can be considered to have sufficient reference value. The peak of the curve is between 96 hours later (the 4th day when the start of culture is taken as 0 day) and 120 hours later (the 5th day when the start of culture is taken as 0 day) (about 114 hours later). The preferred range is up to -10% of the peak, which is about 84 hours later to 143 hours later. Since the lifespan of dendritic cells is about several days to 1 week (168 hours), if the time required for the production using this production method (which refers to the period from collecting monocytes or adding a specified factor for differentiating monocytes into immature dendritic cells until they mature into mature dendritic cells, that is, until adding GM-CSF, IL-4, OK-432, and PGE2 for inducing into mature dendritic cells (it should be noted that the time length from then until complete maturity is about 10 hours to 20 hours, so the process of this time length part can also be included)) is too long, the time available for use as a preparation becomes short. Therefore, in order to complete the preparation within 1 week from the start of culture, it is preferred to apply the pulse from 84 hours later to 132 hours later. More preferably, the pulse is applied in the range of -5% of the peak, that is, from 94 hours later to 132 hours later.

[0168] When dendritic cells are pulsed with α-galactosylceramide before maturation, the preparation produced by performing the pulse 84 to 132 hours after adding monocytes collected from the beneficiary to the culture vessel can exert the maximum effect in the beneficiary's body.

[0169] <Embodiment 6: Summary> Mainly claim 6

[0170] In this Embodiment 6, based on the method for producing the dendritic cell preparation of Embodiment 1, the day when non-adherent cells including monocytes other than the cells adhering to the inner surface of the culture vessel are removed in the non-adherent cell removal step (2) is taken as day 0, and the pulse step (4) is carried out on the 4th to 5th day.

[0171] <Embodiment 6: (4) Pulse step: Pulse of α-galactosylceramide>

[0172] Compared with the pulse timing of α-galactosylceramide in Embodiment 1, in this Embodiment 6, α-galactosylceramide is pulsed from the 4th to 5th day when the day when non-adherent cells including monocytes other than the cells adhering to the inner surface of the culture vessel are removed in the non-adherent cell removal step (2) is taken as day 0, earlier. The α-galactosylceramide used the product number KRN7000 of Kyowa Kirin Co., Ltd. as in Embodiment 1.

[0173] <Embodiment 6: (4) Pulse step: Most suitable pulse timing>

[0174] The dendritic cell preparation produced by the production method of the present invention is expected to stimulate and activate Vα24-NKT cells, produce IFN-γ, and activate various participants in the immune system. For the judgment of the efficacy of the preparation, for the preparation of the present invention, non-adherent cells (lymphocytes (T cells, B cells, NK cells), etc.) removed in the (2) non-adherent cell removal step added in the production method after the preparation is completed are co-cultured, and the ratio of the number of NKT cells producing IFN-γ is investigated. The relationship between the pulse timing and the ratio of the number of NKT cells after the co-culture is investigated to obtain the most suitable pulse timing. The above-mentioned pulse timing is determined in this way. It should be noted that the use of non-adherent cells is to avoid imposing a physical burden on the beneficiary, and there is no other purpose. Therefore, it is also possible to collect peripheral blood from the beneficiary again, add the dendritic cell preparation produced by the production method of the present invention to the collected peripheral blood, and similarly compare and confirm the effects. In addition, the difference in the increase in the proportion of NKT cells relative to all lymphocytes was observed by co-culturing, and it was found from this observation that the proliferation rate of NKT cells (which can be considered self-dividing proliferation) is affected by the dendritic cell preparation of the present invention. Therefore, the step (5) described in claim 1 of the present application is simply recorded as "NKT-stimulating dendritic cell induction step" in the claim, in other words, it can also be said to be "NKT proliferation-stimulating dendritic cell induction step".

[0175] In the present embodiment, starting from when non-adherent cells including monocytes other than the cells adhered to the inner surface of the culture vessel are removed in the (2) non-adherent cell removal step, the time is recorded in units of days hereinafter. After the preparation is completed, in order to confirm the effect of the preparation, non-adherent cells are added and co-cultured. In order to verify the most suitable pulse timing, using the production method based on the production method of Embodiment 1, the collected monocytes are divided into three parts, and three samples of dendritic cell preparations with different pulse timings of only α-galactosylceramide are prepared. Samples (Condition 1) pulsed with α-galactosylceramide (concentration 100 ng / mL) 3 days (72 hours) after the non-adherent cells including monocytes other than the cells adhered to the inner surface of the culture vessel are removed in the (2) non-adherent cell removal step, samples pulsed 5 days (120 hours) later (Condition 2), and samples pulsed 6 days (144 hours) later (Condition 3) are respectively prepared. It should be noted that in this experiment, 24 hours after the non-adherent cells including monocytes other than the cells adhered to the inner surface of the culture vessel are removed in the (2) non-adherent cell removal step, GM-CSF and IL-4 are added as specified factors for differentiating monocytes into immature dendritic cells.

