Method for producing dendritic cell preparations comprising dendritic cells that stimulate NKT cells

Abstract

[Problem] To provide a method for producing a dendritic cell formulation that stimulates NKT cells, from monocytes collected from a recipient (monocytes collected by apheresis or peripheral blood containing monocytes), and that can exhibit the effect more quickly than conventional products. [Solution] This method for producing a dendritic cell formulation comprises: an adhesion step for placing collected monocytes in a culture vessel using a liquid culture medium, and leaving the culture vessel still to cause a portion of the monocytes to adhere to the inner surface of the vessel; a non-adhering cell removal step for removing non-adhering cells including monocytes other than the cells adhering to the inner surface of the culture vessel; a differentiation step for adding a predetermined factor in the culture vessel to cause the monocytes adhering to the inner surface of the culture vessel to differentiate into immature dendritic cells; a pulsing step for performing, with α-galactosylceramide, pulsing in the culture vessel in which the immature dendritic cells are present in a non-adhering state; and an NKT-stimulating dendritic cell induction step for inducing, from the immature dendritic cells, NKT-stimulating dendritic cells that stimulate NKT cells. The dendritic cell formulation is administered back into a recipient from which the blood have been collected.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a dendritic cell preparation consisting of dendritic cells that stimulate NKT cells ("T" stands for thymus) with fewer side effects, which is used for one or more of cancer treatment, cancer prevention, and improving the immune system of healthy individuals. In particular, the present invention relates to a method for producing a dendritic cell preparation based on cells collected from the beneficiary himself / herself, and to blood to which the dendritic cell preparation produced by this method has been added. [Background technology]

[0002] In recent years, cancer immunotherapy or methods for improving the immune system of healthy individuals that target NKT cells have been developed. This therapy does not target cancer cells themselves, but activates NKT cells, the command center of the immune system. The adjuvant effect of activated NKT cells (activating some immune system cells promotes the activation of other immune system cells, activating the immune system comprehensively) produces a strong antitumor effect. Furthermore, because it is not limited to targeting specific antigens, it is expected to be effective against new mutated cancer cells as well as non-cancer pathogens such as bacteria, viruses, and parasites.

[0003] Patent Document 1 describes a method in which monocytes are placed in a first culture vessel and allowed to stand, allowing a portion of the monocytes to settle on the bottom of the vessel, and non-adherent cells other than those adhering to the bottom of the vessel are collected and stored. Of the remaining cells adhering to the bottom, the monocytes are subjected to a specified treatment to differentiate into immature dendritic cells, which are then subjected to a further specified treatment to mature them. α-Galactosylceramide (hereinafter referred to as α-galactosylceramide) is then added to the mature dendritic cells, and dendritic cells that stimulate NKT cells are induced from the mature dendritic cells. These dendritic cells are then placed in a second culture vessel together with the preserved non-adherent cells and cultured, thereby inducing NKT cells from a portion of the non-adherent cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6854290 Summary of the Invention [Problem to be solved by the invention]

[0005] Patent Document 1 describes that when NKT cells are activated, they produce large amounts of IFN-γ (interferon gamma), and that when CD-1d molecules are presented on the surface of antigen-presenting cells (dendritic cells), an activation signal is transmitted via the T cell receptor on the surface of the NKT cells, activating the NKT cells.

[0006] Patent Document 1 discloses a cell composition containing NKT cells and dendritic cells that stimulate NKT cells, and the percentage of NKT cells is shown, but the amount of IFN-γ produced, which produces various effects on cancer cells, is not disclosed, so the effect cannot be said to be clear. Furthermore, it states that induction for stimulating NKT cells is performed after dendritic cells have matured. However, there is no description examining the temporal relationship between the timing of induction and the timing of maturation.

[0007] In order to solve these problems, the present inventors have conducted research and development and discovered a preparation of the present invention that produces a larger amount of IFN-γ and initiates IFN-γ production more quickly than the method of Patent Document 1. As a result of examining the timing of inducing dendritic cells to stimulate NKT cells, the present invention is able to produce a larger amount of IFN-γ by NKT cells than the prior art (Patent Document 1). The produced IFN-γ stimulates and activates both the innate and adaptive immune system pathways in the human body, and therefore, greater effects from both pathways stimulated by the large amount of IFN-γ produced can be expected in cancer treatment and other conditions. This provides a method for producing a dendritic cell preparation consisting of dendritic cells that stimulate NKT cells, which can be expected to have a greater therapeutic effect. [Means for solving the problem]

[0008] First, to summarize the means for solving the problem, the effect is greater when the pulsing timing of the ligand to dendritic cells is performed when the dendritic cells are immature. Furthermore, the timing of pulsing the ligand to immature dendritic cells is not necessarily any time as long as the dendritic cells are immature; even when the dendritic cells are immature, significant effects can be obtained by pulsing at a specific timing. Specific means for solving the problem are described below.

[0009] In order to solve the above-mentioned problems related to dendritic cell preparations, the first invention of the present application is to provide: A method for producing a dendritic cell preparation comprising dendritic cells that stimulate natural killer T (NKT) cells for the purpose of one or more of cancer treatment, cancer prevention, and immune enhancement, comprising: (1) an adhesion step in which monocytes collected from a beneficiary (apheresis or collected monocytes or peripheral blood containing monocytes) are placed in a culture vessel using a liquid medium and allowed to stand to allow some of the monocytes to adhere to the inner surface of the vessel; (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 vessel; (3) a differentiation step in which a predetermined factor is added to the culture vessel to differentiate the monocytes adhered to the inner surface of the culture vessel into immature dendritic cells; (4) a pulsing step of pulsing α-galactosylceramide into a culture vessel in which the immature dendritic cells exist in a non-adherent state; (5) an NKT-stimulatory dendritic cell induction step of inducing NKT-stimulatory dendritic cells, which are dendritic cells that stimulate NKT cells, from immature dendritic cells; The present invention provides a method for producing a dendritic cell preparation that stimulates NKT cells to be infused back into the body of the recipient from whom the monocytes were collected, the method comprising:

[0010] Furthermore, as a second invention, based on the first invention, there is provided a method for producing a dendritic cell preparation, in which the pulsing step (4) of the first invention is carried out on day 3 or 4, with the day on which a predetermined factor is added in the differentiation step (3) being considered day 0.

[0011] Furthermore, as a third invention, based on the first invention, there is provided a method for producing a dendritic cell preparation, in which the pulsing step (4) of the first invention is carried out 60 to 108 hours after adding a predetermined factor in the differentiation step (3).

[0012] Furthermore, as a fourth invention, based on the first invention, there is provided a method for producing a dendritic cell preparation according to claim 1, in which the pulsing step (4) is carried out by the fourth or fifth day, with the day on which monocytes collected from the recipient in the adhesion step (1) are placed in a culture vessel being considered as day 0.

[0013] Furthermore, as a fifth invention, based on the first invention, there is provided a method for producing a dendritic cell preparation, in which the pulsing step (4) is carried out 84 to 132 hours after the monocytes collected from the recipient in the adhesion step (1) are placed in a culture vessel.

[0014] Furthermore, as a sixth invention, based on the first invention, there is provided a method for producing a dendritic cell preparation, in which the pulsing step (4) is performed on the fourth or fifth day, with the day on which non-adherent cells, including monocytes, other than cells adhered to the inner surface of the culture vessel are removed in the non-adherent cell removal step (2), being considered as day 0.

[0015] Furthermore, as a seventh invention, based on the first invention, there is provided a method for producing a dendritic cell preparation, in which the pulsing step (4) is carried out between 84 and 132 hours after the removal of non-adherent cells, including monocytes, other than cells adhered to the inner surface of the culture vessel in the non-adherent cell removal step (2).

[0016] Furthermore, as an eighth invention, based on any one of the first to seventh inventions, there is provided a method for producing a dendritic cell preparation, in which the concentration of α-galactosylceramide pulsed in the pulse step (4) is 300 to 1000 ng / mL.

[0017] Furthermore, as a ninth invention, the blood of a recipient to which a dendritic cell preparation produced by the production method according to any one of the first to seventh inventions has been added is provided.

[0018] Furthermore, as a tenth aspect of the present invention, the blood of a recipient to which a dendritic cell preparation produced by the production method described in the eighth aspect of the present invention has been added is provided. [Effects of the Invention]

[0019] The method of the present invention for producing a dendritic cell preparation that stimulates NKT cells, in which monocytes are collected and then returned to the body of the recipient, can produce a greater amount of IFN-γ by NKT cells than the prior art (Patent Document 1). The produced IFN-γ stimulates and activates both the innate and adaptive immune systems in the human body, and therefore greater effects from both pathways stimulated by the large amount of IFN-γ produced can be expected in cancer treatment and other conditions, providing a method for producing a dendritic cell preparation consisting of dendritic cells that stimulate NKT cells, which can be expected to have a greater therapeutic effect. [Brief explanation of the drawings]

[0020] [Figure 1] Graph showing the relationship between the pulse timing of α-galactosylceramide during the production of a dendritic cell preparation according to embodiment 2 and the ratio of NKT cells after co-culture. [Figure 2a] Graph 1 shows the relationship between the timing of α-galactosylceramide pulses during the production of a dendritic cell preparation according to embodiment 2 and the maximum ratio of NKT cell counts after co-culture under each condition (when the day of addition of a specific differentiation factor is set as day 0). [Figure 2b] Graph 2 shows the relationship between the pulse timing of α-galactosylceramide during the production of a dendritic cell preparation according to embodiment 2 and the maximum ratio of NKT cells after co-culture under each condition (when the start of culture is used as the starting point). [Figure 3] Graph showing the relationship between the pulse timing of α-galactosylceramide during the production of a dendritic cell preparation according to embodiment 3 and the ratio of NKT cells after co-culture. [Figure 4a] Graph 1 showing the relationship between the timing of α-galactosylceramide pulses during the production of a dendritic cell preparation according to embodiment 3 and the maximum ratio of NKT cell counts after co-culture under each condition (when the day of addition of a specific differentiation factor is set as day 0). [Figure 4b]Graph 2 shows the relationship between the pulse timing of α-galactosylceramide during the production of the dendritic cell preparation of embodiment 3 and the maximum ratio of NKT cell counts after co-culture under each condition (when the start of culture is used as the starting point). [Figure 5] Graph showing the relationship between the concentration of α-galactosylceramide pulsed during the production of the dendritic cell preparation of embodiment 4 and the amount of IFN-γ produced after co-culture. [Figure 6] NK cell activity and IFN-γ production when the dendritic cell preparation of the present invention is administered to the human body [Figure 7] Comparison of the effects of the prior art (Patent Document 1) and the present invention (IFN-γ production amount after co-culture) DETAILED DESCRIPTION OF THE INVENTION

[0021] First, before describing the embodiments of the present invention, the body's immunity will be described. In the following description, the effect of the formulation of the present invention on cancer cells will be explained as an example, but similar effects can be obtained on other pathogens, etc. Immunity can be divided into innate immunity, which an organism originally possesses, and adaptive immunity, which an organism acquires later.

[0022] Innate immunity is a type of immunity that humans are born with, and is composed primarily of phagocytes that ingest and eliminate foreign substances such as bacteria and viruses. Phagocytes have receptors that recognize molecules and structures, and are broadly divided into types that process and eliminate foreign substances, and types that transmit signals between cells. Innate immune cells include eosinophils, neutrophils, basophils, macrophages, dendritic cells, and NK (natural killer) cells.

[0023] On the other hand, adaptive immunity is an immunity that remembers pathogens in the body and can effectively eliminate them when encountered again. It takes longer to respond than innate immunity, but has the diversity to react to a variety of pathogens. Adaptive immune cells include B cells and T cells, and T cells are further subdivided into helper T cells, killer T cells, and regulatory T cells. B cells change into plasma cells or memory B cells depending on the condition.

