Method for producing cocktail dendritic cell vaccine

Abstract

To provide a cocktail dendritic cell vaccine with high anti-cancer effect by combining specific and non-specific immunotherapies, from a small amount of a blood component collected in a single blood draw from a patient.SOLUTION: A method for producing a patient-specific cocktail dendritic cell vaccine includes: a preparation step of preparing for blood component sampling from a patient; a blood component sampling step of performing blood component sampling to collect, in a single puncture, a predetermined amount of monocytes from a patient ready for blood component sampling; a division step of dividing, into multiple groups, the monocytes collected only from the patient or immature dendritic cells differentiated from the monocytes; a pulsing step of pulsing a different type of ligands into each group divided in the division step, optionally with different timing starting from the time of the division; and a storage step of storing each group into which the ligands are pulsed.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This invention relates to a method for producing a vaccine that activates the immune system and enhances anti-cancer effects, which is produced from the patient's own cells. A "cancer vaccine" is a vaccine that aims to suppress the progression of cancer by enhancing the patient's "immunity against cancer" and further reduce / eliminate remaining cancer cells in the body. Most "cancer vaccines" currently in use are used for the purpose of cancer treatment, not prevention, and are referred to as "immunotherapy against cancer." [Background technology]

[0002] In recent years, cancer immunotherapy has been recognized as a practical treatment method on a par with other surgical therapies, chemotherapy, and radiation therapy. Cancer immunotherapy is a treatment method that enhances the immune system's aggressiveness against cancer by strengthening the body's immune system or by having immune cells recognize cancer as a target for attack. To date, methods such as BRM therapy, cytokine therapy, cellular immunotherapy, and vaccine therapy have been used. In particular, in the 2000s, vaccine therapy using dendritic cells began to be performed and has shown high effectiveness. Here, each immunotherapy will be briefly explained.

[0003] BRM (Biological Response Modifier) ​​therapy, typified by the Maruyama vaccine, is an early treatment method that uses substances to modify the patient's biological response to tumor cells, etc. Known BRMs include PSK, bestatin, and OK-432. While this treatment method has been shown to be effective against some cancers, it is primarily intended as an adjunctive therapy that is effective when used in conjunction with other treatments that weaken the immune system, such as surgery or chemotherapy. Furthermore, it does not necessarily strengthen the immune system, and there are issues with its weak anti-cancer effects when used alone.

[0004] Cytokine therapy is a treatment method that kills cancer cells or virus-infected cells by directly administering cytokines into the body that proliferate or activate lymphocytes such as T cells and NK cells. For example, cytokine therapy using interleukin-2 (IL-2) is one example. However, this therapy has not achieved the expected results in clinical results and has been associated with serious side effects such as organ dysfunction and fluid retention (in the case of IL-2 administration), and cold-like symptoms and mental disorders (in the case of interferon (IFN) administration).

[0005] Cellular immunotherapy is a treatment that enhances the patient's immune system by activating and expanding immune cells collected from the patient outside the body before returning them to the patient. It is also known as "adoptive immunotherapy (broadly defined as adoptive immunotherapy)." In the narrow sense, adoptive immunotherapy is classified into activated lymphocyte therapy and NK cell therapy depending on the type of immune cells processed outside the body.

[0006] Activated lymphocyte therapy is further classified into activated lymphocyte therapy in the narrow sense (activated T lymphocyte therapy), which involves activating and proliferating T cells outside the body, and activated NK cell therapy, which involves activating and proliferating NK cells.

[0007] Currently, vaccine therapy is divided into peptide vaccines and dendritic cell vaccines. Peptide vaccines are a method in which peptides that serve as cancer markers are administered directly into the body as antigens, and immune cells react to these antigens, recognizing cancer as a target for attack, thereby exerting a cancer-suppressing effect. On the other hand, dendritic cell vaccines are administered into the body by carrying antigens that serve as cancer markers on antigen-presenting cells called dendritic cells, activating killer T cells and helper T cells and increasing their aggressiveness against cancer. Current research results show that dendritic cell vaccines, which present antigens using dendritic cells, are more effective than peptide vaccine therapy, which involves directly administering antigens. The method for producing the cocktail dendritic cell vaccine of the present invention involves dividing monocytes of equivalent condition (with little variation in monocyte quality) collected in a single course of component blood collection to produce multiple types of dendritic cell vaccines of equivalent condition.

[0008] Dendritic cell vaccines are made by culturing monocytes taken from the patient's blood into immature dendritic cells, adding (sometimes called pulsing) a ligand (one example is an antigen), and culturing the cells to turn them into mature dendritic cells, resulting in a dendritic cell vaccine that presents a specific antigen. This dendritic cell vaccine therapy can also be divided into what is called specific immunotherapy and what is called non-specific immunotherapy, depending on the antigen used.

[0009] <Specific immunotherapy> Cancer-specific immunotherapy involves producing an appropriate number of dendritic cells ex vivo, using them as a vaccine to target cancer cells with antigens that identify the cancer. These cells then act as a target for killer T cells and helper T cells, enabling them to attack cancer cells. The use of WT1 (double T-one) peptides as a representative ligand has proven effective against a wide range of cancers. This finding is supported by evidence that the WT1 gene is expressed as an antigen in various solid malignant tumors, including leukemia, lung cancer, colon cancer, pancreatic cancer, bone and soft tissue sarcoma, and malignant glioma. Therefore, the use of vaccines with WT1 peptides as ligands for dendritic cells could potentially be used to treat a wide range of cancers.

[0010] <Non-specific immunotherapy> Non-specific immunotherapy is a treatment method that aims to strengthen the overall immune system. BRM therapy and cytokine therapy are also classified as non-specific immunotherapy, but dendritic cell vaccines use α-galactosylceramide, an NKT cell activating ligand, to activate NKT cells, and have been shown to have a high immune activation effect.

[0011] NKT cells, known as the fourth lymphocyte, are a type of T cell that also possesses the characteristics of NK cells. NKT cells are activated by the presentation of glycolipid antigens and produce a variety of cytokines, which induce both immunostimulatory and suppressive responses. Immunostimulation activates the immune system to strengthen defenses against external invaders, while immunosuppression suppresses immune overreactions and inflammation. Maintaining a good balance between these two functions helps maintain a healthy state. NKT cells can also induce cancer cell death and have various anti-cancer effects, but their direct attack on cancer is limited. Therefore, the anti-cancer effects of NKT cells are expected to be secondary effects mediated by other immune cells.

[0012] Six anti-cancer effects of NKT cells are explained below.

[0013] (1) Dendritic cell maturation As dendritic cells mature, they present cancer antigens to immune cells, promoting attacks against cancer. However, immunosuppressive cells and substances produced by cancer prevent dendritic cells from maturing, reducing their cancer-suppressing effect. By maturing dendritic cells through the effects of cytokines produced by NKT cells, immune function against cancer is enhanced.

[0014] (2) Adjuvant effect It releases a substance called cytokine (IFN-γ), which activates and proliferates various immune cells. It has an inhibitory effect on cancer by activating killer T cells, NK cells, and macrophages.

[0015] (3) Direct killing of cancer cells Activated NKT cells have the ability to induce cell death by apoptosis in cancer cells.