[0176] To confirm the effect of this preparation, the day when non-adherent cells other than the cells adhering to the inner surface of the culture vessel, including monocytes, were removed in the non-adherent cell removal step of (2) was taken as day 0, and non-adherent cells were added on day 7 to start co-culture. The ratio of the number of NKT cells to the number of lymphocytes was investigated three times: on the co-culture start day (day 0 since the start of co-culture), on day 7 since the start of co-culture, and on day 14 since the start of co-culture.

[0177] Figure 3 It is a graph showing the investigation results of the pulse timing conditions (conditions 1 to 3) of the above-mentioned α-galactosylceramide and the ratio of NKT cells producing IFN-γ (NKT cells contained in non-adherent cells) to the total lymphocyte number at each timing after co-culture. The Figure 3 "Since the start of culture" in the conditions on the right side can be interchanged with "since the removal of non-adherent cells other than the cells adhering to the inner surface of the culture vessel, including monocytes, in the non-adherent cell removal step of (2)". Figure 3 It is the result of investigation using a flow cytometer. Under the three conditions, the ratio of the number of NKT cells to the number of lymphocytes reached the maximum on day 7 after the start of co-culture. In addition, among the three conditions, when the pulse was performed on day 5 after the removal of non-adherent cells, the NKT cell number ratio (%) reached the maximum. The relationship between the NKT cell number ratio (%) on day 7 after co-culture, which had reached the maximum, and the pulse timing is shown in Figure 4a the scatter plot. Figure 4aThe approximation curve when approximated by a quadratic equation is described by extrapolating forward and backward. The coefficient of determination of this approximation curve is 1, and it can be considered to have sufficient reference value. The day when non-adherent cells are removed is taken as day 0, and the peak of the curve is between day 4 and day 5 (calculated to be 4.7 days by the approximation formula). The preferred range is up to -10% of the peak value, approximately from day 3.5 to day 6. Since the lifespan of dendritic cells is several days to about one week, if the manufacturing using this manufacturing method (which refers to the period from collecting monocytes or adding a specified factor for differentiating monocytes into immature dendritic cells until they mature into mature dendritic cells, that is, until adding GM-CSF, IL-4, OK-432, and PGE2 for inducing into mature dendritic cells (it should be noted that the time length from then until complete maturity is about 10 hours to 20 hours, so the process of this time length part can also be included)) takes too long, the time available for use as a preparation becomes short. Therefore, if it is also considered to complete the preparation within one week from removing non-adherent cells, the range of -5% of the peak is preferred, that is, between day 3.9 and day 5.5. It is most preferred to perform the pulse on day 5.

[0178] In the case of pulsing α-galactosylceramide to dendritic cells before maturation, as the day when non-adherent cells other than the cells adhering to the inner surface of the culture vessel are removed in the non-adherent cell removal step of (2), the pulse is performed on day 4 or day 5, and thus the preparation produced can exert the maximum effect in the body of the beneficiary.

[0179] <Embodiment 7: Summary> Mainly Claim 7

[0180] In this Embodiment 7, based on the manufacturing method of the dendritic cell preparation of Embodiment 1, the (4) pulsing step is performed 84 hours to 132 hours after removing non-adherent cells other than the cells adhering to the inner surface of the culture vessel in the non-adherent cell removal step of (2).

[0181] <Embodiment 7: (4) Pulsing Step: Pulse of α-Galactosylceramide>

[0182] Compared with the pulse timing of α-galactosylceramide in Embodiment 1, in this Embodiment 7, α-galactosylceramide is pulsed between 84 hours and 132 hours after removing non-adherent cells other than cells adhering to the inner surface of the culture vessel, including monocytes, in the non-adherent cell removal step of (2) earlier. The α-galactosylceramide used the product number KRN7000 of Kyowa Kirin Co., Ltd. in the same manner as in Embodiment 1.

[0183] <Embodiment 7: (4) Pulse step: Optimal pulse timing>

[0184] The dendritic cell preparation produced by the production method of the present invention is expected to stimulate and activate Vα24-NKT cells, produce IFN-γ, and activate various participants in the immune system. As a judgment of the efficacy of the preparation, for the preparation of the present invention, non-adherent cells (lymphocytes (T cells, B cells, NK cells), etc.) removed in the non-adherent cell removal step of (2) added to the production method after the preparation is completed are co-cultured, and the ratio of the number of NKT cells producing IFN-γ is investigated. In the same manner as in the aforementioned Embodiment 6, the relationship between the pulse timing and the ratio of the number of NKT cells after the co-culture is investigated, and the optimal pulse timing is obtained. The aforementioned pulse timing is determined in this way. It should be noted that the use of non-adherent cells is to avoid imposing a physical burden on the beneficiary, and there is no other purpose. Therefore, it is also possible to collect peripheral blood from the beneficiary again, add the dendritic cell preparation produced by the production method of the present invention to the collected peripheral blood, and similarly compare and confirm the effects. In addition, the difference in the increase in the proportion of NKT cells relative to all lymphocytes was observed during co-culture, and it was found from this observation that the proliferation rate of NKT cells (which can be considered self-dividing proliferation) is affected by the dendritic cell preparation of the present invention. Therefore, the step (5) described in claim 1 of the present application is simply described as "NKT-stimulating dendritic cell induction step" in the claim, in other words, it can also be said to be "NKT proliferation-stimulating dendritic cell induction step".