[0024] In addition, there are NKT cells that activate both innate and adaptive immunity. Of the immune cells mentioned above, the cells that this dendritic cell preparation acts on directly are NKT cells, but the adjuvant effect of IFN-γ, which is produced by the activation of NKT cells, also activates NK cells, dendritic cells, T cells (helper T cells, killer T cells), B cells, and other cells.

[0025] Below, we will briefly explain the immune cells and substances related to immune function that are involved in the action of the dendritic cell preparation produced by this method for producing a dendritic cell preparation.

[0026] <Terminology for immune cells and substances related to immune function> <Terminology: antigen> A foreign substance that enters the body serves as a marker for adaptive immune cells to recognize as a target for attack. In the case of cancer antigens, immune cells recognize them as targets for attack based on the protein structure that does not exist in normal cells.

[0027] <Terminology: Antibody> Cancer antibodies are a general term for proteins called immunoglobulins. They bind to cancer antigens on cancer cells and attack the cancer cells through the following three mechanisms: Neutralizing effect: Preventing the growth of cancer cells ADCC activity: It acts as a marker for killer T cells and NK cells and assists in attacking cancer. CDC activity: Complements (a type of protein synthesized in the body and present in serum, which is involved in immune responses such as infection prevention; there are nine types, C1 to C9) bind to antibodies that have bound to cancer antigens on cancer cells, activating complements C1 to C9 in a chain reaction, which then creates holes in the cell membrane of the cancer cells, damaging and killing them.

[0028] <Terminology: Cytokine> Cytokines are proteins secreted by immune cells and are a general term for physiologically active substances involved in intercellular interactions. They transmit signals to target cells, causing various cellular responses such as cell activation, cell proliferation, differentiation, cell death, and functional expression. Interleukins, interferons, and tumor necrosis factors are examples of cytokines.

[0029] <Terminology: Cytokine: Interleukin> More than 30 types of interleukins, a type of cytokine, have now been identified. They are secreted in large quantities by lymphocytes, a type of white blood cell that fights foreign substances in the body, and phagocytes such as macrophages and neutrophils. Interleukins are referred to by numerical names such as interleukin-2 (hereafter, interleukins will be abbreviated as IL. For example, interleukin-4 is IL-4). For example, the sources and functions of some interleukins are described below.

[0030] <Terminology: Cytokine: Interleukin: IL-4> IL-4 is one of the Th2 cytokines, and is produced by activated type 2 helper T cells (Th2 cells) and NKT cells. It promotes the proliferation and differentiation of antigen-presenting cells. It affects B cells, T cells, macrophages, monocytes, etc. Naive T cells differentiate into Th2 cells in response to stimulation by cytokines such as IL-4 and IL-13. When IL-4 acts on activated B cells, a class switch occurs in which the immunoglobulins (antibodies) produced change from IgM to IgE and IgG1. In this way, it is involved in the humoral immune system. NKT cells are activated when they recognize α-galactosylceramide presented by antigen-presenting cells (dendritic cells), and produce IL-4 and IFN-γ.

[0031] <Terminology: Cytokine: Interleukin: IL-12> IL-12 is produced by phagocytes (cells that migrate between tissues in the animal body and are responsible for cellular immunity, such as macrophages, monocytes, and neutrophils) and dendritic cells. It is an NK cell stimulatory factor, and together with IFN-γ, it acts on naive T cells to differentiate into Th1 cells, a type of helper T cell. Th1 cells produce Th1 cytokines such as IFN-γ and are involved in cellular immunity, activating macrophages and killer T cells. α-Galactosylceramide induces dendritic cells (antigen-presenting cells) to produce IL-12. IL-12 produced by antigen-presenting cells acts on NKT cells to produce IFN-γ and, via the Fas ligand on the surface of NKT cells, induces cytotoxicity against target cells expressing the Fas antigen (Fas receptor).

[0032] <Terminology: Cytokine: Interferon> Interferon, a type of cytokine, activates NK cells and macrophages, which attack cancer cells and virus-infected cells, and has the function of suppressing the growth of viruses and tumor cells and promoting the phagocytosis of cancer cells (adjuvant action). It has also been approved by the government as an antiviral drug and anticancer agent, and is used to treat multiple myeloma, brain tumors, and kidney cancer.

[0033] <Terminology: Cytokine: Interferon: IFN-γ> IFN-γ is an important cytokine in both the innate and adaptive immune systems. IFN-γ is primarily produced by NK cells and NKT cells as part of the innate immune response, and is also produced by Th1 cells, killer T cells, macrophages, and other cells after the induction of antigen-specific immunity. It accelerates the processing of large amounts of invading non-self antigens and enhances the cellular immune response. It is an important activator of macrophages, stimulating them to phagocytose and kill bacteria. It also promotes NK cell activity and differentiates naive T cells into Th1 cells.

[0034] <Terminology: Cytokine: Tumor necrosis factor> Tumor necrosis factor (TNF) is a substance secreted by lymphocytes and other cells that causes tumor cell necrosis. It acts on most malignant cells and promotes immune function. In a narrow sense, there are three types: TNF-α, TNF-β (lymphotoxin (LT)-α), and LT-β.

[0035] <Terminology: Cytokine: Tumor necrosis factor: TNF-α> TNF-α is primarily produced by macrophages and was discovered as a cytokine that induces hemorrhagic necrosis in solid tumors. Generally, the term "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 controlled and regulated suicide of cells, or programmed cell death.

[0036] <Terminology: NK cells> NK cells have various receptors that capture abnormal cells, and when they discover cells that differ from the normal cells that make up the human body, such as virus-infected cells or cancer cells (they remember the cells that make up the human body), they recognize them as abnormal and have the ability to damage the abnormal cells.

[0037] <Terminology: Dendritic cells> Dendritic cells are antigen-presenting cells. They acquire the ability to present antigens specific to foreign substances by ingesting (phagocytosis) them (such as pathogenic bacteria, virus-infected cells, or cancer cells), or by receiving antigens from macrophages, etc. They then present these antigens to T cells, causing them to be recognized as targets for attack. Dendritic cells have the phagocytic ability to ingest viruses and cancer cells, thereby acquiring antigens and gaining the ability to present them to other immune cells. Early dendritic cells (immature dendritic cells) have the ability to ingest antigens (phagocytosis), but cannot activate naive T cells (they are said to lack antigen-presenting ability). Mature dendritic cells acquire the antigen-presenting ability to activate T cells through maturation, but lose the ability to ingest antigens (phagocytosis).

[0038] <Terminology: Helper T cells> When antigens are presented to them by dendritic cells, they recognize them as targets for attack and instruct killer T cells to attack. They also produce cytokines to activate B cells. Furthermore, when antigens are presented to them by B cells, they determine whether the antigen is a dangerous foreign substance and communicate the result to B cells. They also assist in the production of antibodies.

[0039] <Terminology: Killer T cells> Killer T cells recognize antigens presented by MHC class I molecules (MHC class I molecules primarily bind peptides generated by the degradation of cytoplasmic proteins by the proteasome (a protein-degrading enzyme complex) (as a marker for identifying the human self). The MHC-I peptide complex then passes through the endoplasmic reticulum membrane and is inserted into the outer surface of the cell membrane. Epitope peptides are bound to the extracellular portion of the MHC-I peptide molecule. In this way, the MHC-I peptide molecule presents intracellular proteins to killer T cells. However, MHC-I peptide molecules can also present peptides formed from foreign proteins, a process known as cross-presentation.), recognize self and non-self, and destroy non-self cells. The ability to recognize self and non-self is conferred by competence education (negative selection) in the thymus, and this ability is utilized by MHC-I peptide molecules. Thus, adaptive immune responses of T cells begin with antigen presentation of antigenic peptides by MHC molecules. Under the direction of helper T cells, killer T cells kill and eliminate cells that are dangerous or unnecessary to the body, such as virus-infected cells and cancer cells.

[0040] <Terminology: B cells> They are stimulated by cytokines produced by helper T cells to produce antibodies. Even if the antigen is eliminated, they act as memory B cells, remembering the antigen and preparing for the next invasion.

[0041] <Terminology: NKT cells> It is a cell that combines the characteristics of both T cells and NK cells. When activated, it produces cytokines (IFN-γ) and also has the function of inducing responses to immunostimulatory and immunosuppressive therapies. Moreover, the most expected effect of the preparation of the present invention is the adjuvant effect, which activates various immune cells in the body and, which is an important point here, has the effect of proliferating them.

[0042] <Function of dendritic cell preparation that stimulates NKT cells> The main effects expected by activating NKT cells with this preparation are the effects on both non-specific immunity and specific immunity, such as promoting the maturation of dendritic cells, adjuvant effects (activation and proliferation of various immune cells), inducing apoptosis of cancer cells, and inhibiting angiogenesis. Apoptosis means the managed and regulated suicide of cells, that is, programmed cell death.

[0043] As described above, by using a dendritic cell preparation that stimulates NKT cells and activates the innate immune system, various immune functions in the body can be activated to attack cancer cells.

[0044] Hereinafter, embodiments will be described in detail. Regarding the substances used during the manufacturing process, they are common to all embodiments. The manufacturer, etc. will be described only at the first appearance.

[0045] <Embodiment 1: Overview> Mainly claim 1 It is a manufacturing method in which monocytes collected from a beneficiary (apheresis or monocytes collected by blood collection, or peripheral blood containing monocytes) are transformed into dendritic cells that stimulate NKT cells to obtain a dendritic cell preparation.

[0046] Hereinafter, in this specification, a "beneficiary" refers to a person who desires to obtain the benefits of the effects of administering a dendritic cell preparation for the purpose of any one or more of cancer treatment, cancer prevention, and improvement of immunity, and who provides their own monocytes as the raw material for the preparation.

[0047] <Embodiment 1: Manufacturing method> First, an outline of the method for producing an NKT cell-stimulating dendritic cell preparation of the present invention will be explained, followed by an explanation of the effects of this preparation.

[0048] <Embodiment 1: (1) Adhesion Step: Preparation of Monocytes> Monocytes collected from a beneficiary (monocytes collected by apheresis or blood collection, or peripheral blood containing monocytes) are prepared. "Preparation" refers to the delivery of monocytes collected from a beneficiary (monocytes collected by apheresis or blood collection, or peripheral blood containing monocytes) to the manufacturing site, but does not include the blood collection itself. In this adhesion process, preparation may also include the delivery of culture vessels and liquid media to be used later to the manufacturing site and preparing them for use. Unless otherwise specified, in this specification, "culture vessel" refers to a culture vessel that contains a culture medium such as a liquid medium.

[0049] "Component blood collection" refers to the process of withdrawing blood from the body, separating the desired blood components and factors using specialized equipment, and then returning the blood to the body. In the present invention, peripheral blood mononuclear cells, including monocytes, are separated as blood components, and approximately 100 cc of blood components are collected. The blood components obtained after the separation of the peripheral blood mononuclear cells, including monocytes, contain plasma and platelets in addition to the desired blood components. Component blood collection places a significant burden on the body, as an anticoagulant is mixed into the blood before the separation of the peripheral blood mononuclear cells, including monocytes, to prevent blood clotting, and the anticoagulant is then returned to the body. Furthermore, the component blood collection process for the separation of the peripheral blood mononuclear cells, including monocytes, takes a long time.

[0050] It is also possible to collect 100-400 mL of peripheral blood instead of apheresis. Although apheresis contains a higher proportion of monocytes, peripheral blood collection is acceptable because it can be collected in a short time without the use of anticoagulants.