[0016] (4) Checkpoint inhibitory effect Immune cells have receptors called immune checkpoints that suppress immune overreactions. Cancer cells exploit this mechanism to produce immunosuppressive cells (cells with immune checkpoint inhibitory functions that produce immune checkpoint molecules or their ligands) to avoid attack by immune cells, but these immunosuppressive cells can be killed, relieving immunosuppression. This promotes the attack of cancer cells by killer T cells, inducing anti-cancer effects.

[0017] (5) Inhibition of angiogenesis It inhibits the formation of new blood vessels that supply cancer tissue with nutrients and oxygen for growth. This prevents cancer cells from obtaining nutrients, which has the effect of suppressing the growth of cancer cells.

[0018] (6) Long-term immunological memory NKT cells can retain the ability to attack cancer cells for a long period of time by differentiating into memory immune-like NKT cells with immune memory function through the induction of CD1d-expressing cells.

[0019] As mentioned above, immunotherapy targeting NKT cells is a relatively new cancer immunotherapy method that is expected to strengthen the immune system overall.

[0020] Thus, various immunotherapies for cancer have been developed and improved, and there are also many methods for producing vaccines. For example, Patent Document 1 describes a high-quality dendritic cell vaccine that contains dendritic cells and zinc at a concentration of 200 ng / mL to 400 ng / mL, and increases the cell viability of the dendritic cells by 10% compared to when the cells are washed with physiological saline. Furthermore, these dendritic cells are matured by adding cancer antigens, including WT1 peptides, and the dendritic cell vaccine, which is a specific immunotherapy, has enhanced anti-cancer effects.

[0021] Furthermore, in Patent Document 2, a dendritic cell vaccine is produced by adding an NKT cell-activating ligand containing α-galactosylceramide to dendritic cells and maturing them. This is a method for producing a dendritic cell vaccine that corresponds to non-specific immunotherapy, activating a wide range of immune cells. [Prior art documents] [Patent documents]

[0022] [Patent Document 1] Patent Publication No. 2018-83849 [Patent Document 2] Patent No. 6447947 Summary of the Invention [Problem to be solved by the invention]

[0023] However, clinical research is underway on both the dendritic cell vaccines disclosed in Patent Document 1 and Patent Document 2, and although their effectiveness has been confirmed as being higher than that of conventional immunotherapy, they do not necessarily achieve the expected effect in all cancer patients. This is because dendritic cell vaccines can be used to treat a variety of cancers and diseases depending on the antigen used. However, it can be said that sufficient effectiveness will not be achieved unless the vaccine contains an antigen that is appropriate for the patient's symptoms.

[0024] For example, when specific immunotherapy is performed using the dendritic cell vaccine described in Patent Document 1, even if the ability of lymphocytes to recognize cancer is increased, the therapeutic effect may not be fully achieved because the patient's overall immune strength is weakened, or the patient may die from complications caused by other diseases.

[0025] Furthermore, when non-specific immunotherapy is performed using the dendritic cell vaccine of Patent Document 2, the patient's immune function is improved, but this does not necessarily mean that an anti-cancer effect is achieved. This is because if the patient's immune system does not recognize cancer as a target to attack, a sufficient anti-cancer effect cannot be achieved, and as a result, the therapeutic effect on cancer may not be achieved.

[0026] In other words, it is important that two actions occur simultaneously: activating and proliferating the various immune cells that are responsible for the body's immune system, and then allowing the specific immune system to clearly recognize the cancer cells that are the target of attack. Furthermore, reducing the burden on cancer patients is also important for ultimately activating the immune system, yet conventional cancer therapies often impose a heavy physical burden on patients. It is important to note that cancer patients who use immunotherapy often have advanced stages of cancer and are often limited in time and physical strength. To generate dendritic cell vaccines, the patient's own monocytes must be collected and differentiated, necessitating apheresis. However, apheresis involves first collecting blood from the patient's body, separating monocytes using apheresis equipment, and then returning them to the patient. This process takes a long time, and mixing anticoagulants into the blood can cause side effects. For this reason, it is desirable to maximize the effectiveness of the blood collected in a single blood collection session and ensure sufficient recovery to allow for subsequent treatment.

[0027] In light of this situation, the present invention aims to provide a cocktail dendritic cell vaccine that has a synergistic therapeutic effect on various cancer patients and offers hope of recovery by activating and proliferating (this is an important point) various immune cells that are responsible for the body's immune system through non-specific immunotherapy, and then allowing the specific immune system to clearly recognize the cancer cells that are the target of attack through specific immunotherapy. [Means for solving the problem]

[0028] The present invention is a method for producing a cocktail dendritic cell vaccine that activates a patient's immune system. The first invention relates to a method for producing a cocktail dendritic cell vaccine specifically for a patient, comprising: a preparation step for preparing to collect blood components from the patient; a blood component collection step for collecting a predetermined amount of monocytes from the patient who is ready for blood component collection by a single puncture (meaning that the collection of a predetermined amount of blood is completed in one course; multiple punctures are not prohibited); a division step for dividing monocytes collected exclusively from the patient or immature dendritic cells differentiated from monocytes into multiple groups; a pulsing step for pulsing each group divided in the division step with a different type of ligand, optionally at different times starting from the time of division; and a storage step for storing each group pulsed with the ligand.

[0029] Furthermore, the cocktail dendritic cell vaccine of the second invention, in addition to the features of the first invention, includes an immunity / disease information acquisition step prior to the preparation step, in which one or more of immune information, which is information relating to the patient's immune strength, the patient's personal biological information, and the patient's disease information is acquired, and a ligand selection step in which the ligand to be pulsed in the pulsing step is selected according to the acquired immunity / disease information, thereby producing a cocktail dendritic cell vaccine dedicated to the patient.

[0030] Furthermore, the cocktail dendritic cell vaccine of the third invention, in addition to the second invention, further comprises a division ratio determination step in which the division ratio in the division step is determined based on the acquired immune strength and disease information, thereby producing a cocktail dendritic cell vaccine.

[0031] In addition, the cocktail dendritic cell vaccine of the fourth invention is produced by adding to the second or third invention a storage period determination step in which the storage period for each group in the storage step is determined based on the acquired immune status and disease information.

[0032] Furthermore, the cocktail dendritic cell vaccine of the fifth invention, in addition to any one of the first to fourth inventions, further comprises an observation result obtaining step of observing each group pulsed with the ligand after the pulsing step and obtaining observation results, thereby producing a cocktail dendritic cell vaccine.

[0033] Furthermore, the cocktail dendritic cell vaccine of the sixth invention is a cocktail dendritic cell vaccine produced by, in addition to any one of the first to fifth inventions, determining the storage period for each group in the storage step based on the observation results obtained in the observation result obtaining step.

[0034] Furthermore, the cocktail dendritic cell vaccine of the seventh invention is a method for producing a cocktail dendritic cell vaccine, which, in addition to any one of the first to sixth inventions, includes a freezing / refrigerating storage step in which at least one of each group divided between the division step and the pulsing step is frozen or refrigerated.