[0185] In the present embodiment, the time is recorded in hours starting from the removal of non-adherent cells including monocytes other than the cells adhering to the inner surface of the culture vessel in the non-adherent cell removal step (2). After the preparation of the preparation, in order to confirm the effect of the preparation, non-adherent cells are added and co-cultured. In order to verify the most suitable pulse timing, using the production method based on the production method of Embodiment 1, the collected monocytes are divided into three portions, and three samples of dendritic cell preparations with different pulse timings of only α-galactosylceramide are prepared. Samples are prepared by pulsing α-galactosylceramide (concentration 100 ng / mL) 72 hours (3 days) after the removal of non-adherent cells (Condition 1), by pulsing 120 hours (5 days) after the removal, and by pulsing 144 hours (6 days) after the removal (Condition 3). It should be noted that in this experiment, 24 hours after the removal of non-adherent cells, GM-CSF and IL-4 were added as the specified factors for differentiating monocytes into immature dendritic cells.

[0186] In order to confirm the effect of this preparation, non-adherent cells are added and co-culture is started 168 hours after the removal of non-adherent cells. The ratio of the number of NKT cells to the number of lymphocytes is investigated three times: on the day of the start of co-culture (day 0 from the start of co-culture), on the 7th day from the start of co-culture, and on the 14th day from the start of co-culture.

[0187] Figure 3 It is a graph showing the results of the investigation of the pulse timing conditions (Conditions 1 to 3) of the above-mentioned α-galactosylceramide and the ratio of the NKT cells producing IFN-γ (NKT cells contained in non-adherent cells) to the total number of lymphocytes at each timing after co-culture. Figure 3 In the conditions on the right side, "from the start of culture" is interchanged with "from the removal of non-adherent cells including monocytes other than the cells adhering to the inner surface of the culture vessel in the non-adherent cell removal step (2)". Figure 3 These are the results of the investigation using a flow cytometer. Under the three conditions, the ratio of the number of NKT cells to the number of lymphocytes reached the maximum on the 7th day after the start of co-culture. In addition, among the three conditions, these are the results of the investigation using a flow cytometer. When pulsing is performed on the 5th day after the removal of non-adherent cells, the ratio (%) of the number of NKT cells on the 7th day after the start of co-culture reaches the maximum. The relationship between the ratio (%) of the number of NKT cells on the 7th day after co-culture, which has reached the maximum, and the pulse timing is shown in Figure 4b a scatter plot. Figure 4bThe approximation curve when approximated by a quadratic equation is described by extrapolating forward and backward. The coefficient of determination of this approximation curve is 1, and it can be considered to have sufficient reference value. The peak of the curve is between 96 hours (the 4th day when the non-adherent cells are removed is taken as day 0) and 120 hours (the 5th day when the non-adherent cells are removed is taken as day 0) (about 114 hours). The preferred range is up to -10% of the peak, which is about 84 hours to 143 hours. Since the lifespan of dendritic cells is several days to about 1 week (168 hours), if the production using this production method (which means from mononuclear cell blood collection or from the addition of a specified factor for differentiating mononuclear cells into immature dendritic cells until they mature into mature dendritic cells, that is, until the addition of GM-CSF, IL-4, OK-432, and PGE2 for inducing into mature dendritic cells (it should be noted that the time length from then until complete maturity is about 10 hours to 20 hours, so the process of this time length part can also be included)) takes too long, the time available for use as a preparation becomes short. Therefore, in order to complete the preparation within 1 week from the start of cultivation, it is preferred to perform the pulse from 84 hours to 132 hours. More preferably, the pulse is performed in the range of -5% of the peak, that is, from 94 hours to 132 hours.

[0188] When α-galactosylceramide is pulsed to dendritic cells before maturity, the pulse is performed from 84 hours to 132 hours after removing non-adherent cells, and the preparation thus produced can exert the maximum effect in the beneficiary's body.

[0189] <Embodiment 8: Summary> Mainly Claim 8

[0190] In order to induce dendritic cells that stimulate Vα24-NKT cells, namely NKT-stimulating dendritic cells, the concentration of α-galactosylceramide pulsed to immature dendritic cells is set to 300 ng / mL to 1000 ng / mL. In the following embodiments, the timing of pulsing α-galactosylceramide is recorded with the day when monocytes collected from the beneficiary are added to the culture vessel as day 0. In this embodiment, based on Embodiment 5, the timing of pulsing α-galactosylceramide is set to 120 hours after the start of culture. It should be noted that the timing of pulsing can also be between 84 hours and 132 hours after the start of culture, or as described in Embodiment 4, with the day when mononuclear cells are added to the culture vessel and culture is started as day 0, and pulsing is performed 4 days to 5 days later. Or as described in Embodiments 2 and 3, with the day when a specified factor is added in the differentiation process as day 0, pulsing is performed on the 3rd day to the 4th day, or pulsing is performed from 60 hours to 108 hours after the addition of the specified factor. Or as described in the aforementioned Embodiments 6 and 7, with the day when non-adherent cells are removed from the culture vessel as day 0, pulsing is performed on the 4th day to the 5th day, or pulsing is performed between 84 hours and 132 hours after the removal of non-adherent cells.