[0051] <Embodiment 1: (1) Adhesion step: leaving still> Next, mononuclear cells are collected from the collected peripheral blood mononuclear cells, including monocytes, by density centrifugation using Ficoll®. (Ficoll® is a part of Ficoll-Paque® and is used to separate blood into its components (e.g., red blood cells, white blood cells, etc.).) The collected mononuclear cells are placed in a culture vessel containing a liquid medium. It is desirable to place the mononuclear cells collected from the recipient in a culture vessel containing a medium (preferably a liquid medium) within approximately 72 hours of collection. The start of culture is the time when the mononuclear cells, including monocytes, are placed in a culture vessel containing a medium (preferably a liquid medium). Unless otherwise specified, the expression "monocytes are placed in a culture vessel" below refers to the placement in a culture vessel containing a medium (preferably a liquid medium). The cells are then left to stand for approximately 30 minutes in a sterilized, constant-temperature environment at 37°C and incubated. Unless otherwise specified, subsequent steps are performed in a sterilized, constant-temperature environment at 37°C. The time for standing may be longer than 30 minutes, but if it is shorter than 30 minutes, a sufficient number of monocytes will not adhere to the inner surface of the container, so it is preferable to stand for at least about 30 minutes.

[0052] As used herein, "adhesion" to the inner surface of a culture vessel does not mean intentionally pressing cells against the inner surface of the culture vessel or using some kind of substance to act as an adhesive and attach the cells so that they cannot easily separate from the inner surface of the culture vessel. "Adhesion" refers to the state in which cells come into contact with the inner surface and then maintain that contact without the application of external forces other than gravity.

[0053] <Embodiment 1: (2) Non-adherent cell removal step: removal of non-adherent cells> After leaving the cells to stand (e.g., for 30 minutes), non-adherent cells, including monocytes that do not adhere to the inner surface of the culture vessel and float in the liquid medium, are removed (transferred to another vessel). Cells that adhere to the inner wall of the vessel (monocyte fraction) remain in the culture vessel containing the liquid medium.

[0054] The removed non-adherent cells, including monocytes, are preferably cultured separately or cryopreserved. The removed non-adherent cells (including NKT cells) can be used to verify the efficacy of the formulation by co-culturing them with the manufactured formulation. After removing the non-adherent cells, a detachment step may be performed in which cells adhered to the inner wall of the container (monocyte fraction) are detached and suspended in a liquid medium. During the above-mentioned standing, monocytes mainly adhere to the inner surface of the container, and after detachment, mainly monocytes float in the liquid medium.

[0055] <Embodiment 1: (3) Differentiation step: Differentiation of monocytes into immature dendritic cells> To the culture vessel containing the liquid medium from which non-adherent cells have been removed, granulocyte-macrophage colony-stimulating factor (GM-CSF) 50 ng / mL (preferably approximately 30 ng / mL to 70 ng / mL; concentrations below this range are too weak, resulting in slow differentiation, while concentrations above this range result in a plateau in differentiation rate, making it unreasonable from a cost perspective) and interleukin 4 (IL-4) 50 ng / mL (preferably approximately 30 ng / mL to 70 ng / mL; concentrations below this range are too weak, resulting in slow differentiation, while concentrations above this range result in a plateau in differentiation rate, making it unreasonable from a cost perspective) are added as specific factors for differentiating monocytes into immature dendritic cells. After addition, the culture is continued. Monocytes are stimulated by the addition of GM-CSF and IL-4, resulting in differentiation of the cultured monocytes into immature dendritic cells. The substances used to stimulate differentiation are preferably, but not limited to, the aforementioned GM-CSF and IL-4. By adding a stimulating substance, the culture vessel becomes a state in which immature dendritic cells are present in much greater numbers (predominantly present) than undifferentiated monocytes or mature dendritic cells. Differentiated immature dendritic cells account for the majority of the cells in the culture vessel, preferably 80% or more, and more preferably 90% or more.

[0056] <Embodiment 1: (4) Pulse Step: Pulse of α-Galactosylceramide> As described above, α-galactosylceramide is pulsed into a liquid medium in a culture vessel containing a majority of immature dendritic cells. The α-galactosylceramide used was KRN7000, a product of Kyowa Kirin Co., Ltd. By pulsing with α-galactosylceramide, immature dendritic cells become immature dendritic cells capable of stimulating NKT cells after maturation. α-galactosylceramide is a glycolipid antigen necessary for activating NKT cells. A 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 through the phagocytosis of various foreign substances. However, the present invention is characterized in that α-galactosylceramide is imparted (pulsed) to dendritic cells at an immature stage, thereby imparting antigen-presenting ability to mature dendritic cells. The inventors of the present application have confirmed that, although prior art Patent Document 1 describes that pulsing mature dendritic cells with α-galactosylceramide imparts antigen presentation ability, according to the present invention, pulsing at least immature dendritic cells is more effective in presenting antigens to and activating NKT cells.

[0057] <Embodiment 1: (5) NKT-stimulating dendritic cell induction step: dendritic cell maturation> As mentioned above, after pulsing with α-galactosylceramide, GM-CSF (5 ng / mL: 3 ng / mL to 7 ng / mL is preferable. Less than this will be too weak a stimulus and slow maturation, and more than this will cause the maturation rate to plateau and is therefore unreasonable from a cost perspective) and IL-4 (5 ng / mL: 3 ng / mL to 7 ng / mL is preferable. Less than this will be too weak a stimulus and slow maturation, and more than this will cause the maturation rate to plateau and is therefore unreasonable from a cost perspective). and OK-432 (10ng / mL: approximately 7ng / mL to 13ng / mL is preferable. Less than this will be too weak a stimulus, slowing maturation, while more than this will cause the maturation rate to plateau, making it unreasonable from a cost perspective), and PGE2 (50ng / mL: approximately 30ng / mL to 70ng / mL is preferable. Less than this will be too weak a stimulus, slowing maturation, while more than this will cause the maturation rate to plateau, making it unreasonable from a cost perspective) are added to the liquid medium in the culture vessel. These four ingredients should be added on the sixth day after the start of culture. Stimulated by the added ingredients, immature dendritic cells mature into mature dendritic cells. OK-432 is the trade name Picibanil, manufactured by Chugai Pharmaceutical Co., Ltd., and PGE2 is prostaglandin E2 (hereinafter referred to as PGE2). The substances used to stimulate maturation are preferably, but not limited to, the above-mentioned GM-CSF, IL-4, OK-432 and PGE2.

[0058] <Embodiment 1: (5) NKT-Stimulating Dendritic Cell Induction Step: Final Test> After the mature dendritic cells are produced, sterility tests, endotoxin tests, and mycoplasma tests are performed to confirm whether they are safe to inject into humans. Furthermore, surface marker measurements are performed to evaluate the quality of the dendritic cells. These quality confirmation tests may be supplemented with other tests as appropriate. Furthermore, if the dendritic cells are not to be administered to the recipient immediately or are planned to be administered in several divided doses, the produced dendritic cells can be collected as needed and stored in a cryopreservation solution at 0.5 x 10 7 0.5 x 10 cells / ml / vial (0.5 x 10 cells / ml / vial if whole blood is used as raw material) 6The dendritic cell preparation is prepared to a concentration of 10 or more cells / ml / vial and then frozen and stored under liquid nitrogen. A vial is a type of container for injections, and is classified as a sealed container in the Japanese Pharmacopoeia. To achieve the desired effect, it is preferable to use a concentration equal to or higher than the above. This completes the method for producing the dendritic cell preparation of the present invention that stimulates NKT cells.

[0059] <Embodiment 1: Effect of dendritic cell preparations that stimulate NKT cells> When the NKT cell-stimulating dendritic cell preparation of the present invention is administered to a recipient from whom blood has been drawn, the preparation stimulates and activates Vα24-NKT cells in the recipient's body, causing them to produce IFN-γ, a type of cytokine, resulting in an adjuvant effect. NK cells are strongly activated by this adjuvant effect and attack cancer cells or cells other than normal cells through a cytotoxic response. Activated NKT cells also activate the adaptive immune system by promoting the maturation of immature dendritic cells in the body. By activating both the innate and adaptive immune systems, the preparation is effective not only for cancer treatment but also for cancer prevention and disease prevention by improving the immune system of healthy individuals.

[0060] Next, the effects of the NKT cell-stimulating dendritic cell preparation of the present invention when it is returned to the body of a recipient from whom monocytes were collected to produce said preparation will be described.

[0061] <Embodiment 1: Action of the preparation: Return to the body by intravenous infusion> The preparation, which contains immature dendritic cells that have been pulsed with α-galactosylceramide and then matured, is then administered back into the recipient's body by intravenous infusion or other methods.

[0062] <Embodiment 1: Effect of the Present Preparation: Activation of Vα24-NKT Cells> The dendritic cells of the present invention pass through the blood vessels of the body and bind the glycolipid antigen α-galactosylceramide to a ligand called CD1d, presenting it to Vα24-NKT cells in the body. Vα24-NKT cells in the body are activated when α-galactosylceramide binds to their receptors (receiving antigen presentation).

[0063] <Embodiment 1: Effect of the present formulation: Activated NKT cells: TNF-α production, inhibition of angiogenesis> Activated Vα24-NKT cells produce TNF-α (tumor necrosis factor-α), which stimulates immature dendritic cells (immature DCs). At the same time, TNF-α inhibits angiogenesis in cancer cells.

[0064] <Embodiment 1: Action of the present formulation: Maturation of dendritic cells and activation of the adaptive immune system> Immature dendritic cells phagocytose neoantigens, which are cancer antigens released by cancer cells or dead cancer cells. Alternatively, immature dendritic cells stimulated by TNF-α from activated Vα24-NKT cells phagocytose neoantigens. Dendritic cells that phagocytose neoantigens mature, also due to the effects of TNF-α stimulation. Killer T cells and helper T cells receive antigen presentation from mature dendritic cells as a stimulus. Helper T cells that receive the antigen presentation stimulate B cells with cytokines. The stimulated B cells produce and release cancer cell-specific antibodies.

[0065] <Embodiment 1: Effects of the Present Preparation: Apoptosis and Cytotoxicity> NK cells are activated by IFN-γ produced by activated Vα24-NKT cells. These activated NK cells and Vα24-NKT cells then attack non-human cancer cells (or pathogens) by killing them (cytotoxic activity) or promoting apoptosis (innate immune system). Meanwhile, antibodies released from the B cells bind to cancer cells. Killer T cells attack the cancer cells (or pathogens) by creating holes in their cell membranes, causing necrosis (cytotoxic activity), or by producing substances such as TNF-β (tumor necrosis factor, a cytokine) to promote apoptosis (adaptive immune system). Attack by activated NK cells and Vα24-NKT cells (innate immune system) occurs 1–2 days after intravenous infusion of this formulation, while attack by killer T cells (adaptive immune system) takes place after antigen presentation by dendritic cells, taking approximately 7–14 days.

[0066] Killer T cells and helper T cells that attack cancer cells cannot survive for long periods of time, but some of these T cells exist in lymph nodes and other locations as memory killer T cells and memory helper T cells (they are thought to be maintained through replication and replacement of memory T cells). Even after cancer cells have disappeared, if cancer cells that express the same antigen reappear, the memory helper T cells will immediately issue instructions to immune cells to attack because they retain their memory of the previous attack, allowing the killer T cells to respond quickly. In addition to T cells, B cells that have released antibodies also exist as memory B cells, and can quickly release antibodies in the event of a recurrence.