[0035] Furthermore, the cocktail dendritic cell vaccine of the eighth invention, in addition to any one of the first to seventh inventions, further comprises a ligand generation step of generating a ligand based on the body tissue of an individual patient, and the pulsing step is configured to pulse the ligand generated in the ligand generation step, thereby producing a cocktail dendritic cell vaccine.

[0036] Furthermore, the cocktail dendritic cell vaccine of the ninth invention, in addition to any one of the first to eighth inventions, includes a filling step of filling each ligand-pulsed group that has been stored after a storage period into an injection device (including but not limited to a syringe; the same applies below) for introducing each group into the patient's body on a group-by-group basis, thereby producing a cocktail dendritic cell vaccine.

[0037] Furthermore, the cocktail dendritic cell vaccine of the tenth invention is a cocktail dendritic cell vaccine produced according to any one of the first to ninth inventions, characterized in that the ligands pulsed in the pulse step are two or more ligands selected from the group consisting of WT1 peptide, α-galactosylceramide, and neoantigens. [Effects of the Invention]

[0038] The method for producing a cocktail dendritic cell vaccine of the present invention has the excellent effect of being able to produce multiple dendritic cell vaccines that are expected to be highly effective in treating cancer using limited blood components. [Brief explanation of the drawings]

[0039] [Figure 1] Summary of the method for producing the cocktail dendritic cell vaccine of the present invention [Figure 2]FIG. 1 shows an example of the immune effect of a dendritic cell vaccine produced by the method for producing a cocktail dendritic cell vaccine of the present invention. [Figure 3] 1 is a flowchart showing the method for producing a cocktail dendritic cell vaccine according to embodiment 1. [Figure 4] 1 is a flowchart showing the method for producing a cocktail dendritic cell vaccine according to embodiment 2. [Figure 5] 1 is a flowchart showing the method for producing a cocktail dendritic cell vaccine according to embodiment 3. [Figure 6] 1 is a flowchart showing the method for producing a cocktail dendritic cell vaccine according to embodiment 4. [Figure 7] 1 is a flowchart showing the method for producing a cocktail dendritic cell vaccine according to embodiment 5. [Figure 8] 1 is a flowchart showing the method for producing a cocktail dendritic cell vaccine according to embodiment 7. [Figure 9] 10 is a flowchart showing the method for producing a cocktail dendritic cell vaccine according to embodiment 8. [Figure 10] 10 is a flowchart showing the method for producing a cocktail dendritic cell vaccine according to embodiment 9. [Figure 11] A diagram showing an example of a table for determining a division ratio in a division step. [Figure 12] Figure 1 showing an example of the storage period determined in the storage steps [Figure 13] Figure 2 shows an example of the storage period determined in the storage steps [Figure 14] Table ranking the anti-cancer effects of dendritic cell vaccine-induced immunity DETAILED DESCRIPTION OF THE INVENTION

[0040] First, before describing the embodiments of the present invention, the body's immunity will be described. Immunity can be divided into innate immunity, which organisms have from the start, and adaptive immunity, which organisms acquire later.

[0041] 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 play a role in transmitting signals within the cell. Immune cells include eosinophils, neutrophils, basophils, macrophages, dendritic cells, and NK (natural killer) cells.

[0042] On the other hand, adaptive immunity is a mechanism that remembers pathogens that have infected the body and allows it to effectively eliminate them when encountered again. Although it takes longer to respond than innate immunity, it has the diversity to react to a variety of pathogens. 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 can change into plasma cells or memory B cells depending on the condition.

[0043] Furthermore, some NKT cells have properties of both innate and adaptive immunity. Of the immune cells mentioned above, the cells that this cocktail dendritic cell vaccine directly acts on are NKT cells, helper T cells, and killer T cells, but the activation of these cells also activates NK cells, macrophages, dendritic cells, B cells, and other cells.

[0044] Below, we will briefly explain the immune cells and substances related to immune function that are involved in the action of the dendritic cell vaccine produced by this cocktail dendritic cell vaccine production method.

[0045] <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.

[0046] <Terminology: Antibody> Cancer antibodies are a general term for proteins called immunoglobulins. They bind to cancer cells and attack them 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: Activates complement and damages cancer cells

[0047] <Terminology: Cytokine> A general term for proteins secreted by immune cells and 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. Interferons and interleukins are classified as cytokines.

[0048] <Terminology: Interleukin> Interleukins are a type of cytokine, and more than 30 types have been identified to date. They are secreted in large quantities by lymphocytes, a type of white blood cell that fights foreign substances in the body, and by phagocytes such as macrophages and neutrophils.

[0049] <Terminology: Interferon> Interferon, a type of cytokine, activates NK cells and macrophages, which attack cancer cells and virus-infected cells, and suppresses the growth of viruses and tumor cells. It has also been approved by the government as an antiviral and anticancer drug, and is used to treat multiple myeloma, brain tumors, and kidney cancer.

[0050] <Terminology: NK cells> NK cells have various receptors that capture abnormal cells and have the ability to damage cells that they recognize as abnormal, such as virus-infected cells or cancer cells.

[0051] <Terminology: Macrophage> Macrophages are a type of white blood cell that are activated by signals from living tissues or environmental stress, and phagocytose foreign substances such as bacteria. They also act as antigen-presenting cells that present the phagocytosed foreign substances as antigens to helper T cells, and when they ingest antigens, they produce cytokines (IL-12). They are also activated by cytokines (IFN-γ) produced by helper T cells.

[0052] <Terminology: Dendritic cells> They are antigen-presenting cells that take in foreign substances and present antigens to T cells, allowing them to be recognized as targets for attack. Early dendritic cells are called immature dendritic cells and have the ability to take in antigens (phagocytosis).

[0053] <Terminology: Helper T cells> When presented with an antigen, it recognizes it as a target for attack and instructs killer T cells to attack. It also produces substances called cytokines to activate B cells, and together with B cells, it determines whether a foreign substance is dangerous and assists in the production of antibodies.

[0054] <Terminology: Killer T cells> Killer T cells recognize antigens presented by MHC class I molecules and kill the cells. Thus, the T cell response, which is part of adaptive immunity, begins with the presentation of antigen peptides by MHC molecules. Under the direction of helper T cells, they kill and remove cells that are dangerous or unnecessary to the body, such as virus-infected cells and cancer cells.

[0055] <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.

[0056] <Terminology: NKT cells> These cells have the characteristics of both T cells and NK cells. When activated, they produce cytokines (IFN-γ) and induce responses to immunostimulatory and suppressive therapy. The most anticipated effect of this invention is its adjuvant action, which activates various immune cells in the body and, importantly, promotes their proliferation.

[0057] <Outline of Cocktail Dendritic Cell Vaccine> Next, with reference to Figures 1 and 2, an outline of the method for producing the cocktail dendritic cell vaccine of the present invention and the mechanism by which this cocktail dendritic cell vaccine improves immunity will be explained.