[0191] <Embodiment 8: (4) Pulsing step: Optimal pulse amount>

[0192] In this Embodiment 8, it is manufactured based on the manufacturing method of the aforementioned Embodiment 5, but the difference from Embodiment 5 is that the concentration of α-galactosylceramide pulsed is set to 300 ng / mL to 1000 ng / mL. In this Embodiment 8, α-galactosylceramide is pulsed 120 hours after the timing when monocytes collected from the beneficiary are added to the culture vessel, that is, the start of culture.

[0193] The dendritic cell preparation manufactured by the manufacturing method of the present invention can be expected to stimulate and activate Vα24-NKT cells to produce IFN-γ, thereby activating various participants in the immune system. As a judgment of the efficacy of the preparation, for the preparation of the present invention, non-adherent cells (lymphocytes (T cells, B cells, NK cells, NKT cells), etc.) removed and separately cultured and preserved in the (2) non-adherent cell removal step in the manufacturing method are added after the preparation is completed and co-cultured, and the relationship between the concentration of α-galactosylceramide pulsed and the amount (concentration) of IFN-γ produced by NKT cells in the non-adherent cells cultured after co-culture is investigated and confirmed as the effect. In this way, the range of the aforementioned pulse amount is determined.

[0194] In this embodiment, the time is recorded in hours starting from the point when the mononuclear cells collected from the beneficiary are added to the culture container and the culture is started. After the preparation of the preparation, in order to confirm the effect of the preparation, non-adherent cells are added and co-cultured. In order to verify the most suitable pulse concentration, the collected mononuclear cells are divided into 4 parts, and 4 samples of dendritic cell preparations with different pulse timing of α-galactosylceramide are prepared. 120 hours after the mononuclear cells collected from the beneficiary are added to the culture container and the culture is started, α-galactosylceramide is pulsed under condition 1: concentration 0 ng / mL, condition 2: 100 ng / mL, condition 3: 300 ng / mL, and condition 4: 1000 ng / mL, respectively.

[0195] In order to confirm the effect of this preparation (evaluation of IFN-γ production concentration), non-adherent cells are added and co-culture is started 168 hours after the start of the culture. The day when the co-culture starts is taken as day 0, and the IFN-γ production amount is investigated for the samples of each condition on day 0 (the day when the co-culture starts), day 2, day 4, day 7, and day 14.

[0196] In the measurement of IFN-γ, it is carried out by the ELISA method using ELISA MAX Deluxe Set Human IFN-γ from BIOLEGEND.

[0197] Figure 5 It is a graph showing the relationship between the concentration of α-galactosylceramide pulsed 120 hours after the mononuclear cells collected from the beneficiary are added to the culture container and the culture is started, and the concentration of IFN-γ measured at the time points of day 0 (the same day), day 2, day 4, day 7, and day 14 after the start of co-culture with non-adherent cells. The horizontal axis is the number of days of co-culture, and the vertical axis is the IFN-γ concentration (pg / mL). As described above, in conditions 1 to 4, the concentration of the pulsed α-galactosylceramide is divided into 4 stages in the range of 0 to 1000 ng / mL for the experiment. As Figure 5As shown, in Condition 1 (0 ng / mL) and Condition 2 (100 ng / mL), there is a peak in the production concentration of IFN-γ on the 7th day since the start of co-culture. In Condition 3 (300 ng / mL) and Condition 4 (1000 ng / mL), there is a peak in the production concentration on the 4th day, and the peak value is the same as that in Condition 2 (100 ng / mL). In Condition 3 (300 ng / mL) and Condition 4 (1000 ng / mL), the result is that the peak in the production concentration at the same pulse concentration as the concentration condition (100 ng / mL) of Embodiment 1 disclosed in the prior art (Patent Document 1, the same below) is advanced by 3 days, indicating that it has a fast onset. Thus, when the concentration of pulsed α-galactosylceramide is less than 300 ng / mL, the production peak of IFN-γ is delayed, and even when pulsed at a concentration greater than 1000 ng / mL, the result is the same as that at 1000 ng / mL, causing waste of α-galactosylceramide. Therefore, it can be known that the most suitable concentration of α-galactosylceramide is 300 ng / mL or more and 1000 ng / mL or less.

[0198] In addition, in the comparative experiment with the prior art (Patent Document 1) described below, when pulsing α-galactosylceramide at the same concentration, the present invention that pulses immature dendritic cells can obtain a production concentration of IFN-γ approximately 4 times that of the prior art (Patent Document 1) that pulses mature dendritic cells. Compared with the prior art, more IFN-γ can be produced, and it is expected to further activate the immune system and play a greater anti-cancer role.

[0199] According to this embodiment, the preparation of this manufacturing method can advance the production peak of IFN-γ in the beneficiary's body under the same conditions as the peak of other conditions, and it is expected to play an anti-cancer role earlier. In addition, a higher production concentration of IFN-γ can be obtained compared with the prior art, and it is also expected to play a greater anti-cancer role.