[0067] <Effects> By producing dendritic cells that stimulate NKT cells, when they are returned to the recipient, it is expected that NK cells will be activated through the innate immune system pathway, and by stimulating dendritic cells, helper T cells, killer T cells, and B cells will be indirectly activated. By improving overall immunity rather than just targeting specific cancers, it is possible to prevent beneficiaries weakened by cancer from contracting diseases other than cancer, and even if they do contract them, it is expected to be milder and lead to a faster recovery. In addition to cancer treatment, it is also effective in preventing cancer and other diseases by improving immunity. Furthermore, because this dendritic cell preparation is cultured from the recipient's own cells and is made exclusively for the recipient, it is a highly safe preparation with no rejection reactions.

[0068] <Embodiment 2: Overview> Mainly claim 2 In this embodiment 2, based on the method for producing a dendritic cell preparation of embodiment 1, the (4) pulse step is performed on day 3 or 4, with the day on which a specific factor was added in the (3) differentiation step being considered day 0.

[0069] <Embodiment 2: (4) Pulse Step: Pulse of α-Galactosylceramide> In this embodiment 2, the timing of α-galactosylceramide pulsing is earlier than in embodiment 1, and α-galactosylceramide is pulsed into the liquid medium in a culture vessel containing a predominance of immature dendritic cells on either day 3 or day 4, with the day on which GM-CSF and IL-4 were added as predetermined factors in the differentiation step being considered day 0. As in embodiment 1, α-galactosylceramide used was model KRN7000 manufactured by Kyowa Kirin Co., Ltd.

[0070] <Embodiment 2: (4) Pulse Process: Optimal Pulse Timing> Dendritic cell preparations produced by the manufacturing method of the present invention are expected to stimulate and activate Vα24-NKT cells, thereby producing cytokines such as IFN-γ and activating various immune system players. To assess the efficacy of the preparation, non-adherent cells (lymphocytes (T cells, B cells, NK cells, NKT cells, etc.) removed in the non-adherent cell removal step (2) of the manufacturing method and separately cultured and stored) are added to the preparation after completion of the preparation, and co-cultured to examine the ratio of IFN-γ-producing NKT cells to the total number of lymphocytes. The relationship between the pulse timing and the NKT cell ratio after the co-culture is examined to determine the optimal pulse timing. The pulse timing was determined in this manner. The use of non-adherent cells is intended to avoid physical strain on the recipient and has no other purpose. Therefore, peripheral blood may be newly collected from the recipient, and the dendritic cell preparation produced by the manufacturing method of the present invention may be added to the collected peripheral blood to similarly compare and confirm efficacy. Furthermore, co-culture was performed to observe the difference in the increase in the proportion of NKT cells relative to total lymphocytes, and what became clear from this observation was that the proliferation rate of NKT cells (considered to be self-dividing proliferation) is influenced by the dendritic cell preparation of the present invention. Therefore, although step (5) in claim 1 of the present application is simply described as an "NKT-stimulating dendritic cell induction step," it can also be said as an "NKT proliferation-stimulating dendritic cell induction step."

[0071] In this embodiment, the day on which a specific factor (e.g., GM-CSF and IL-4) was added to differentiate monocytes into immature dendritic cells was designated as day 0, and time is expressed in days hereafter. After the formulation was completed, non-adherent cells were added and co-cultured to confirm the efficacy of the formulation. To verify the optimal pulse timing, collected monocytes were divided into thirds, and three samples of dendritic cell formulations with different α-galactosylceramide pulse timings were prepared. Each sample was pulsed with α-galactosylceramide (concentration 100 ng / mL) on day 2 (48 hours) after the addition of the specific factor (condition 1), pulsed on day 4 (96 hours) (condition 2), and pulsed on day 5 (120 hours) (condition 3).

[0072] To confirm the effectiveness of this formulation, non-adherent cells were added and co-culture was initiated on day 6, with the day of addition of a specific factor to induce monocytes to become immature dendritic cells being designated day 0. The ratio of NKT cell counts to lymphocyte counts was investigated three times: on the day co-culture began (day 0), on day 7, and on day 14.

[0073] Figure 1 shows the results of the α-galactosylceramide pulse timing conditions (conditions 1–3) and the ratio of IFN-γ-producing NKT cells (those contained in non-adherent cells) to the total lymphocyte count at each timing after co-culture. This figure was obtained using a flow cytometer. For all three conditions, the ratio of NKT cells to lymphocytes was highest on day 7 after the start of co-culture. Furthermore, among the three conditions, the NKT cell ratio (%) after co-culture was highest when the pulse was performed on day 4, where day 0 is the day when a specific factor was added to differentiate monocytes into immature dendritic cells. Figure 2a shows the relationship between the maximum NKT cell ratio (%) on day 7 after co-culture and the pulse timing (day), where day 0 is the day of specific factor addition. Figure 2a also shows the approximate curve, extrapolated both forward and backward, based on a quadratic fit. The coefficient of determination for this approximate curve is 1, which is considered sufficient for reference. The peak of the curve is between days 3 and 4 of the pulse timing (calculated from the approximate equation as day 3.7). The preferred range is approximately days 2.5 to 5, when the concentration reaches -10% of the peak value. Because the lifespan of dendritic cells is approximately several days to a week, the production process (from monocyte collection or the addition of specific factors to differentiate monocytes into immature dendritic cells to maturation into mature dendritic cells, i.e., the addition of GM-CSF, IL-4, OK-432, and PGE2 to induce mature dendritic cells) requires a long time. (Note: Since the time required for complete maturation after this is approximately 10 to 20 hours, this time period may also be included.) In this experiment, the timing of the α-galactosylceramide pulse differed between conditions 1, 2, and 3, but GM-CSF, IL-4, OK-432, and PGE2 were added simultaneously.) Therefore, if the production process using this method takes too long, the period during which the product can be used will be shortened. Therefore, considering that the preparation should be completed within one week from the start of culture, the pulse should be performed between days 3 and 4.5, which is also the range where the concentration is down to -5% of the peak value, and it is best to pulse on day 3 or 4. The most preferred is to pulse on day 4.

[0074] When α-galactosylceramide is pulsed into immature dendritic cells, the resulting formulation can be maximized in the recipient's body by pulsing it on either the 3rd or 4th day after adding certain factors (e.g., GM-CSF and IL-4) to differentiate monocytes into immature dendritic cells.

[0075] <Embodiment 3: Overview> Mainly claim 3 In this third embodiment, based on the method for producing a dendritic cell preparation of the first embodiment, the (4) pulse step is carried out between 60 and 108 hours after the addition of a predetermined factor in the (3) differentiation step.

[0076] <Embodiment 3: (4) Pulse Step: Pulse of α-Galactosylceramide> In this embodiment 3, the α-galactosylceramide pulse timing is earlier than that in embodiment 1, and α-galactosylceramide is pulsed between 60 and 108 hours after the addition of GM-CSF and IL-4 as predetermined factors in the differentiation step. As in embodiment 1, α-galactosylceramide was used, model name KRN7000 by Kyowa Kirin Co., Ltd.

[0077] <Embodiment 3: (4) Pulse Process: Optimal Pulse Timing> Dendritic cell preparations produced by the manufacturing method of the present invention are expected to stimulate and activate Vα24-NKT cells, thereby producing cytokines such as IFN-γ and activating various immune system players. To assess the efficacy of the preparation, non-adherent cells (lymphocytes (T cells, B cells, NK cells, etc.)) removed in the non-adherent cell removal step (2) of the manufacturing method are added to the completed preparation after co-culture, and the proportion of IFN-γ-producing NKT cells is examined. The relationship between the pulse timing and the proportion of NKT cells after co-culture is examined, and the optimal pulse timing is determined in the same manner as in Example 2. The pulse timing was determined in this manner. The use of non-adherent cells is intended to avoid placing a physical burden on the recipient, and has no other purpose. Therefore, peripheral blood may be newly collected from the recipient, and the dendritic cell preparation produced by the manufacturing method of the present invention may be added to the collected peripheral blood to similarly compare and confirm the effects. Furthermore, co-culture was performed to observe the difference in the increase in the proportion of NKT cells relative to total lymphocytes, and what became clear from this observation was that the proliferation rate of NKT cells (considered to be self-dividing proliferation) is influenced by the dendritic cell preparation of the present invention. Therefore, although step (5) in claim 1 of the present application is simply described as an "NKT-stimulating dendritic cell induction step," it can also be said as an "NKT proliferation-stimulating dendritic cell induction step."

[0078] In this embodiment, the time when a specific factor (e.g., GM-CSF and IL-4) was added to differentiate monocytes into immature dendritic cells was defined as time 0, and time is expressed in units of hours hereafter. After the preparation was completed, non-adherent cells were added and co-cultured to confirm the effectiveness of the preparation. To verify the optimal pulse timing, blood-collected monocytes were divided into thirds using a production method based on the production method of Embodiment 1, and three types of dendritic cell preparation samples were prepared, differing only in the timing of α-galactosylceramide pulse. Each sample was pulsed with α-galactosylceramide (concentration 100 ng / mL) 48 hours (day 2) after the addition of the specific factor (condition 1), 96 hours (day 4) (condition 2), and 120 hours (day 5) (condition 3).

[0079] To confirm the effectiveness of this formulation, non-adherent cells were added 144 hours (day 6) after the addition of a specific factor to induce monocytes to become immature dendritic cells, and co-culture was initiated. The ratio of NKT cells to lymphocytes was investigated three times: on the day co-culture began (day 0), on day 7, and on day 14.

[0080] Figure 1 shows the results of the α-galactosylceramide pulse timing conditions (conditions 1–3) and the ratio of IFN-γ-producing NKT cells (those contained in non-adherent cells) to the total lymphocyte count at each timing after co-culture. This figure was obtained using a flow cytometer. For all three conditions, the ratio of NKT cells to lymphocytes was highest on day 7 after the start of co-culture. Furthermore, among the three conditions, the NKT cell ratio (%) was highest when the pulse was administered 96 hours (day 4) after the addition of the specified factor to differentiate monocytes into immature dendritic cells. The relationship between the maximum NKT cell ratio (%) on day 7 after co-culture and the pulse timing is shown in the scatter plot in Figure 2b. Figure 2b also shows the approximate curve, extrapolated both before and after the quadratic fit. The coefficient of determination for this approximate curve is 1, which is considered sufficient for reference. The peak of the curve is between 70 and 96 hours after the pulse timing (90 hours calculated from the approximate equation).

[0081] The preferred range is approximately 60 to 118 hours, which is 10% of the peak value. Because the lifespan of dendritic cells is approximately several days to a week (up to 168 hours), the production process (from monocyte collection or the addition of specific factors to differentiate monocytes into immature dendritic cells to their maturation into mature dendritic cells, i.e., the addition of GM-CSF, IL-4, OK-432, and PGE2 to induce mature dendritic cells) can be extended. (Note: Since the time required for complete maturation is approximately 10 to 20 hours, this time period may also be included.) In this experiment, the timing of α-galactosylceramide pulsing differed between Conditions 1 and 3, but GM-CSF, IL-4, OK-432, and PGE2 were added simultaneously.) Therefore, pulsing between 60 and 108 hours after the start of culture is preferred, considering the need to complete the preparation within one week of the start of culture. More preferably, the pulse is carried out between 70 hours and 108 hours, which is the range where the peak value is -5%.

[0082] When α-galactosylceramide is pulsed into immature dendritic cells, the resulting formulation can be maximized in the recipient's body by pulsing between 60 and 108 hours after adding specific factors (e.g., GM-CSF and IL-4) to differentiate monocytes into immature dendritic cells.

[0083] <Embodiment 4: Overview> Mainly claim 4 In this embodiment 4, based on the manufacturing method of the dendritic cell preparation of embodiment 1, the (4) pulsing step is carried out by the 4th or 5th day, with the day on which the monocytes collected from the recipient in the adhesion step (1) are placed in the culture vessel being considered as day 0.