[0058] FIG. 1 is a diagram showing an outline of the method for producing a cocktail dendritic cell vaccine of the present invention. This cocktail dendritic cell vaccine is a vaccine cultivated from the patient's own cells and is made exclusively for the patient, so there is no rejection reaction and it is an extremely safe vaccine. First, monocytes are collected from the patient by apheresis. "Component blood collection" involves removing blood from the body using a medical tube or other device, separating the desired blood components and factors from the blood using specialized equipment, and then returning the blood to the body. In this invention, monocytes are separated as blood components, and approximately 100 cc of blood components are collected. The blood components after monocyte separation contain not only monocytes, but also plasma and platelets. Component blood collection places a significant burden on the body, as an anticoagulant is mixed into the blood before monocyte separation to prevent blood clotting, and the anticoagulant is then returned to the body, and the component blood collection for monocyte separation takes a long time.

[0059] Next, monocytes from the collected blood are cultured with the addition of cytokines, etc., to induce differentiation into immature dendritic cells. The cultured immature dendritic cells are then separated into two or more containers, one of which is added with a ligand used for specific immunotherapy, and the other with a ligand used for non-specific immunotherapy, and both are cultured to mature into dendritic cells. In this way, the dendritic cells cultured with the addition of antigen are produced as a dendritic cell vaccine targeting a specific disease.

[0060] The quality of the cultured dendritic cell vaccine is visually confirmed using a microscope or the like. The quality of a dendritic cell vaccine is determined by factors such as the survival rate of dendritic cells, the morphology of the dendritic cells (whether they have many branches, etc.), and whether they are contaminated with bacteria or fungi. Once the quality and safety of the dendritic cell vaccine has been confirmed, it is stored in a storage container and then administered according to the patient's condition. The above is an outline of the method for producing the cocktail dendritic cell vaccine of the present invention. As described above, the method for producing a cocktail dendritic cell vaccine of the present invention makes it possible to produce a combination of multiple dendritic cell vaccines that are expected to have a high anti-cancer effect from blood components collected from a patient through a single puncture. Although the term "cocktail dendritic cell vaccine" is used, the term "cocktail" in this invention refers to two or more types of dendritic cell vaccines administered to a patient. The treatment of administering the vaccine to a patient may involve pre-mixing two or more types of dendritic cell vaccines and then administering them, or multiple types of dendritic cell vaccines may be administered individually. Furthermore, when three or more types of dendritic cell vaccines are used, an administration method in which two or more types of dendritic cell vaccines are administered as a mixture, and the remaining dendritic cell vaccines are administered individually may also be used.

[0061] <Effects of cocktail dendritic cell vaccine> FIG. 2 shows an example of using two types of dendritic cell vaccines produced by the method for producing a cocktail dendritic cell vaccine of the present invention. The thickest arrows (0201, 0210) in the figure indicate the direct effects of two different types of dendritic cell vaccines: one pulsed with the ligand α-galactosylceramide, and the other pulsed with the WT1 peptide. The second thickest arrows (0202, 0203) indicate the particularly strong effect of activated NKT cells compared to other activated NKT cells. The thin arrows indicate the effects of immune cells in the body in response to the effects of the dendritic cell vaccine. The ellipses represent the immune cells depicted within them, and the triple ellipses represent immune cells that are particularly enhanced by the cocktail dendritic cell vaccine. The central polygon represents cancer cells. The dashed lines surrounding the cancer cells indicate the immunosuppressive effect of cancer cells, forming a barrier against immune function. The illustration of blood vessels indicates the acquisition of nutrients from blood vessels through angiogenesis. Next, each function will be explained with reference to the numbering.

[0062] Effect of α-galactosylceramide-pulsed dendritic cell vaccine: Activation of NKT cells: 0201 Dendritic cell vaccines pulsed with α-galactosylceramide activate NKT cells, which exert various effects on immune function. The five main effects are explained below.

[0063] <Function of activated NKT cells 1: Promoting dendritic cell maturation: 0202> Activated NKT cells produce cytokines and promote the maturation of immature dendritic cells in the body.

[0064] <Function of activated NKT cells 2: Adjuvant effect: 0203> 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 nonspecific immune activity pathways, resulting in a 1 + 1 > 2 effect, i.e., a synergistic effect. This is one of the features of the present invention.

[0065] <Function of activated NKT cells 3: Induction of apoptosis in cancer cells: 0204> Activated NKT cells produce a type of serine protease that breaks down the DNA of cancer cells, causing apoptosis of the cancer cells.

[0066] <Function of activated NKT cells 4: Checkpoint inhibitory effect: 0205> Activated NKT cells act to kill immunosuppressive cells that cancer cells have prepared to protect themselves from attacks by the body's immune cells. This releases various forms of immunosuppression, significantly improving the immune status within cancer tissue. For example, it enables killer T cells to more effectively attack cancer cells. In this way, the checkpoint inhibitory effect based on nonspecific immune activity further amplifies the cancer-attacking ability of killer T cells based on specific immune activity. This is one of the features of the present invention, which, like the adjuvant effect, achieves the 1+1>2 effect, i.e., a synergistic effect.

[0067] <Function of activated NKT cells 5: Inhibition of angiogenesis: 0206> Activated NKT cells inhibit the angiogenesis that cancer cells need to obtain nutrients, making it difficult for the cells to obtain nutrients and suppressing their growth.

[0068] <Action of activated macrophages: Cancer cell phagocytosis by macrophages: 0224> Activated macrophages phagocytose cancer cells and cancer dead cells.

[0069] <Function of dendritic cell vaccine pulsed with WT1 peptide: Cancer antigen presentation function: 0210> Dendritic cell vaccine 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 below by producing cytokines, and promote B cells to produce antibodies.

[0070] <Function of activated helper T cells: Instruction for attacking cancer cells: 0211> Activated helper T cells produce cytokines (INF-γ) and instruct killer T cells to attack cancer cells based on the information contained in the cytokines. In addition, the effects of cytokines activate NK cells, B cells and macrophages, enhancing the attack on cancer.

[0071] <Function of activated helper T cells: Instruction for antibody production against cancer antigen (e.g., WT1): 0212> 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 the cytokines.

[0072] <Function of activated killer T cells: Attack on cancer cells by killer T cells: 0213> The activated killer T cells, which are the main force for directly attacking cancer cells, recognize and bind to WT1 expressed on cancer cells, create holes in the cell membrane, and cause cancer cells to necrotize. Furthermore, substances such as TNF-β (tumor necrosis factor, a type of cytokine) are produced to induce apoptosis of cancer cells. Thus, 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.

[0073] <Action of activated killer T cells: Destruction of new blood vessels: 0214> 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 the cancer cells but also the newly formed blood vessels. This destroys the newly formed blood vessels, reducing the supply of nutrients to the cancer cells and suppressing their proliferation. This effect is also one of the factors that enable the cocktail dendritic cell vaccine of the present invention to exhibit the 1+1>2 effect.

[0074] <Activated NK cell action: NK cell attack on cancer cells: 0220> Activated NK cells exert a cytotoxic response against cancer cells and attack them.

[0075] <Action 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 their cell surface. In other words, they perform an antigen-presenting function, presenting antigens from cancer cells. These presented antigens are transmitted to dendritic cells, enabling the dendritic cells to further present the antigens.