[0200] <Embodiment 9: Summary> Mainly Claims 9 and 10

[0201] The blood of a beneficiary to whom any one of the dendritic cell preparations manufactured by the manufacturing methods shown in Embodiments 1 to 8 is added.

[0202] As described in Embodiment 1 above, when this preparation is added to the blood of a beneficiary, it stimulates NKT cells and activates the innate immune system (immunity enhancement), and then there is also an effect of stimulating and activating the acquired immune system. It has an effect of enhancing immunity on the blood of the beneficiary.

[0203] It is also possible to take out the beneficiary's blood outside the body, add this preparation and process it. By returning the processed blood (including this preparation) to the beneficiary, an improvement in the beneficiary's immunity can be expected. Alternatively, it is also possible to return this preparation to the beneficiary's body and add it to the blood in the body to achieve an improvement in immunity. It should be noted that it is also possible to use a substance obtained by adding blood that can be transfused to the beneficiary to the beneficiary's blood, that is, the beneficiary's quasi-blood, to replace the above-mentioned beneficiary's blood.

[0204] <Results of Application to the Human Body>

[0205] The dendritic cell preparation manufactured according to the conditions of Embodiment 4 of the present invention (culturing starts on the blood collection day and non-adherent cells are removed, GM-CSF and IL-4 are added 24 hours after the start of culturing, and α-galactosylceramide is pulsed at 300 ng / mL on the 5th day after the start of culturing) was administered to 2 beneficiaries. How the dendritic cell preparation of the present invention exerts its effect in the body is as described above. It activates NKT cells and activates cells involved in various other immunities by producing IFN-γ. In particular, it has a great influence on NK cells. Therefore, the NK activity value and the amount of IFN-γ after effective administration were studied. The results are shown in Figure 6 . The results of administration to 2 beneficiaries, one by subcutaneous injection (upper part of the table) and the other by intravenous drip (lower part of the table). The NK activity value is a value indicating the activation of NK cells. The NK activity increased after the 3rd day after administration and was still higher than the value before administration on the 7th day. The lower limit of measurement of IFN-γ is 0.1, and it is usually 0.1 or less. Regarding the measurement results of IFN-γ, it was 0.1 on the first day (day 0), reached a peak on the 2nd day after administration, and the numbers of both increased significantly. It shows that this dendritic cell preparation achieved the activation of the target NKT cells and a rapid increase in the production amount of IFN-γ, and also activated NK cells.

[0206] <Comparison with the Prior Art: Overview>

[0207] In the prior art, there is no description in the specification about the production amount (production concentration) of IFN-γ. Therefore, the inventor of the present invention made a cell composition by imitating the manufacturing method of the cell composition (corresponding to the dendritic cell preparation of the present application) described in Patent Document 1 as the prior art, and conducted an experiment to compare the IFN-γ production concentration after co-culture with non-adherent cells containing NKT cells.

[0208] <Comparison with the Prior Art: Preparation of Comparative Examples>

[0209] To reproduce the prior art, the mononuclear cells obtained by apheresis were allowed to stand in the liquid medium in a culture vessel for 2 hours, and the non-adherent cells (lymphocytes (T cells, B cells, NK cells, NKT cells), etc.) were separated and cryopreserved. The day when the cells were allowed to stand and began to adhere was designated as day 0. On the next day (day 1), GM-CSF and IL-4 were added to the culture medium of the adherent cells to induce their differentiation into immature dendritic cells. On day 5, GM-CSF, IL-4, PGE-2 and OK-432 were added to the immature dendritic cells to mature them. On day 6, which was the next day, the mature dendritic cells were pulsed with α-galactosylceramide (concentration 300 ng / mL).

[0210] <Comparison with the prior art: Method for confirming the effect of the comparative example>

[0211] On day 7, co-culture of the aforementioned mature dendritic cells and the non-adherent cells recovered on the first day was started. On the day when co-culture was started (day 0), and on days 2, 4, 7, and 14 when the start of co-culture was designated as day 0, the IFN-γ production concentration (pg / mL) produced by NKT cells in the non-adherent cells was measured.

[0212] <Comparison with the prior art: Comparison of the effects of the comparative example and the embodiment of the present application>

[0213] The IFN-γ production concentration after co-culture of dendritic cells prepared as a comparative example by imitating the prior art as described above (manufacturing method described in Patent Document 1, wherein the concentration of the pulsed α-galactosylceramide is 300 ng / mL) with non-adherent cells, and the IFN-γ production concentration after co-culture of the dendritic cell preparation manufactured according to Condition 3 of Embodiment 8 of the present application (the concentration of the pulsed α-galactosylceramide is 300 ng / mL) were compared. It should be noted that in order to confirm the effects of the comparative example and the preparation manufactured according to Condition 3 of Embodiment 8 of the present application, dendritic cells and non-adherent cells containing NKT cells from the same beneficiary with the difference in cell number suppressed within a range where no significant difference is produced were used.