[0084] <Embodiment 4: (4) Pulse Step: Pulse of α-Galactosylceramide> In this embodiment 4, the α-galactosylceramide pulse timing is earlier than in embodiment 1, and α-galactosylceramide is pulsed between day 4 and day 5, with the day on which monocytes collected from the recipient are placed in a culture vessel being considered day 0. As in embodiment 1, α-galactosylceramide used was Kyowa Kirin Co., Ltd., model name KRN7000.

[0085] <Embodiment 4: (4) Pulse Process: Optimal Pulse Timing> Dendritic cell preparations produced by the manufacturing method of the present invention are expected to stimulate and activate Vα24-NKT cells, produce IFN-γ, and activate various immune system players. To assess the efficacy of the preparation, non-adherent cells (lymphocytes (T cells, B cells, NK cells, etc.)) removed in the non-adherent cell removal step (2) of the manufacturing method are added to the completed preparation after co-culture, and the proportion of IFN-γ-producing NKT cells is examined. The relationship between the pulse timing and the proportion of NKT cells after co-culture is examined, and the optimal pulse timing is determined. The pulse timing was determined in this manner. The use of non-adherent cells is intended to avoid placing a physical burden on the recipient, and has no other purpose. Therefore, peripheral blood may be newly collected from the recipient, and the dendritic cell preparation produced by the manufacturing method of the present invention may be added to the collected peripheral blood to similarly compare and confirm efficacy. Furthermore, co-culture was performed to observe the difference in the increase in the proportion of NKT cells relative to total lymphocytes, and what became clear from this observation was that the proliferation rate of NKT cells (considered to be self-dividing proliferation) is influenced by the dendritic cell preparation of the present invention. Therefore, although step (5) in claim 1 of the present application is simply described as an "NKT-stimulating dendritic cell induction step," it can also be said as an "NKT proliferation-stimulating dendritic cell induction step."

[0086] In this embodiment, the time is expressed in days, starting from the time when monocytes collected from the recipient were placed in a culture vessel and culture was initiated. After the preparation was completed, non-adherent cells were added and co-cultured to confirm the effectiveness of the preparation. To verify the optimal pulse timing, blood-collected monocytes were divided into three parts using a manufacturing method based on the manufacturing method of Embodiment 1, and three types of dendritic cell preparation samples were prepared, differing only in the timing of α-galactosylceramide pulse. The monocytes collected from the recipient were pulsed with α-galactosylceramide (concentration 100 ng / mL) three days (72 hours) after being placed in the culture vessel (Condition 1), five days (120 hours) after being placed in the culture vessel (Condition 2), and six days (144 hours) after being pulsed (Condition 3). In this experiment, GM-CSF and IL-4 were added as predetermined factors for differentiating monocytes into immature dendritic cells 24 hours after placing the monocytes collected from the recipient in the culture vessel.

[0087] To confirm the effectiveness of this formulation, the day that monocytes collected from the recipients were placed in the culture vessel was set as day 0, and non-adherent cells were added on day 7 to begin co-culture. The ratio of NKT cell counts to lymphocyte counts was investigated three times: on the day co-culture began (day 0), on day 7, and on day 14.

[0088] Figure 3 shows the results of the α-galactosylceramide pulse timing conditions (conditions 1–3) and the ratio of IFN-γ-producing NKT cells (those contained in non-adherent cells) to the total lymphocyte count at each timing after co-culture. These results were obtained using a flow cytometer. Under all three conditions, the ratio of NKT cells to lymphocytes was highest on day 7 after the start of co-culture. Furthermore, among the three conditions, the NKT cell ratio (%) was highest when pulsed on day 5 after the start of culture. Figure 4a shows the relationship between the maximum NKT cell ratio (%) on day 7 after co-culture and the pulse timing. Figure 4a also shows the approximate curve, extrapolated forward and backward, based on a quadratic fit. The coefficient of determination for this approximate curve is 1, which is considered sufficient for reference. The peak of the curve is between days 4 and 5, with day 0 as the start of culture (4.7 days according to the approximate formula). The preferred range is approximately days 3.5 to 6, which is -10% of the peak value. Because the lifespan of dendritic cells is approximately several days to a week, the process of this production (from monocyte collection or the addition of specific factors to differentiate monocytes into immature dendritic cells to maturation into mature dendritic cells, i.e., the addition of GM-CSF, IL-4, OK-432, and PGE2 to induce mature dendritic cells; note that the subsequent time required for complete maturation is approximately 10 to 20 hours, and this time period may also be included) takes too long. In this experiment, the timing of the α-galactosylceramide pulse differed between Conditions 1 and 3, but GM-CSF, IL-4, OK-432, and PGE2 were added simultaneously), shortening the usable life of the formulation. Therefore, considering that the formulation must be completed within one week from the start of culture, the preferred time is between days 3.9 and 5.5, which is the range where the peak is -5% of the peak. Pulsing on day 5 is the most preferable.

[0089] When α-galactosylceramide is pulsed onto immature dendritic cells, the preparation can be made to exert its maximum effect in the recipient's body by pulsing it on the fourth or fifth day, with the day on which monocytes collected from the recipient are placed in a culture vessel being considered day 0.

[0090] <Embodiment 5: Overview> Mainly claim 5 In this embodiment 5, based on the manufacturing method of the dendritic cell preparation of embodiment 1, the (4) pulsing step is carried out 84 to 132 hours after the mononuclear cells collected from the recipient in the adhesion step (1) are placed in the culture vessel.

[0091] <Embodiment 5: (4) Pulse Step: Pulse of α-Galactosylceramide> In this embodiment 5, the α-galactosylceramide pulse timing is earlier than that in embodiment 1, and α-galactosylceramide is pulsed between 84 and 132 hours after monocytes collected from the recipient are placed in a culture vessel. As in embodiment 1, α-galactosylceramide, model KRN7000 manufactured by Kyowa Kirin Co., Ltd., was used.

[0092] <Embodiment 5: (4) Pulse Process: Optimal Pulse Timing> Dendritic cell preparations produced by the manufacturing method of the present invention are expected to stimulate and activate Vα24-NKT cells, produce IFN-γ, and activate various immune system players. To assess the efficacy of the preparation, non-adherent cells (lymphocytes (T cells, B cells, NK cells, etc.)) removed in the non-adherent cell removal step (2) of the manufacturing method are added to the completed preparation after co-culture, and the proportion of IFN-γ-producing NKT cells is investigated. As in the fourth embodiment, the relationship between the pulse timing and the proportion of NKT cells after co-culture is investigated, and the optimal pulse timing is determined. The pulse timing was determined in this manner. The use of non-adherent cells is intended to avoid placing a physical burden on the recipient, and has no other purpose. Therefore, peripheral blood may be newly collected from the recipient, and the dendritic cell preparation produced by the manufacturing method of the present invention may be added to the collected peripheral blood to similarly compare and confirm the effects. Furthermore, co-culture was performed to observe the difference in the increase in the proportion of NKT cells relative to total lymphocytes, and what became clear from this observation was that the proliferation rate of NKT cells (considered to be self-dividing proliferation) is influenced by the dendritic cell preparation of the present invention. Therefore, although step (5) in claim 1 of the present application is simply described as an "NKT-stimulating dendritic cell induction step," it can also be said as an "NKT proliferation-stimulating dendritic cell induction step."

[0093] In this embodiment, the time is expressed as follows, starting from the time when monocytes collected from the recipient were placed in a culture vessel and culture was initiated. After the preparation was completed, non-adherent cells were added and co-cultured to confirm the effectiveness of the preparation. To verify the optimal pulse timing, a production method based on the production method of Embodiment 1 was used to divide collected monocytes into three parts, and three types of dendritic cell preparation samples were prepared, differing only in the timing of α-galactosylceramide pulse. Each sample was pulsed with α-galactosylceramide (concentration 100 ng / mL) 72 hours (3 days) after the monocytes collected from the recipient were placed in the culture vessel (start of culture) (Condition 1), 120 hours (5 days) after the monocytes were placed in the culture vessel (start of culture). In this experiment, GM-CSF and IL-4 were added as predetermined factors for differentiating monocytes into immature dendritic cells 24 hours after the monocytes were placed in the culture vessel.

[0094] To confirm the effectiveness of this formulation, mononuclear cells collected from recipients were placed in a culture vessel, and then non-adherent cells were added 168 hours later to begin co-culture. The ratio of NKT cells to lymphocytes was investigated three times: on the day co-culture began (day 0), on day 7, and on day 14.

[0095] Figure 3 shows the results of the α-galactosylceramide pulse timing conditions (conditions 1–3) and the ratio of IFN-γ-producing NKT cells (those contained in non-adherent cells) to the total lymphocyte count at each timing after co-culture. This was determined using a flow cytometer. Under all three conditions, the ratio of NKT cells to lymphocytes was highest on day 7 after the start of co-culture. Among the three conditions, this was also determined using a flow cytometer. The NKT cell ratio (%) on day 7 after the start of co-culture was highest when the pulse was administered on day 5 after the start of co-culture. The relationship between the maximum NKT cell ratio (%) on day 7 after the start of co-culture and the pulse timing is shown in the scatter plot in Figure 4b. Figure 4b also shows the approximate curve, extrapolated before and after the quadratic fit. The coefficient of determination for this approximate curve is 1, which is considered sufficient for reference. The peak of the curve is between 96 hours (day 4, assuming day 0 as the start of culture) and 120 hours (day 5, assuming day 0 as the start of culture) (approximately 114 hours). The preferred range is approximately 84 to 143 hours, which is up to -10% of the peak value. Because the lifespan of dendritic cells is approximately several days to one week (168 hours), the production process described here (from monocyte collection or the addition of specific factors to differentiate monocytes into immature dendritic cells to their maturation into mature dendritic cells, i.e., the addition of GM-CSF, IL-4, OK-432, and PGE2 to induce mature dendritic cells) can be extended to approximately 10 to 20 hours before complete maturation. In this experiment, the timing of α-galactosylceramide pulsing differed between Conditions 1 and 3, but GM-CSF, IL-4, OK-432, and PGE2 were added simultaneously. Therefore, pulsing between 84 and 132 hours after the start of culture is preferable to complete the preparation within one week. Pulsing between 94 and 132 hours, which is the range where the peak activity is -5%, is even more preferable.

[0096] When α-galactosylceramide is pulsed into immature dendritic cells, the preparation can be made to exert its maximum effect in the recipient's body by pulsing it between 84 and 132 hours after the monocytes collected from the recipient are placed in a culture vessel.

[0097] <Embodiment 6: Overview> Mainly claim 6 In this embodiment 6, based on the method for producing a dendritic cell preparation of embodiment 1, the (4) pulsing step is performed on the fourth or fifth day, with the day on which non-adherent cells, including monocytes other than cells adhered to the inner surface of the culture vessel, are removed in the (2) non-adherent cell removal step being set as day 0.

[0098] <Embodiment 6: (4) Pulse Step: Pulse of α-Galactosylceramide> In this embodiment 6, the timing of α-galactosylceramide pulsing is earlier than in embodiment 1, and α-galactosylceramide is pulsed between day 4 and day 5, with the day on which non-adherent cells including monocytes other than cells adhered to the inner surface of the culture vessel are removed in the non-adherent cell removal step (2) being set as day 0. As in embodiment 1, α-galactosylceramide used was Kyowa Kirin Co., Ltd., model name KRN7000.