[0076] <Cancer antigen presentation by dendritic cells: Cancer antigen presentation by dendritic cells: 0221> Immature dendritic cells take up cancer antigens, mature, and differentiate into mature dendritic cells, which present cancer antigens to helper T cells and killer T cells. This effect is an anti-cancer effect that normally occurs in the body, but the action of activated NKT cells results in a greater anti-cancer effect than usual. This cancer antigen is presented when macrophages phagocytose the patient's own cancer cells, so it is expected to be more effective than dendritic cell vaccines that use artificial antigens as ligands. This effect is also one of the factors that enable the cocktail dendritic cell vaccine of the present invention to exhibit the 1+1>2 effect.

[0077] <Function of activated B cells: Cancer antibody production: 0223> 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).

[0078] As mentioned above, by combining non-specific immunotherapy and specific immunotherapy, the effects of both can be synergistic, allowing cancer cells to be attacked using a variety of immune functions. Therefore, a higher effect can be expected than when each therapy is performed alone.

[0079] Figure 14 is a table showing empirical rankings of the anti-cancer effects of each homogeneous dendritic cell vaccine produced by the present invention. Rank A is a rating indicating a primary anti-cancer effect resulting from a direct attack on cancer cells. Rank B is a rating indicating a lower anti-cancer effect than Rank A, and a secondary effect such as suppressing cancer cell proliferation. Rank S indicates an enhanced anti-cancer effect resulting from a direct attack on cancer cells by activated immune cells.

[0080] This rating of anticancer effects is based on experience and assumes that the expected effects are actually demonstrated in patients. If the ligand of the specific immune dendritic cell vaccine does not match the patient's cancer cells, the anti-cancer effect may not be obtained, but this case will not be considered. In addition, in the case of non-specific immune cell vaccines, the anti-cancer effect is exerted through various immune cells, and the degree of effectiveness varies depending on the characteristics of the patient's immune cells. In this case, the vaccine is rated based on the empirically obtained average effectiveness.

[0081] FIG. 14 shows a comparison of the combined efficacy ratings of the non-specific immune cell vaccine and the specific immune cell vaccine with the efficacy rating of the cocktail dendritic cell vaccine of the present invention. Of the anti-cancer effects of non-specific immune cell vaccines and specific immune cell vaccines, killer T cells overlap, so if one rating is included in the total, the effect of rating A would be 5 and the effect of rating B would be 5. In contrast, the anti-cancer effects of cocktail dendritic cell vaccines are rated as follows: S rating is 6, A rating is 1, and B rating is 1, which empirically indicates that the effects are higher than the sum of the effects of non-specific immune cell vaccines and specific immune cell vaccines. This is due to the enhanced activity of killer T cells and NK cells through the adjuvant effect, and the effective anti-cancer effect of killer T cells through checkpoint inhibition. <Embodiment 1: Mainly corresponds to claim 1 Overview>

[0082] In this embodiment, monocytes are obtained from a patient by component blood collection, differentiated into immature dendritic cells, and then divided into multiple groups to produce different types of vaccines.By utilizing immune activity that acts simultaneously through multiple routes, we provide a method for producing a cocktail dendritic cell vaccine that has a synergistic effect in attacking cancer cells compared to when each of the multiple routes acts alone. <Embodiment 1: Configuration mainly corresponding to claim 1>

[0083] Embodiment 1 of the present invention is a method for producing a cocktail dendritic cell vaccine exclusive to a patient, comprising: a preparation step of preparing to collect blood components from a patient; a blood component collection step of collecting a predetermined amount of monocytes from a patient who is ready for blood component collection by a single puncture (meaning that the collection of a predetermined amount of blood is completed in one course; multiple punctures are not precluded); a division step of dividing monocytes collected only from the patient or immature dendritic cells differentiated from monocytes into multiple groups; a pulsing step of pulsing each group divided in the division step with a different type of ligand, optionally at different times starting from the time of division; and a storage step of storing each group pulsed with a ligand. <Embodiment 1: Mainly corresponds to claim 1 Configuration Preparation Steps>

[0084] The "preparation step" is a step in which preparations are made for component blood collection from a patient. When collecting blood, monocytes, the source of dendritic cells, are collected, but it is necessary to ensure that a sufficient number of monocytes can be collected. In addition, we check whether there are any risks due to illness and determine whether the patient is in a condition that can withstand blood component collection.

[0085] Monocytes are a type of white blood cell that, like other white blood cells, are produced in the bone marrow and account for 3-6% of white blood cells. When monocytes enter the body's tissues, they transform into macrophages and dendritic cells. Therefore, the more monocytes present, the more dendritic cells can be cultivated. <White blood cell count>

[0086] Therefore, white blood cell counts are checked to see if they are sufficient, and if they are below the required standard, medication is administered to raise the white blood cell count to at least 3000 / μL, preferably 4000 / μL or higher. <Management of bleeding risk>

[0087] In apheresis, blood is drawn outside the body, the components are separated, and then the blood is returned to the body, a process that is repeated. For this reason, an anticoagulant is mixed into the blood to prevent it from clotting inside the blood component collection device. If the platelet count is lower than that of a healthy person, or if there is bleeding in the affected area due to blood coagulation abnormalities or cancer, there is a risk of bleeding not stopping. For this reason, it is necessary to treat areas that may be bleeding and ensure the platelet count is maintained. <Ensuring physical strength>

[0088] Because blood component collection involves a significant burden, patients must be physically strong enough to withstand the process. Patients with advanced cancer are likely to be physically weak, so they should be provided with sufficient nutrition and hydration beforehand through intravenous drips, etc. <Embodiment 1 mainly corresponds to claim 1 Configuration component blood collection step>

[0089] The "component blood collection step" is a step in which a predetermined amount of monocytes is collected from a patient who is ready for component blood collection by a single puncture (meaning that the predetermined amount of blood is collected in one course; multiple punctures are not prohibited).

[0090] "Apheresis" is the process of drawing blood from a patient's body (blood withdrawal), separating the desired components using an apheresis machine, and returning the remaining separated blood to the body (blood return). This process is repeated until the desired amount of the desired component is collected. Therefore, a single puncture includes the puncture with a needle connected to a tube for drawing blood and the puncture with a needle connected to a tube for returning the blood to the body.

[0091] Monocytes are obtained in the component blood collection used to produce the cocktail dendritic cell vaccine of the present invention. The typical method involves inserting a blood collection needle into the patient's left upper limb to collect blood, and then inserting another needle into the right upper limb, after which the processed blood is returned. The collected blood is mixed with an anticoagulant such as sodium citrate and separated into individual blood components using a centrifuge. The layer containing monocytes is extracted from this and collected in a culture bag. The remaining components are mixed again and returned to the body. This process is repeated for about three hours to process about 4 liters of whole blood from the body. Therefore, the time required for apheresis varies depending on the amount of monocytes required, but it usually takes more than one hour and a half.

[0092] In this blood component collection step, monocytes are separated as the target component, and about 100 cc of blood (which may contain plasma, platelets, etc.) consisting mainly of monocytes is collected. 7 Preferably, the sample contains at least 100 monocytes.

[0093] The blood collection tube and needle can be used in a shorter time if they are used for blood removal and blood transfer, but a single needle can also be used for both blood removal and blood return, allowing for blood to be removed and returned. In this case, the blood removal, separation, and blood transfer operations are performed sequentially. As long as this method can collect monocytes from the patient's blood, there are no particular limitations on the method.