[0214] <Comparison with the prior art: Comparison of the effects of the comparative example and the embodiment of the present application: IFN-γ production concentration>

[0215] Figure 7Shows the comparison results of IFN-γ production concentrations. The horizontal axis represents the number of days elapsed since the start of co-culture: 0 days (the first day of start), 2 days, 4 days, 7 days, and 14 days, and the vertical axis represents the IFN-γ production concentration (pg / mL). The IFN-γ production concentrations of the comparative example (prior art) and Condition 3 of Embodiment 8 of the present application both increased on the 4th day. In the prior art, the production concentration on the 7th day was slightly higher than that on the 4th day. Condition 3 of Embodiment 8 of the present application showed a result of an IFN-γ production concentration that was approximately 4 times higher than that of the prior art.

[0216] <Comparison with the prior art: Comparison of the effects of the comparative example and the embodiments of the present application: IFN-γ production peak>

[0217] Patent Document 1, which is the prior art, describes that the most preferred concentration range of α-galactosylceramide is 80 to 120 ng / mL, and an experiment was conducted at 100 ng / mL in Embodiment 1 of Patent Document 1. In Patent Document 1, no effect verification was performed regarding the production of IFN-γ. In the experiments of the inventors of the present application, in Condition 2 of Embodiment 8 of the present application, pulsed with the same α-galactosylceramide concentration (100 ng / mL) as in Embodiment 1 of Patent Document 1, the production peak of IFN-γ was on the 7th day after co-culture. Therefore, although not described in Patent Document 1, the inventors of the present application found that by not only optimizing the pulsed timing of α-galactosylceramide but also preferably the pulsed concentration, the range in which the effect can be exerted faster than the prior art can be narrowed.

[0218] <Comparison with the prior art: Excellent effects of the present invention>

[0219] Compared with the comparative example (prior art), in the present invention, by pulsing immature dendritic cells with α-galactosylceramide and optimizing the pulsed timing and its concentration, the range in which IFN-γ can be rapidly produced can be narrowed, and the IFN-γ production concentration can be significantly increased.

[0220] <Claims scheme for the treatment method for US application>: (Provisional)

[0221] Regarding the treatment method for beneficiaries who desire any one or more of cancer treatment, cancer prevention, and immune enhancement using the dendritic cell preparation that stimulates NKT cells produced by the manufacturing method of the present invention, it can be described as follows.

[0222] A treatment method, which is a treatment method based on the prepared preparation, the treatment method uses a dendritic cell preparation containing dendritic cells that stimulate natural killer T (NKT) cells, wherein,

[0223] A dendritic cell preparation for the purpose of any one or more of cancer treatment, cancer prevention, and immune enhancement, which contains dendritic cells that stimulate natural killer T (NKT) cells, is produced by the following production method. The production method is characterized by including:

[0224] (1) Collection step: Collect monocytes or peripheral blood containing monocytes from a beneficiary by apheresis or blood collection.

[0225] (2) Adhesion step: Add the collected monocytes to a culture container using a liquid medium, let it stand still, and allow a part of the monocytes to adhere to the inner surface of the container.

[0226] (3) Non - adherent cell removal step: Remove non - adherent cells containing monocytes other than the cells adhering to the inner surface of the culture container.

[0227] (4) Differentiation step: Add a specified factor to the culture container, and differentiate the monocytes adhering to the inner surface of the culture container into immature dendritic cells.

[0228] (5) Pulsing step: Pulse α - galactosylceramide to the culture container in which the aforementioned immature dendritic cells exist in a non - adherent state; and

[0229] (6) NKT - stimulated dendritic cell induction step: Induce dendritic cells that stimulate NKT cells, namely NKT - stimulated dendritic cells, from immature dendritic cells.

[0230] The treatment method has a (7) Return step: Return the aforementioned dendritic cell preparation to the body of the beneficiary from whom the monocytes were collected.

[0231] (Provisional)

[0232] <Limiting condition scheme of the aforementioned claim scheme>(Provisional)

[0233] Regarding the aforementioned treatment method, any one or more of the following limiting conditions can be added.

[0234] (a) A treatment method, wherein, in the pulsing step, the pulsing of α - galactosylceramide is 84 hours to 132 hours after (4 days to 5 days after taking the addition of monocytes as day 0) when the monocytes are added to the culture container using a liquid medium in the adhesion step.

[0235] (b) A treatment method, wherein, in the pulsing step, the concentration of the pulsed α - galactosylceramide is 300 - 1000 ng / mL.

[0236] (c) A treatment method, wherein, in the return step, returning the aforementioned preparation to the body of the beneficiary is after 156 hours and later (7 days and later when the day of adding monocytes is regarded as day 0) from the time of adding monocytes to a culture container using a liquid medium in the adhesion step.

[0237] <Other Embodiment: Mixed Preparation>

[0238] The following describes the effect when returning the mixed (cocktail) preparation of other embodiments to the body of the beneficiary. In the example of this description, the beneficiary has cancer cells in the body. In this other embodiment, the collected monocytes are separately packed and added to a culture container separately from the preparation of the present invention, and a preparation obtained by using a WT1 peptide as a ligand instead of α-galactosylceramide pulsed to immature dendritic cells in the present invention is prepared in parallel in another container, and is configured to coexist with the preparation of the present invention in the body of the beneficiary.