[0099] <Embodiment 6: (4) Pulse Process: Optimal Pulse Timing> Dendritic cell preparations produced by the manufacturing method of the present invention are expected to stimulate and activate Vα24-NKT cells, produce IFN-γ, and activate various immune system players. To assess the efficacy of the preparation, non-adherent cells (lymphocytes (T cells, B cells, NK cells, etc.)) removed in the non-adherent cell removal step (2) of the manufacturing method are added to the completed preparation after co-culture, and the proportion of IFN-γ-producing NKT cells is examined. The relationship between the pulse timing and the proportion of NKT cells after co-culture is examined, and the optimal pulse timing is determined. The pulse timing was determined in this manner. The use of non-adherent cells is intended to avoid placing a physical burden on the recipient, and has no other purpose. Therefore, peripheral blood may be newly collected from the recipient, and the dendritic cell preparation produced by the manufacturing method of the present invention may be added to the collected peripheral blood to similarly compare and confirm efficacy. Furthermore, co-culture was performed to observe the difference in the increase in the proportion of NKT cells relative to total lymphocytes, and what became clear from this observation was that the proliferation rate of NKT cells (considered to be self-dividing proliferation) is influenced by the dendritic cell preparation of the present invention. Therefore, although step (5) in claim 1 of the present application is simply described as an "NKT-stimulating dendritic cell induction step," it can also be said as an "NKT proliferation-stimulating dendritic cell induction step."

[0100] In this embodiment, the time is expressed in days, starting from the time when non-adherent cells, including monocytes other than those adhered to the inner surface of the culture vessel, were removed in the non-adherent cell removal step (2). After the preparation was completed, non-adherent cells were added and co-cultured to confirm the effectiveness of the preparation. To verify the optimal pulse timing, blood collected monocytes were divided into three parts using a production method based on the production method of Embodiment 1, and three types of dendritic cell preparation samples were prepared, differing only in the timing of α-galactosylceramide pulse. Each sample was pulsed with α-galactosylceramide (concentration 100 ng / mL) three days (72 hours) after the removal of non-adherent cells, including monocytes other than those adhered to the inner surface of the culture vessel, in the non-adherent cell removal step (2). (Condition 1) was pulsed three days (120 hours) after the removal of non-adherent cells, including monocytes other than those adhered to the inner surface of the culture vessel, (Condition 2) was pulsed, and (Condition 3) was pulsed six days (144 hours). In this experiment, 24 hours after the removal of non-adherent cells, including monocytes other than those adhered to the inner surface of the culture vessel, in the non-adherent cell removal step (2), GM-CSF and IL-4 were added as specific factors for differentiating monocytes into immature dendritic cells.

[0101] To confirm the effectiveness of this formulation, the day when non-adherent cells, including monocytes other than those adhered to the inner surface of the culture vessel, were removed in the non-adherent cell removal step (2) was set as day 0, and non-adherent cells were added on day 7 to begin co-culture. The ratio of NKT cell counts to lymphocyte counts was investigated three times: on the day co-culture began (day 0), on day 7, and on day 14.

[0102] Figure 3 shows the results of the α-galactosylceramide pulse timing conditions (conditions 1–3) and the ratio of IFN-γ-producing NKT cells (those contained in non-adherent cells) to the total lymphocyte count at each timing after co-culture. The condition "from the start of culture" in the right column of Figure 3 can be interpreted as "after the removal of non-adherent cells, including monocytes, other than those adhered to the inner surface of the culture vessel in the non-adherent cell removal step (2)." Figure 3 shows the results of a flow cytometer study. Under all three conditions, the ratio of NKT cells to lymphocytes was highest on day 7 after the start of co-culture. Furthermore, among the three conditions, the NKT cell ratio (%) was highest when pulsed on day 5 after removal of non-adherent cells. The relationship between the maximum NKT cell ratio (%) on day 7 after co-culture and the pulse timing is shown in Figure 4a as a scatter plot. Figure 4a also shows the approximate curve, extrapolated both before and after the quadratic fit. The coefficient of determination for this approximate curve is 1, which is considered sufficient for reference. The peak of the curve is between days 4 and 5, with the day of nonadherent cell removal being considered day 0 (4.7 days according to the approximation formula). The preferred range is approximately days 3.5 to 6, which corresponds to a decrease of 10% of the peak value. Because the lifespan of dendritic cells is approximately a few days to a week, the process of production using this method (from monocyte collection or the addition of specific factors to differentiate monocytes into immature dendritic cells to their maturation into mature dendritic cells, i.e., the addition of GM-CSF, IL-4, OK-432, and PGE2 to induce mature dendritic cells) can be extended (note that the time required for complete maturation is approximately 10 to 20 hours, so this time period may also be included). In this experiment, the timing of the α-galactosylceramide pulse differed between conditions 1 and 3, but the timing of the addition of GM-CSF, IL-4, OK-432, and PGE2 was simultaneous for all dendritic cells) shortened the usable life of the formulation. Therefore, taking into consideration the need to complete the preparation within one week after removal of non-adherent cells, the pulse is preferably performed between days 3.9 and 5.5, which is also the range where the peak is -5%. Most preferably, the pulse is performed on day 5.

[0103] When α-galactosylceramide is pulsed onto immature dendritic cells, the preparation can be made to exert its maximum effect in the recipient's body by pulsing it on the fourth or fifth day, which is the day on which non-adherent cells, including monocytes other than those adhered to the inner surface of the culture vessel, are removed in the non-adherent cell removal step (2).

[0104] <Embodiment 7: Overview> Mainly claim 7 In this embodiment 7, based on the method for producing a dendritic cell preparation of embodiment 1, the (4) pulsing step is carried out between 84 and 132 hours after the removal of non-adherent cells, including monocytes, other than cells adhered to the inner surface of the culture vessel in the (2) non-adherent cell removal step.

[0105] <Embodiment 7: (4) Pulse Step: Pulse of α-Galactosylceramide> In this embodiment 7, the α-galactosylceramide pulse timing is earlier than in embodiment 1, and α-galactosylceramide is pulsed between 84 and 132 hours after the removal of non-adherent cells, including monocytes, other than cells adhered to the inner surface of the culture vessel in the non-adherent cell removal step (2). α-galactosylceramide used was model KRN7000 manufactured by Kyowa Kirin Co., Ltd., as in embodiment 1.

[0106] <Embodiment 7: (4) Pulse Process: Optimal Pulse Timing> Dendritic cell preparations produced by the manufacturing method of the present invention are expected to stimulate and activate Vα24-NKT cells, produce IFN-γ, and activate various immune system players. To assess the efficacy of the preparation, non-adherent cells (lymphocytes (T cells, B cells, NK cells, etc.)) removed in the non-adherent cell removal step (2) of the manufacturing method are added to the completed preparation after co-culture, and the proportion of IFN-γ-producing NKT cells is investigated. As in the sixth embodiment, the relationship between the pulse timing and the proportion of NKT cells after co-culture is investigated, and the optimal pulse timing is determined. The pulse timing was determined in this manner. The use of non-adherent cells is intended to avoid placing a physical burden on the recipient, and has no other purpose. Therefore, peripheral blood may be newly collected from the recipient, and the dendritic cell preparation produced by the manufacturing method of the present invention may be added to the collected peripheral blood to similarly compare and confirm the effects. Furthermore, co-culture was performed to observe the difference in the increase in the proportion of NKT cells relative to total lymphocytes, and what became clear from this observation was that the proliferation rate of NKT cells (considered to be self-dividing proliferation) is influenced by the dendritic cell preparation of the present invention. Therefore, although step (5) in claim 1 of the present application is simply described as an "NKT-stimulating dendritic cell induction step," it can also be said as an "NKT proliferation-stimulating dendritic cell induction step."

[0107] In this embodiment, the time is expressed as follows, starting from the time when non-adherent cells, including monocytes, other than those adhered to the inner surface of the culture vessel were removed in the non-adherent cell removal step (2). After the preparation was completed, non-adherent cells were added and co-cultured to confirm the effectiveness of the preparation. To verify the optimal pulse timing, blood collected monocytes were divided into three using a production method based on the production method of Embodiment 1, and three types of dendritic cell preparation samples were prepared, differing only in the timing of α-galactosylceramide pulse. Each sample was pulsed with α-galactosylceramide (concentration 100 ng / mL) 72 hours (3 days) after removal of non-adherent cells (Condition 1), 120 hours (5 days) after removal of non-adherent cells (Condition 2), and 144 hours (6 days) after removal of non-adherent cells (Condition 3). In this experiment, GM-CSF and IL-4 were added 24 hours after removal of non-adherent cells as specific factors for differentiating monocytes into immature dendritic cells.

[0108] To confirm the effectiveness of this formulation, non-adherent cells were removed and then added to the cells 168 hours later to initiate co-culture. The ratio of NKT cells to lymphocytes was investigated three times: on the day co-culture began (day 0), on day 7, and on day 14.

[0109] Figure 3 shows the results of the α-galactosylceramide pulse timing conditions (conditions 1–3) and the ratio of IFN-γ-producing NKT cells (those contained in non-adherent cells) to the total lymphocyte count at each timing after co-culture. The condition "from the start of culture" in the right column of Figure 3 can be interpreted as "after the removal of non-adherent cells, including monocytes, other than those adhered to the inner surface of the culture vessel in the non-adherent cell removal step (2)." Figure 3 shows the results of a flow cytometry study. Under all three conditions, the ratio of NKT cells to lymphocytes was highest on day 7 after the start of co-culture. Among the three conditions, this was also measured using a flow cytometer. The NKT cell ratio (%) on day 7 after the start of co-culture was highest when the pulse was performed on day 5 after the removal of non-adherent cells. The relationship between the maximum NKT cell ratio (%) on day 7 after the start of co-culture and the pulse timing is shown in Figure 4b, a scatter plot. Figure 4b also shows the approximate curves obtained by quadratic approximation, extrapolated both before and after the curve. The coefficient of determination for this fitted curve is 1, which is considered to be sufficient for reference. The peak of the curve is between 96 hours (day 4, assuming that the day of removal of non-adherent cells is day 0) and 120 hours (day 5, assuming that the day of removal of non-adherent cells is day 0) (approximately 114 hours). The preferred range is approximately 84 to 143 hours, which is up to -10% of the peak value. Because the lifespan of dendritic cells is approximately several days to one week (168 hours), the process of this method (from monocyte collection or the addition of specific factors to differentiate monocytes into immature dendritic cells to their maturation into mature dendritic cells, i.e., the addition of GM-CSF, IL-4, OK-432, and PGE2 to induce mature dendritic cells (note that the time required for complete maturation after this is approximately 10 to 20 hours, so this time period may also be included)) requires a long time. In this experiment, the timing of α-galactosylceramide pulsing differed between Conditions 1 and 3, but GM-CSF, IL-4, OK-432, and PGE2 were added simultaneously), shortening the usable lifespan of the formulation. Therefore, to complete the formulation within one week after the start of culture, it is preferable to pulse between 84 and 132 hours.More preferably, the pulse should be performed between 94 and 132 hours after the start of the test, which is within the range of -5% of the peak.

[0110] When α-galactosylceramide is pulsed into premature dendritic cells, the resulting formulation can exert its maximum effect in the recipient's body by pulsing it between 84 and 132 hours after the removal of non-adherent cells.