[0094] Furthermore, if it becomes possible to generate monocytes from stem cells or iPS cells in the future, it is acceptable to culture them from stem cells or iPS cells obtained from a patient using this method. The present invention includes any method that allows the acquisition of the monocytes required for subsequent treatment with a single puncture, taking into consideration the patient's physical strength, etc. <Embodiment 1 mainly corresponds to claim 1 Configuration Division Step>

[0095] The "division step" is a step in which monocytes collected from the patient's blood or immature dendritic cells differentiated from monocytes are divided into multiple groups. The immature dendritic cells differentiated from monocytes are divided into separate culture vessels (e.g., petri dishes, beakers, measuring cylinders, etc.) at a predetermined ratio. This ratio is determined based on the ratio at which a dendritic cell vaccine pulsed with a specific ligand is ultimately produced. Based on this ratio, the cells are pulsed with a ligand in the pulsing step described below. At least one of the culture vessels containing the divided immature dendritic cells is pulsed with an NKT-activating ligand, and the ratio is 3% or more but not more than 30% of the total. When dividing at the monocyte stage, the cells may first be divided into equal parts and then matured under the same conditions for a certain period of time to become immature dendritic cells. At this stage, the two may be reunited and then further divided. This has the effect of homogenizing the divided immature dendritic cells. The advantage of dividing monocytes is that in the unlikely event of contamination, the contaminated fraction becomes unusable, but the remaining fraction can be used. In addition, the conditions for maturing monocytes into immature dendritic cells may vary slightly depending on the patient's immune strength, and it is possible to create immature dendritic cells in good condition by trial and error with multiple conditions.

[0096] NKT cells are said to account for only 0.1% of T cells in peripheral blood, making them extremely rare. Therefore, vaccines aimed at activating NKT cells, which are considered nonspecific immunotherapy, may be wasted even if administered in large doses. In other words, the efficacy of dendritic cell vaccines pulsed with NKT-activating ligands tends to plateau relative to the dose administered. Therefore, it is preferable to allocate more of these cells to specific immunotherapy.

[0097] Furthermore, in the "division step," the cells may be divided in the state of monocytes and differentiated into immature dendritic cells. It is preferable to divide the cells into a container that allows for direct culture, such as a petri dish. The division ratio in this case is the same as when the cells are divided in the state of immature dendritic cells. However, splitting at the immature dendritic cell stage is preferable because it minimizes the variability in the quality of the cells in the multiple splits. This is because cells tend to mature at different rates in the culture vessel, and splitting at the early monocyte stage can result in inconsistent effects of ligand pulsing. <Embodiment 1 mainly corresponds to claim 1. Configuration: division step; first culture substep>

[0098] The division step includes a "first culture substep." In this substep, monocytes collected from the patient's blood are cultured in an incubator for approximately 5 to 7 days to differentiate into immature dendritic cells. The monocytes are placed in a serum-free medium optimized for dendritic cells, and stimulatory cytokines (e.g., interleukins (IL4) and GM-CSF)) are added. This culture induces differentiation into immature dendritic cells. A key feature of live immature dendritic cells is that after two days of culture, downstream regulation of CD14 surface expression, increased dextran reabsorption, and increased response to MIP-1a can be detected. These can be used as markers for observing the differentiation of immature dendritic cells. Various research and proposals for improving efficiency and stabilization of the culture method have been conducted, so any method that can efficiently and stably generate immature dendritic cells from monocytes is acceptable. It is preferable to perform the first culture step in a single culture vessel to avoid variations in culture. However, if the cells are split into monocytes and cultured after splitting, each group is cultured separately. It is also possible to vary the conditions for culturing the split monocytes to control the quality of the immature dendritic cells. Culture conditions can also be determined depending on the type of ligand to ensure that the ligand to be pulsed later is effective. The effectiveness of the ligand must be determined by comprehensively taking into account factors such as antigen-presenting ability, maturation rate, and control of the properties of immature dendritic cells that will be contained in the patient's body. Culture conditions can also include temperature, type of medium, nutrients provided, and time. <Embodiment 1 corresponds mainly to claim 1. Configuration: Pulse Step>

[0099] The "pulse step" is a step in which different types of ligands are pulsed to each group divided in the division step, possibly at different timings starting from the time of division. A "ligand" is a substance that forms a protein complex with a biological substance and fulfills a biological purpose. In this case, it activates the immune system by specifically binding to various receptors on immune cell membranes. Ligands are composed of amino acids, peptides, proteins, glycolipids, etc. "Pulse" here refers to the addition of an antigen-containing chemical to the culture medium to promote culture. Specifically, this is the process of adding a ligand, such as a peptide or α-galactosylceramide, to immature dendritic cells, culturing them, and then maturing them to produce mature dendritic cells. Note that the rate at which immature dendritic cells mature may vary depending on the type of ligand used. Therefore, pulsing at different times, starting from the time of division, is necessary. This is because the initiation points of specific immunotherapy and nonspecific immunotherapy must be approximately the same to expect a synergistic effect of both. Furthermore, it is preferable to keep the storage period (frozen storage period) as short as possible to minimize the amount of more active mature dendritic cells or dysfunctional mature dendritic cells.

[0100] There are two types of pulsing ligands, one of which is an NKT cell ligand, which is classified as a non-specific immunotherapy and is also called NKT cell targeted therapy. The other is classified as specific immunotherapy, which uses cancer antigens as ligands.

[0101] An "NKT cell-activating ligand" is a glycolipid that activates NKT cells. NKT cells are activated by the presentation of NKT cell-activating ligands by antigen-presenting cells such as dendritic cells. Activated NKT cells produce large amounts of interferon (IFN-γ) and can activate various immune cells. Furthermore, because NKT cell receptors are common to all individuals and do not target cancer cells, a consistent effect can be expected. In recent years, α-galactosylceramide has attracted attention as an NKT cell-activating ligand, but research and development is also underway on ligands with even greater efficacy than α-galactosylceramide. Therefore, if new NKT cell-activating ligands are developed in the future, they may be used.

[0102] Cancer antigens are proteins (including peptides) with a structure unique to cancer cells, a structure not found in normal cells. When presented as an antigen to T cells by antigen-presenting cells such as dendritic cells, the T cells are activated and, using the protein structure of the cancer antigen as a marker, recognize the cancer cells as targets for attack. Therefore, if the amino acid structure of the artificial cancer antigen presented by dendritic cells matches the amino acid structure of the cancer cells affecting the patient, it will be effective, but if it does not match, it will be ineffective. Against this backdrop, a cancer antigen called WT1 peptide has shown relatively high efficacy in recent years.

[0103] The WT1 peptide is an artificial cancer antigen with a protein structure commonly found in various cancers, so it can be used in most cancer patients and is expected to be effective to a certain extent. For this reason, using the WT1 peptide as a ligand is very effective for cancers that are not specific to the individual.