[0239] The preparation using α-galactosylceramide as a ligand activates the innate immune system of the beneficiary, realizes an improvement in immunity, and also indirectly activates the acquired immune system. In the case of a preparation using a WT1 peptide present in various cancers as a ligand, the acquired immune system targeting cancer cells is directly activated.

[0240] <Effect of the Mixed Preparation>

[0241] This other embodiment uses the following two preparations: a preparation manufactured by the manufacturing method of the present invention; and a preparation obtained by a manufacturing method similar to that of the present invention, and as a drug pulsed to immature dendritic cells, not pulsed with α-galactosylceramide but pulsed with a WT-1 peptide. Since non-specific immunotherapy (preparation (ligand: α-galactosylceramide)) has been described, the following mainly describes the effect of the preparation targeting the acquired immune system and using a WT1 peptide as a ligand as a different part. A barrier (due to immunosuppressive effects) from the immune function has formed around the cancer cells in the body of the beneficiary.

[0242] <Effect of the Preparation Pulsed with α-Galactosylceramide: Activation of NKT Cells>

[0243] The preparation pulsed with α-galactosylceramide activates NKT cells. Activated NKT cells exert various effects on immune function.

[0244] <Effect of Activated NKT Cells: Adjuvant Effect>

[0245] Activated NKT cells activate NK cells, macrophages, and cytotoxic T cells by producing cytokines (IFN-γ), enhancing the attack on cancer. Additionally, various immune cells are activated. Cytotoxic T cells and helper T cells are activated by pulsing dendritic cells with WT1 peptides with specific immunological activity, and the activated NK cells and activated B cells are simultaneously favorably affected by the non-specific immunological activity pathway, resulting in a synergistic effect greater than simple addition. This is one of the characteristics of this other embodiment.

[0246] <Function of activated NKT cells: Apoptosis induction effect on cancer cells>

[0247] Activated NKT cells cause apoptosis of cancer cells by producing one of the serine proteases to break down the DNA of cancer cells.

[0248] <Function of activated macrophages: Cancer cell phagocytosis based on macrophages>

[0249] Activated macrophages phagocytose cancer cells and cancer dead cells.

[0250] <Function based on proliferation and activation of macrophages: Antigen presentation effect based on macrophages>

[0251] Activated NKT cells proliferate and activate macrophages. The proliferated and activated macrophages fragment the cancer cells ingested by phagocytosis and expose them on the cell surface. That is, they exert the antigen presentation effect of presenting the antigens of cancer cells. Macrophages present this antigen to helper T cells.

[0252] <Cancer antigen presentation effect of dendritic cells: Cancer antigen presentation effect based on dendritic cells>

[0253] Immature dendritic cells ingest cancer antigens and mature, differentiating into mature dendritic cells and presenting cancer antigens to helper T cells and cytotoxic T cells. This effect is an anti-cancer effect that usually occurs in the body, and through the action of activated NKT cells, an anti-cancer effect greater than normal can be obtained. The cancer antigen is the antigen presented by dendritic cells and macrophages phagocytosing the patient's own cancer cells, so a higher effect can be expected compared to preparations using artificial antigens as ligands. This effect is also one of the reasons for the synergistic effect of the mixed preparation of the present invention.

[0254] <Function of the preparation pulsed with WT1 peptide: Cancer antigen presentation effect>

[0255] The preparation obtained from pulsed WT1 peptide presents the WT1 peptide, which is an antigen of cancer, to helper T cells and cytotoxic T cells and activates them. Thus, cytotoxic T cells recognize cancer cells as targets for attack, helper T cells activate cytotoxic T cells, and further produce cytokines. Thus, as described later, NK cells are activated and B cells are promoted to produce antibodies.

[0256] <Function of activated helper T cells: Indication of cancer cell attack>

[0257] Activated helper T cells produce cytokines (INF-γ) and indicate to cytotoxic T cells to attack cancer cells based on the information contained in these cytokines. In addition, due to the effects of cytokines, NK cells, B cells, and macrophages are activated, thus enhancing the attack effect on cancer.

[0258] <Function of activated helper T cells: Indication of antibody production against cancer antigen (e.g., WT1)>

[0259] Activated helper T cells produce cytokines and indicate to B cells to produce antibodies against cancer antigen (e.g., WT1) based on the information contained in these cytokines.

[0260] <Function of activated cytotoxic T cells: Cancer cell attack effect based on cytotoxic T cells>

[0261] Activated cytotoxic T cells function as the main force directly attacking cancer cells. They recognize and bind to WT1 expressed in cancer cells, create pores in the cell membrane, and cause cancer cells to necrosis. In addition, substances such as TNF-β (tumor necrosis factor, a type of cytokine) are produced to induce apoptosis of cancer cells. In this way, the direct attack of cytotoxic T cells on cancer cells is a potent anti-cancer effect that attacks cancer cells from both the outside and the inside.

[0262] <Function of activated NK cells: Cancer cell attack effect based on NK cells>

[0263] Newly formed blood vessels due to cancer cells express the WT1 gene. Cytotoxic T cells presented with the cancer antigen (WT1) not only attack cancer cells but also attack newly formed blood vessels. Thus, the newly formed blood vessels are damaged, the nutrient supply to cancer cells decreases, and the proliferation of cancer cells is inhibited. This effect is also one of the reasons for the synergistic effect of the mixed preparation of other embodiments of the present invention.