[0111] <Embodiment 8: Overview> Mainly claim 8 The concentration of α-galactosylceramide pulsed into immature dendritic cells to induce NKT-stimulatory dendritic cells, which are dendritic cells that stimulate Vα24-NKT cells, is 300 ng / mL to 1000 ng / mL. Hereinafter, in this embodiment, the timing of α-galactosylceramide pulse is described, with the day on which monocytes collected from the recipient are placed in a culture vessel as day 0. In this embodiment, the timing of α-galactosylceramide pulse is 120 hours after the start of culture, based on the fifth embodiment. The pulse timing may be between 84 and 132 hours after the start of culture, or, as described in the fourth embodiment, the pulse may be 4 or 5 days after the start of culture, with the day on which monocytes are placed in a culture vessel and the start of culture as day 0. Alternatively, as described in the second and third embodiments, the pulse may be 3 or 4 days after the addition of a predetermined factor in the differentiation step, with day 0 being the day on which the factor is added, or it may be 60 to 108 hours after the addition of the predetermined factor. Alternatively, as in the sixth and seventh embodiments, the pulse may be performed on the fourth or fifth day, with the day on which the non-adherent cells were removed from the culture vessel being day 0, or between 84 and 132 hours after the non-adherent cells were removed.

[0112] <Embodiment 8: (4) Pulse Process: Optimal Pulse Amount> In the present embodiment 8, the cells are produced in accordance with the production method of the above-mentioned embodiment 5, but differ from embodiment 5 in that the concentration of α-galactosylceramide to be pulsed is 300 ng / mL to 1000 ng / mL. In the present embodiment 8, α-galactosylceramide was pulsed 120 hours after the start of culture, which is the timing when monocytes collected from the recipient were placed in the culture vessel.

[0113] Dendritic cell preparations produced by the manufacturing method of the present invention are expected to activate various immune system players by stimulating and activating Vα24-NKT cells to produce IFN-γ. To assess the efficacy of the preparation, non-adherent cells (lymphocytes (T cells, B cells, NK cells, NKT cells, etc.)) removed in the non-adherent cell removal step (2) of the manufacturing method and cultured and stored separately are added to the preparation after completion of the preparation, followed by co-culture. The relationship between the concentration of α-galactosylceramide used for pulsing and the amount (concentration) of IFN-γ produced by NKT cells in the cultured non-adherent cells after co-culture is investigated, and the effectiveness is confirmed. The range of the pulse dose described above was determined in this manner.

[0114] In this embodiment, the time is expressed in hours starting from the time when monocytes collected from the recipient were placed in a culture vessel and culture was initiated. After the preparation was completed, non-adherent cells were added and co-cultured to confirm the effectiveness of the preparation. To verify the optimal pulse concentration, collected monocytes were divided into four groups, and four samples of dendritic cell preparations with different α-galactosylceramide pulse timing were prepared. 120 hours after the time when monocytes collected from the recipient were placed in a culture vessel and culture was initiated, α-galactosylceramide was pulsed at the following concentrations: Condition 1: 0 ng / mL, Condition 2: 100 ng / mL, Condition 3: 300 ng / mL, and Condition 4: 1000 ng / mL. To confirm the effectiveness of this formulation (evaluation of IFN-γ production levels), non-adherent cells were added 168 hours after the start of culture, and co-culture was initiated. The amount of IFN-γ produced in samples under each condition was investigated on day 0 (the day co-culture began), 2 days, 4 days, 7 days, and 14 days, which was designated as day 0.

[0115] IFN-γ was measured by ELISA using BIOLEGEND's ELISA MAX Deluxe Set Human IFN-γ.

[0116] Figure 5 shows the relationship between the concentration of α-galactosylceramide pulsed onto monocytes collected from recipients 120 hours after the start of culture in a culture vessel and the IFN-γ concentration measured on days 0 (the day of co-culture), 2, 4, 7, and 14 after the start of co-culture with non-adherent cells. The horizontal axis represents the number of days of co-culture, and the vertical axis represents the IFN-γ concentration (pg / mL). As mentioned above, in conditions 1 to 4, the pulsed α-galactosylceramide concentration was varied between 0 and 1000 ng / mL. As shown in Figure 5, under conditions 1 (0 ng / mL) and 2 (100 ng / mL), the IFN-γ production concentration peaked on day 7 after the start of co-culture. Under conditions 3 (300 ng / mL) and 4 (1000 ng / mL), the production concentration peaked on day 4, with the peak value being equivalent to that under condition 2 (100 ng / mL). Conditions 3 (300 ng / mL) and 4 (1000 ng / mL) resulted in a peak production concentration three days earlier than the concentration condition (100 ng / mL) of embodiment 1 disclosed in the prior art (Patent Document 1, the same applies hereinafter), demonstrating earlier efficacy. Thus, pulsing α-galactosylceramide concentrations below 300 ng / mL delayed the peak IFN-γ production, while pulsing concentrations above 1000 ng / mL resulted in the same results as pulsing 1000 ng / mL, resulting in wasted α-galactosylceramide. Therefore, the optimal α-galactosylceramide concentration was found to be between 300 ng / mL and 1000 ng / mL. Furthermore, in a comparative experiment with the prior art (Patent Document 1) described below, when the same concentration of α-galactosylceramide was pulsed, the present invention, which pulses immature dendritic cells, achieved approximately four times the IFN-γ production concentration compared to the prior art (Patent Document 1), which pulses mature dendritic cells. This allows for the production of more IFN-γ than the prior art, which is expected to further activate the immune system and exert a greater anti-cancer effect.

[0117] The present invention is expected to accelerate the peak production of IFN-γ in the recipient's body while maintaining the same peak value as under other conditions, and to exert an anticancer effect more quickly. Furthermore, a higher IFN-γ production concentration can be obtained than in the prior art, and a greater anticancer effect can be expected.

[0118] <Embodiment 9: Overview> Mainly claims 9 and 10 The blood is that of a recipient to which has been added any one of the dendritic cell preparations produced by the production methods shown in Embodiments 1 to 8.

[0119] As explained in the first embodiment, adding the present preparation to the recipient's blood stimulates NKT cells to activate the innate immune system (improving immunity), and subsequently stimulates and activates the adaptive immune system, thereby improving immunity in the recipient's blood.

[0120] The recipient's blood may be extracted from the body and processed by adding this product. By returning the processed blood (including this product) to the beneficiary, it is expected that the beneficiary's immune system will be improved. Alternatively, the product may be returned to the beneficiary's body and added to the blood in the body to improve the immune system. In addition, the beneficiary's blood may be replaced with beneficiary's quasi-blood, which is the beneficiary's blood plus blood that can be transfused to the beneficiary.

[0121] <Results of application to the human body> A dendritic cell preparation manufactured under the conditions of embodiment 4 of the present invention (culture was initiated on the day of blood collection, non-adherent cells were removed, GM-CSF and IL-4 were added 24 hours after the start of culture, and α-galactosylceramide was pulsed at 300 ng / mL on the fifth day) was administered to two beneficiaries. The effectiveness of the dendritic cell preparation of the present invention in the body has been described above. It activates NKT cells and activates various other immune-controlling cells through the production of IFN-γ. As it has a particularly large effect on NK cells, the NK activity value and IFN-γ amount after effective administration were examined. The results are shown in Figure 6. The preparation was administered to two beneficiaries, one by subcutaneous injection (top row of the table) and the other by intravenous drip (bottom row of the table). The NK activity value was higher because NK cells were activated. These are values ​​that indicate the level of NK activity. NK activity increased from the third day after administration onwards, and on the seventh day it was still higher than before administration. The lower limit of IFN-γ measurement is 0.1, and normally it is below 0.1. The IFN-γ measurement result was 0.1 on the first day (day 0), but peaked on the second day after administration, with both values ​​increasing significantly. This shows that this dendritic cell preparation achieved its intended goal of activating NKT cells, rapidly increasing the amount of IFN-γ produced, and also activating NK cells.

[0122] <Comparison with prior art: Overview> The prior art does not describe the amount of IFN-γ produced (production concentration). Therefore, the inventors of the present invention prepared a prototype cell composition following the method for producing a cell composition (corresponding to the dendritic cell preparation of the present application) described in prior art Patent Document 1, and conducted an experiment to compare the IFN-γ production concentration after co-culture with non-adherent cells including NKT cells.

[0123] <Comparison with prior art: Preparation of comparative examples> To replicate the prior art, mononuclear cells from a blood component were placed in liquid medium in a culture vessel for two hours, and non-adherent cells (lymphocytes (T cells, B cells, NK cells, NKT cells, etc.)) were separated and cryopreserved. The day when the culture of adherent cells began after this placement was designated as day 0. The following day (day 1), GM-CSF and IL-4 were added to the culture medium for the adherent cells, causing them to differentiate into immature dendritic cells. On day 5, GM-CSF, IL-4, PGE-2, and OK-432 were added to the immature dendritic cells, causing them to mature. On the following day, day 6, the mature dendritic cells were pulsed with α-galactosylceramide (concentration 300 ng / mL).

[0124] <Comparison with prior art: Method for confirming the effect of comparative examples> On day 7, co-culture of the mature dendritic cells with the non-adherent cells collected on day 1 was initiated. The concentration of IFN-γ (pg / mL) produced by NKT cells in the non-adherent cells was measured on the day the co-culture was initiated (day 0), and on days 2, 4, 7, and 14, with the day the co-culture was initiated designated as day 0.

[0125] <Comparison with Prior Art: Comparison of Effects between Comparative Examples and the Present Embodiment> As described above, the IFN-γ production concentration after co-culture of dendritic cells and non-adherent cells, which were prepared as a comparative example according to the prior art (in accordance with the manufacturing method described in Patent Document 1, except that the pulsed α-galactosylceramide was used at a concentration of 300 ng / mL), was compared with the IFN-γ production concentration after co-culture of a dendritic cell preparation prepared under Condition 3 of Embodiment 8 of the present application (pulsed α-galactosylceramide at a concentration of 300 ng / mL) and non-adherent cells. To confirm the effects of the comparative example and the preparations prepared under Condition 3 of Embodiment 8 of the present application, dendritic cells and non-adherent cells containing NKT cells derived from the same recipient were used, with the difference in cell number kept within a range that would not result in a significant difference.

[0126] <Comparison with Prior Art: Comparison of Effects between Comparative Examples and the Present Embodiment: IFN-γ Production Concentration> Figure 7 shows the results of comparing IFN-γ production concentrations. The horizontal axis shows the number of days since the start of co-culture: 0 days (first day), 2 days, 4 days, 7 days, and 14 days, and the vertical axis shows the IFN-γ production concentration (pg / mL). In both the comparative example (prior art) and condition 3 of embodiment 8 of the present application, the IFN-γ production concentration was high on day 4. In the prior art, the production concentration was slightly higher on day 7 than on day 4. Condition 3 of embodiment 8 of the present application resulted in an IFN-γ production concentration that was approximately four times higher than that of the prior art.

[0127] <Comparison with Prior Art: Comparison of Effects Between Comparative Examples and the Present Embodiment: IFN-γ Production Peak> Prior art Patent Document 1 describes the most preferred α-galactosylceramide concentration range as 80 to 120 ng / mL, and in Embodiment 1 of Patent Document 1, an experiment was conducted using 100 ng / mL. Patent Document 1 does not verify the effectiveness of IFN-γ production. In the inventor's experiments, under Condition 2 of Embodiment 8 of the present application, in which an α-galactosylceramide concentration (100 ng / mL) similar to that of Embodiment 1 of Patent Document 1 is pulsed, IFN-γ production peaks on day 7 after co-culture. Therefore, although not described in Patent Document 1, the inventor discovered that by optimizing not only the α-galactosylceramide pulse timing but also the pulse concentration, it is possible to narrow down the range in which the effect is achieved earlier than in the prior art.

[0128] <Comparison with the prior art: Superior effects of the present invention> Compared to the comparative example (prior art), the present invention narrowed the range in which IFN-γ can be produced quickly and significantly improved the concentration of IFN-γ produced by pulsing immature dendritic cells with α-galactosylceramide and optimizing the timing and concentration of the pulse.