[0104] There are many artificial cancer antigens, not just WT1. However, there is also a method of culturing or identifying proteins extracted from the patient's own cancer cells or body tissues to artificially generate antigens. In this case, since the antigen is based on the patient's own cancer cells or similar, the amino acid structure of the antigen matches, and therefore greater effectiveness can be expected. Such ligands, known as neoantigens, are also beginning to be put into practical use.

[0105] "Neoantigens" are called neoantigens and are artificially generated by identifying the protein structures expressed in cells that have undergone genetic changes not found in normal cells. To identify these, it is necessary to analyze new cancer tissue, which is a stage before genetic changes occur. Neoantigens vary from patient to patient and can also change depending on the stage of cancer progression. Therefore, it is most effective to use neoantigens that are as newly identified as possible.

[0106] As described above, research into cancer antigens that can be used as ligands is progressing daily, and even newly discovered ligands that belong to specific immunotherapy can be used as ligands in the method for producing a cocktail dendritic cell vaccine of the present invention.

[0107] As described above, various ligands can be used in the pulsing step, but at least one group of immature dendritic cells divided in the dividing step is treated with a ligand corresponding to non-specific immunotherapy, and the remaining group is treated with a ligand corresponding to specific immunotherapy. In other words, immature dendritic cells divided in the dividing step are used so as to contain both ligands corresponding to non-specific immunotherapy and ligands corresponding to specific immunotherapy. <Embodiment 1 mainly corresponds to claim 1. Configuration: Pulse step; Second culture sub-step>

[0108] The pulse step includes a second culture substep in which immature dendritic cells are pulsed with a ligand and cultured to mature them into mature dendritic cells. The immature dendritic cells take up the added ligand as an antigen and are cultured for approximately one to three days to mature. The proportion of mature dendritic cells in the total after culture may be 70% or more, preferably 80% or more, although the culture may be scheduled so that some immature dendritic cells remain. After being returned to the patient's body, these immature dendritic cells function as an immune system appropriate to the patient's condition and can be used to strengthen the immune system that was not fully prepared during diagnosis or treatment planning. <Embodiment 1: Mainly corresponds to claim 1 Configuration Storage step>

[0109] The "storage step" is a step in which each group pulsed with the ligand is stored. In the pulse step, the cells are matured into mature dendritic cells, and the resulting liquid, which serves as a dendritic cell vaccine, is injected into a storage container such as a vial for each group and stored. That is, a storage container is produced that is filled with a number of dendritic cell vaccines equal to or greater than the number of times the vaccine is to be administered to a patient. This vaccine is stored frozen at -80°C or below until administration, but this is not limited to frozen storage, and if the vaccine can be refrigerated, it may be stored in the refrigerator. Furthermore, if the dendritic cell vaccine is to be administered to a patient immediately after production, it may be stored in the refrigerator or at room temperature. The storage step does not necessarily have to be performed on all divided groups, but may be performed on only some of the groups. Furthermore, each group of divided and matured mature dendritic cells may be reintegrated and stored in a state where multiple types of mature dendritic cells are mixed. In particular, when the storage step is performed by cryogenic freezing, it is possible to consolidate the entire group before storing it, so that minute variations in quality deterioration of mature dendritic cells due to storage do not occur among multiple mature dendritic cell groups. <Embodiment 1: Mainly corresponds to claim 1. Configuration: Preservation step: Storage container dividing sub-step>

[0110] The storing step may include a "storage container dividing sub-step." The storage container is divided into a single dose or less for administration to a patient and stored in a storage container such as a vial. The reason for specifying a dose of less than one dose is that the optimal dose of dendritic cell vaccine varies from patient to patient. For example, one patient may need 1 x 10 6 Although efficacy was observed with doses equivalent to 1 × 10 cells, one patient received 1 × 10 7 In some cases, the effect will not be seen unless an amount equivalent to the number of cells is administered. Also, since the vaccine is used up once thawed, any remaining valuable dendritic cell vaccine will be wasted. For these reasons, one storage container should contain the minimum expected amount, and only the number of containers necessary to secure a single dose should be thawed and used. As described above, a plurality of dendritic cell vaccines are produced by the method for producing a cocktail dendritic cell vaccine of the present invention. <Embodiment 1: Explanation of flow mainly corresponding to claim 1>

[0111] FIG. 3 is a flow chart showing the process for producing the cocktail dendritic cell vaccine of the present invention. The details of each step are the same as those explained in the configuration, and the explanation of this flowchart will provide a rough outline of the flow. First, in preparation step 0301, preparations are made to enable component blood collection from the patient to be treated. Specifically, the white blood cell count is checked, and if it is lower than a predetermined value, a drug to increase the white blood cell count is administered and the patient's condition is monitored. Nutrients and hydration are also administered to ensure that the patient is in a healthy state where component blood collection is not a problem. Thereafter, apheresis is performed in apheresis step 0302. The components collected in apheresis are monocytes, and approximately 100 cc of blood containing monocytes as the main component is collected. Next, in division step 0303, immature dendritic cells differentiated from monocytes collected from the patient are divided into multiple groups at a predetermined ratio. Differentiation of monocytes into immature dendritic cells is achieved by placing monocytes in a culture medium, adding cytokines as stimulatory factors, and culturing for about five days. Next, in the ligand pulsing step 0304, a different ligand is added to each group of culture medium containing immature dendritic cells divided into multiple groups, and the cells are cultured. Here, the immature dendritic cells take up the ligand and mature into mature dendritic cells over a period of about 2 to 3 days. Finally, in storage step 0305, the mature dendritic cells that have been divided and cultured into a plurality of groups are divided into single-dose portions, transferred to storage containers, and frozen and stored at -80°C or below. The above is the flow of production of the cocktail dendritic cell vaccine of the present invention. <Embodiment 1 mainly corresponds to claim 1 Effect>

[0112] A cocktail dendritic cell vaccine consisting of multiple dendritic cell vaccines, including non-specific and specific immune dendritic cell vaccines, can be produced from a single blood draw. Culturing from a single blood draw ensures the homogeneity of the dendritic cells in the multiple dendritic cell vaccines, ensuring a synergistic effect. Homogeneity is further enhanced when cocktail dendritic cell vaccines are produced by dividing extracted monocytes into multiple groups after allowing them to grow to a certain extent and pulsing them with different ligands. This is because cells exhibit significant variation in growth rate in the early stages of growth, but this variation gradually decreases as growth progresses. Furthermore, blood component collection places a significant burden on patients and cannot be performed repeatedly within a short period of time. Therefore, by generating multiple dendritic cell vaccines from a single blood collection, simultaneous administration can be ensured without wasting time. Therefore, patients can achieve a high anti-cancer effect through the synergistic effect of improved immunity and improved anti-cancer activity. <Embodiment 2: Mainly corresponds to claim 2 Overview>

[0113] In embodiment 2, in addition to the procedures of embodiment 1, information on the patient's immune status and disease is obtained before the preparation step, and the optimal ligand is selected depending on the patient's condition. <Configuration of Embodiment 2, mainly corresponding to Claim 2>

[0114] In addition to the procedures of embodiment 1, embodiment 2 includes an immunity / disease information acquisition step that acquires, before the preparation step, one or more of immunity information, which is information related to the patient's immunity, the patient's personal biological information, and the patient's disease information, and a ligand selection step that selects the ligand to be pulsed in the pulsing step based on the acquired immunity / disease information, thereby producing a cocktail dendritic cell vaccine. Details of each component will be omitted for components that overlap with embodiment 1, and only the additional steps will be described. <Embodiment 2 corresponds mainly to claim 2. Configuration: Immunity and disease information acquisition step>

[0115] The "immunity and disease information acquisition step" is a step of acquiring one or more of immune information, which is information about the patient's immunity, the patient's personal biological information, and the patient's disease information, before the preparation step. "Immunity and disease information" refers to information related to a patient's immune system, and is made up of immune information obtained from blood tests, etc., as well as disease information obtained from various test information such as blood tests, tumor markers, cancer tissue biopsies, X-rays, CT scans, and MRIs, including information related to cancer, cancer complications, and diseases other than cancer.