[0264] <Function of activated NK cells: Cancer cell attack effect based on NK cells>

[0265] In addition to IFN-γ produced by activated NKT cells, IFN-γ produced by helper T cells is added, and NK cells activated after being stimulated show a cytotoxic response to cancer cells and attack the cancer cells.

[0266] <Function of Activated B Cells: Cancer Antibody Production>

[0267] Activated B cells produce antibodies against cancer antigens (such as WT1) and attack cancer cells. This antibody inhibits the proliferation of cancer cells by binding to the cancer cells, or serves as a target for other immune cells (such as NK cells and cytotoxic T cells), thereby having the effect of promoting the attack, etc.

[0268] As described above, by returning the preparation with α-galactosylceramide as a ligand and the preparation with WT1 peptide as a ligand in a manner that they coexist in the body of the beneficiary, compared with using only the preparation of the present invention (the preparation with α-galactosylceramide as a ligand), the acquired immune system targeting cancer cells can be activated faster, and by directly activating both the innate immune system and the acquired immune system, a synergistic effect that is not simply additive can be obtained. Therefore, in the case of cancer treatment, a faster and greater effect can be expected compared with using the preparation alone.

[0269] <Effect>

[0270] According to the method for manufacturing a dendritic cell preparation that stimulates NKT cells by returning to the body of a beneficiary from whom monocytes have been collected according to the present invention, conditions have been found that can stimulate NKT cells to produce more IFN-γ compared with the prior art (Patent Document 1), and the concentration range of α-galactosylceramide that can produce IFN-γ more rapidly has been narrowed, so that the effects from both the innate immune system and the acquired immune system in cancer treatment and the like can be further expected, and an excellent effect of being able to produce a dendritic cell preparation that can be expected to have a higher therapeutic effect can be exerted. In addition, the aforementioned dendritic cell preparation can also exert the effect of enhancing immunity for healthy individuals. Moreover, by manufacturing with the monocytes of the beneficiary himself / herself, a safe preparation that does not need to worry about rejection can be obtained.

Claims

1. A method for producing a NKT cell-stimulating dendritic cell preparation, wherein the NKT cell-stimulating dendritic cell preparation is used to return to the body of a beneficiary from whom monocytes have been collected to stimulate NKT cells, wherein the method is a method for producing a dendritic cell preparation containing dendritic cells that stimulate natural killer T (NKT) cells for the purpose of any one or more of cancer treatment, cancer prevention, and immunity improvement. It is characterized in that include: (1) an adhesion step of adding monocytes collected from a beneficiary (monocytes obtained by blood collection or blood collection, or peripheral blood containing monocytes) to a culture container using a liquid culture medium, and allowing the culture container to stand to allow a portion of the monocytes to adhere to the inner surface of the container; (2) a non-adherent cell removal step of removing non-adherent cells including monocytes other than cells adhered to the inner surface of the culture container; (3) a differentiation step of adding a predetermined factor to the culture container to differentiate the monocytes adhered to the inner surface of the culture container into immature dendritic cells; (4) a pulsing step of pulsing α-galactosylceramide into the culture container where the immature dendritic cells are dominant; and (5) The NKT-stimulated dendritic cell induction step is to induce dendritic cells that stimulate NKT cells, namely, NKT-stimulated dendritic cells, from immature dendritic cells.

2. The method for producing a dendritic cell preparation according to claim 1, in, The day when the predetermined factor was added in the differentiation step (3) was defined as day 0, and the pulse step (4) was performed from day 3 to day 4.

3. The method for producing a dendritic cell preparation according to claim 1, in, The pulse step (4) is performed between 60 hours and 108 hours after the addition of the predetermined factor in the differentiation step (3).

4. The method for producing a dendritic cell preparation according to claim 1, in, The day when the mononuclear cells collected from the beneficiary are added to the culture container in the adhesion step (1) is defined as day 0, and the pulse step (4) is performed on the 4th to 5th day.

5. The method for producing a dendritic cell preparation according to claim 1, in, The pulse step (4) is performed between 84 hours and 132 hours after the mononuclear cells collected from the beneficiary are added to the culture container in the adhesion step (1).

6. The method for producing a dendritic cell preparation according to claim 1, in, The day when non-adherent cells including monocytes other than cells adhering to the inner surface of the culture container were removed in the non-adherent cell removal step (2) was defined as day 0, and the pulse step (4) was performed on the 4th to 5th day.

7. The method for producing a dendritic cell preparation according to claim 1, in, The pulse step (4) is performed between 84 hours and 132 hours after the non-adherent cells including monocytes other than cells adhered to the inner surface of the culture container are removed in the non-adherent cell removal step (2).

8. The method for producing a dendritic cell preparation according to any one of claims 1 to 7, in, The concentration of α-galactosylceramide pulsed in the pulse step (4) is 300 to 1000 ng / mL. 9 . Beneficiary's blood, to which the dendritic cell preparation produced by the production method according to claim 1 is added.

10. Beneficiary's blood, to which the dendritic cell preparation produced by the production method according to claim 8 is added.

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