[0129] <Proposed claim describing a treatment method for US application>: (tentative) A treatment for a beneficiary who wishes to receive one or more of cancer treatment, cancer prevention, and immune enhancement using a dendritic cell preparation that stimulates NKT cells produced by the production method of the present invention can be described as follows. A method of treatment with a formulation manufactured by (1) a collection step of collecting monocytes or peripheral blood containing monocytes from a beneficiary by apheresis or blood collection; (2) an adhesion step in which the collected monocytes are placed in a culture vessel using a liquid medium and allowed to stand to allow some of the monocytes to adhere to the inner surface of the vessel; (3) a non-adherent cell removal step of removing non-adherent cells, including monocytes, other than cells adhered to the inner surface of the culture vessel; (4) a differentiation step in which a predetermined factor is added to the culture vessel to differentiate the monocytes adhered to the inner surface of the culture vessel into immature dendritic cells; (5) a pulsing step of pulsing α-galactosylceramide into a culture vessel in which the immature dendritic cells exist in a non-adherent state; (6) an NKT-stimulatory dendritic cell induction step of inducing NKT-stimulatory dendritic cells, which are dendritic cells that stimulate NKT cells, from immature dendritic cells; and producing a dendritic cell preparation comprising dendritic cells that stimulate natural killer T (NKT) cells for the purposes of cancer treatment, cancer prevention, and / or immune enhancement, by a production method comprising the steps of: (7) an infusion step of the dendritic cell preparation back into the recipient from whom the monocytes were collected; A therapeutic method using a dendritic cell preparation comprising dendritic cells that stimulate natural killer T (NKT) cells.

[0130] (tentative) <Proposed limitations on the proposed claims> (tentative) The above-mentioned treatment method may be limited to one or more of the following conditions. (a) A treatment method in which, in the pulse step, α-galactosylceramide is pulsed 84 to 132 hours after the monocytes are placed in a culture vessel using a liquid medium in the adhesion step (4 or 5 days after the monocytes are placed, with day 0 being the day they are placed). (b) A treatment method in which the concentration of α-galactosylceramide to be pulsed in the pulse step is 300 to 1000 ng / mL. (c) In the return step, the preparation is returned to the recipient's body 156 hours or more after the monocytes are placed in the culture vessel using the liquid medium in the adhesion step (7 days or more, assuming that the time when the monocytes are placed is day 0). This is a treatment method.

[0131] <Another embodiment: Cocktail preparation> The effect of a cocktail preparation of another embodiment when administered back into the body of a recipient is described below. In this example, the recipient has cancer cells in their body. In this embodiment, collected monocytes are divided and placed in a culture vessel, separate from the preparation of the present invention. A preparation in which WT1 peptide is used as a ligand instead of α-galactosylceramide, which is pulsed as a ligand to immature dendritic cells in the present invention, is prepared in a separate vessel in parallel, and is configured to be present in the body of the recipient simultaneously with the preparation of the present invention.

[0132] Preparations using α-galactosylceramide as a ligand activate the recipient's innate immune system, improving immunity and indirectly activating the adaptive immune system. Preparations using the WT1 peptide, which is present in various cancers, as a ligand directly activate the adaptive immune system, targeting cancer cells.

[0133] <Effects of cocktail preparations> This alternative embodiment utilizes two types of formulations: one manufactured by the manufacturing method of the present invention, and the other manufactured by a similar method, but pulsing immature dendritic cells with WT-1 peptide instead of α-galactosylceramide. Nonspecific immunotherapy (preparation (ligand: α-galactosylceramide)) has already been explained, so the following mainly describes the effects of a formulation with WT1 peptide as a ligand, which directly targets the adaptive immune system. Cancer cells within the recipient's body form a barrier around them from immune function (due to immunosuppressive effects).

[0134] <Effects of α-galactosylceramide-pulsed preparations: NKT cell activation> Preparations pulsed with α-galactosylceramide activate NKT cells, which exert various effects on immune function.

[0135] <Action of activated NKT cells: Adjuvant effect> Activated NKT cells produce cytokines (IFN-γ), which activate NK cells, macrophages, and killer T cells, enhancing their ability to attack cancer. They also activate various immune cells. Killer T cells and helper T cells are activated by pulsing dendritic cells with the WT1 peptide, which exhibits specific immune activity, as a ligand. These activated NK cells and activated B cells are simultaneously favorably influenced by the pathway of nonspecific immune activity, resulting in a synergistic effect that is greater than the simple sum of the two. This is one of the features of this embodiment.

[0136] <The action of activated NKT cells: Induction of apoptosis in cancer cells> Activated NKT cells produce a type of serine protease that breaks down the DNA of cancer cells, causing apoptosis of the cancer cells.

[0137] <Action of activated macrophages: Cancer cell phagocytosis by macrophages> Activated macrophages phagocytose cancer cells and dead cancer cells.

[0138] <Actions by proliferating and activated macrophages: Antigen presentation by macrophages> Activated NKT cells cause the proliferation and activation of macrophages. The proliferated and activated macrophages fragment the cancer cells they have ingested through phagocytosis and display them on the cell surface. In other words, they perform an antigen-presenting function, presenting the antigens of the cancer cells. The macrophages then present these antigens to helper T cells.

[0139] <Cancer antigen presentation by dendritic cells: Cancer antigen presentation by dendritic cells> Immature dendritic cells take up cancer antigens, mature, and differentiate into mature dendritic cells, presenting cancer antigens to helper T cells and killer T cells. This effect is an anti-cancer effect that can normally occur in the body, but due to the action of activated NKT cells, a higher anti-cancer effect can be obtained than usual. Since this cancer antigen is the antigen presented after dendritic cells and macrophages phagocytize the patient's own cancer cells, a higher effect can be expected compared to preparations using artificial antigens as ligands. This effect is also one of the factors contributing to the synergistic effect of the cocktail preparation of the present invention.

[0140] <Action of the preparation pulsed with WT1 peptide: Cancer antigen presentation effect> The preparation pulsed with WT1 peptide presents and activates the WT1 peptide, which is a cancer antigen, to helper T cells and killer T cells. As a result, killer T cells recognize cancer cells as targets for attack, helper T cells activate killer T cells, and further activate NK cells as described later by producing cytokines, and promote B cells to produce antibodies.

[0141] <Action of activated helper T cells: Instruction for attacking cancer cells> Activated helper T cells produce cytokines (INF-γ) and instruct killer T cells to attack cancer cells based on the information contained in those cytokines. Also, the effect of cytokines activates NK cells, B cells, and macrophages, enhancing the attack on cancer.

[0142] <Action of activated helper T cells: Instruction for antibody production against cancer antigen (e.g., WT1)> Activated helper T cells produce cytokines and instruct B cells to produce antibodies against cancer antigens (e.g., WT1) based on the information contained in those cytokines.

[0143] <Action of activated killer T cells: Attack on cancer cells by killer T cells> Activated killer T cells recognize and bind to WT1 expressed on cancer cells, creating holes in the cell membrane and causing necrosis of the cancer cells. They also produce substances such as TNF-β (tumor necrosis factor, a type of cytokine) that induce apoptosis in cancer cells. In this way, the direct attack of killer T cells on cancer cells is a powerful anti-cancer effect that attacks cancer cells from both the outside and the inside.

[0144] <Action of activated killer T cells: Destruction of new blood vessels> The WT1 gene is expressed in blood vessels newly formed by cancer cells, and killer T cells presented with the cancer antigen (WT1) attack not only cancer cells but also newly formed blood vessels. This destroys the newly formed blood vessels, reducing the supply of nutrients to cancer cells and suppressing their proliferation. This action is also one of the factors that causes the cocktail preparation of this embodiment to have a synergistic effect.

[0145] <The action of activated NK cells: NK cells attack cancer cells> NK cells are stimulated and activated by IFN-γ produced by helper T cells in addition to IFN-γ produced by activated NKT cells, and then exhibit a cytotoxic response against cancer cells, attacking them.

[0146] <Function of activated B cells: production of cancer antibodies> Activated B cells produce antibodies against cancer antigens (e.g., WT1) and attack cancer cells. These antibodies have the effect of suppressing cancer cell growth by binding to cancer cells and promoting attack by serving as a marker for other immune cells (e.g., NK cells and killer T cells).

[0147] As described above, by simultaneously introducing a formulation with α-galactosylceramide as the ligand and a formulation with WT1 peptide as the ligand into the recipient's body, it is possible to activate the adaptive immune system targeting cancer cells more quickly than by using only the formulation of the present invention (the formulation with α-galactosylceramide as the ligand). By directly activating both the innate and adaptive immune systems, a synergistic effect can be achieved, rather than simply adding them together. Therefore, when used for cancer treatment, this formulation is expected to have a faster and more effective effect than using it alone.

[0148] <Effects> The present invention's method for producing a dendritic cell preparation that stimulates NKT cells, in which monocytes are collected and then returned to the recipient's body, has the following advantages over the prior art (Patent Document 1): By identifying conditions that stimulate NKT cells to produce greater amounts of IFN-γ and narrowing the range of α-galactosylceramide concentrations that result in faster IFN-γ production, the present invention can produce a dendritic cell preparation that can be expected to have greater therapeutic effects through both the innate and adaptive immune systems in cancer treatment and other conditions. Furthermore, the dendritic cell preparation can also be effective in improving the immune system of healthy individuals. Furthermore, by producing the dendritic cell preparation from the recipient's own monocytes, a safe preparation can be obtained without the risk of rejection.

Claims

1. A method for producing a dendritic cell preparation containing human dendritic cells that stimulate natural killer T cells (hereinafter referred to as "NKT cells") for the purpose of one or more of cancer treatment, cancer prevention, and immune enhancement in humans, comprising: (1) an adhesion step of placing human monocytes collected from a human recipient in a culture vessel using a liquid medium and allowing the vessel to stand to allow some of the monocytes to adhere to the inner surface of the vessel; (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 vessel; (3) a differentiation step of adding GM-CSF and IL-4 to the culture vessel to differentiate the human monocytes adhered to the inner surface of the culture vessel into human immature dendritic cells; (4) a pulsing step of pulsing α-galactosylceramide at a concentration of 300 to 1000 ng / mL into a culture vessel in which the human immature dendritic cells predominantly exist; (5) a step of inducing NKT-stimulated dendritic cells, which are human dendritic cells that stimulate NKT cells, from human immature dendritic cells pulsed with α-galactosylceramide by maturation; a method for producing an NKT cell-stimulating dendritic cell preparation for injecting the monocytes back into the body of a human recipient from whom the monocytes were collected to stimulate NKT cells, the method comprising:

2. The method for producing a dendritic cell preparation according to claim 1, wherein the pulsing step (4) is carried out on the third or fourth day, with the day on which GM-CSF and IL-4 are added in the differentiation step (3) being considered as day 0.

3. The method for producing a dendritic cell preparation according to claim 1, wherein the pulsing step (4) is carried out between 60 and 108 hours after the addition of GM-CSF and IL-4 in the differentiation step (3).

4. 2. The method for producing a dendritic cell preparation according to claim 1, wherein the pulsing step (4) is carried out on the fourth or fifth day, with the day on which the monocytes collected from the recipient in the adhesion step (1) are placed in the culture vessel being considered as day 0.

5. The method for producing a dendritic cell preparation according to claim 1, wherein the pulsing step (4) is carried out between 84 and 132 hours after the human monocytes collected from the human recipient in the adhesion step (1) are placed in a culture vessel.

6. 2. The method for producing a dendritic cell preparation according to claim 1, wherein the pulsing step (4) is performed on the fourth or fifth day, with the day on which non-adherent cells, including monocytes, other than cells adhered to the inner surface of the culture vessel are removed in the non-adherent cell removal step (2), being considered as day 0.

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

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