[0116] Blood tests can check conditions such as white blood cell count, immunoglobulin levels, whether or not you have immunodeficiency, complement levels, and whether or not you have an autoimmune disease. The white blood cell count measures the percentage of lymphocytes and monocytes, making it an important parameter in the production of this immunologically active vaccine. Immunoglobulins include IgG, IgA, and IgM. Abnormal values ​​may indicate a disease other than cancer, and pre-emptive treatment may be considered. Immunodeficiency is assessed by testing for HIV1 and HIV2 antibodies. If a problem is detected, T cells and B cells, which have key immune functions, are not functioning normally, and treatment to improve the immunodeficiency is necessary. Tumor markers test the levels of characteristic proteins and other substances produced by different types of cancer. Typical examples include CEA, AFP, BCA225, BFP, CA15-3, and CA19-9, and can indicate whether cancer metastasis is suspected. If the type of cancer is known, it is possible to configure the system so that the pulsed ligand is selected according to that type. Alternatively, it is possible to custom-make the pulsed ligand according to the location of the cancer in the patient. Biopsies of cancer tissue are also similar and involve the selection of ligands. X-rays, CT scans, MRI scans, etc. are used to check the progression of the original cancer disease and to identify areas suspected of metastasis using tumor markers, etc., using actual images.

[0117] Such information on immunity and disease may be used, for example, as an indicator for selecting the ligand to be used, or for determining the administration schedule for the cocktail dendritic cell vaccine, including the administration interval and duration.

[0118] The step of acquiring immune status and disease information may be performed after administration of a dendritic cell vaccine produced by the cocktail dendritic cell vaccine production method of the present invention. For example, this information may be acquired by confirming the induction of killer T cells or by checking for an increase in IFN-γ levels. When a specific immune dendritic cell vaccine is administered, the induction of killer T cells can be confirmed as an indicator of efficacy. Furthermore, when a nonspecific immune dendritic cell vaccine is administered, the increase in IFN-γ levels can be evaluated by examining this value to assess the degree of efficacy. For example, three or more divided groups may be created, and after the first two dendritic cell vaccines are administered to the patient, immature dendritic cells kept as spares may be pulsed with a new ligand based on the evaluation results to mature them, thereby producing additional new dendritic cell vaccines.

[0119] On the other hand, the amount of dendritic cell vaccine required to be administered to a patient for efficacy varies from patient to patient. Specifically, whether or not specific immune dendritic cell vaccines induce killer T cells varies from patient to patient. Furthermore, with nonspecific immune dendritic cell vaccines, once IFN-γ reaches a predetermined level, further administration is not necessary until the efficacy declines. For these reasons, it is conceivable to determine the amount of dendritic cell vaccine to be produced by administering a dendritic cell vaccine produced by the cocktail dendritic cell vaccine production method of the present invention, while monitoring the patient's immune response. <Embodiment 2: Mainly corresponds to claim 2. Configuration: Ligand selection step>

[0120] The "ligand selection step" is a step in which the ligand to be pulsed in the pulse step is selected according to the acquired immune and disease information. First, specific immunotherapy uses cancer antigens as ligands, but there are also many artificially produced cancer antigens. Table 1 below lists representative artificial cancer antigens. [Table 1] As such, there are many cancer antigens that can be used as ligands, depending on the location of the cancer, and which ligand will be effective for a patient must be determined based on the patient's test results (location of the cancer, stage of the cancer, tumor marker values, etc.).

[0121] α-Galactosylceramide is known as a ligand used in nonspecific immunotherapy. Research is also underway to develop ligands with even greater efficacy than α-Galactosylceramide. If an effective ligand emerges in the future, it will be included as a potential ligand for use in this cocktail dendritic cell vaccine, as long as it activates NKT cells. <Embodiment 2: Mainly corresponds to claim 2. Explanation of the flowchart>

[0122] 4 is a flowchart showing the procedure of embodiment 2. Explanation of steps that overlap with embodiment 1 will be omitted, and only the different steps will be explained. First, in immunity / disease information acquisition step 0406, necessary tests (for example, blood tests, tumor markers, CT scans, MRIs, etc.) are performed depending on the patient's condition, and immunity / disease information relating to the patient's immunity and diseases is acquired. Thereafter, similarly to the first embodiment, a preparation step 0401, a component blood collection step 0402, and a division step 0403 are performed. In parallel with this, before the pulse step 0404, an optimal ligand is selected based on immune and disease information in a ligand selection step 0407. For example, an artificial ligand may be selected depending on the site of cancer in the patient. Thereafter, the pulse step 0404 and the storage step 0405 are carried out in the same manner as in the first embodiment, but the ligand pulsed in the pulse step 0404 is the one selected in the ligand selection step 0407 . The above is the method for producing the cocktail dendritic cell vaccine of embodiment 2. <Embodiment 2: Mainly corresponds to claim 2 Effect>

[0123] As described above, the cocktail dendritic cell vaccine of embodiment 2 allows for the selection and production of an optimal ligand depending on the site and type of cancer, resulting in a stronger anticancer effect. Furthermore, in cases where a patient is suffering from a disease other than cancer, such as a viral or bacterial infection, the antigen of that virus or bacteria is used as a ligand in a portion of the split immature dendritic cells. This allows for the production of a cocktail dendritic cell vaccine that can be used to treat multiple diseases simultaneously. <Embodiment 3: Mainly corresponds to claim 3 Overview>

[0124] In addition to the features of embodiment 2, embodiment 3 determines the ratio of immature dendritic cells to be divided in the division step based on the immune and disease information acquired in the immune and disease information acquisition step. <Embodiment 3: Configuration mainly corresponding to claim 3>

[0125] In addition to the configuration of embodiment 2, embodiment 3 produces a cocktail dendritic cell vaccine by further including a division ratio determination step for determining the division ratio for division in the division step based on the acquired immune status and disease information. Regarding the details of each component, explanation of components that overlap with embodiment 2 will be omitted, and only the additional steps will be explained. <Embodiment 3: Mainly corresponds to claim 3 Configuration: Division ratio determination step>

[0126] The "division ratio determination step" is a step for determining the division ratio of immature dendritic cells in the division step. The method for producing the cocktail dendritic cell vaccine of the present invention comprises a dendritic cell vaccine using a ligand corresponding to specific immunotherapy and a dendritic cell vaccine using an NKT cell-activating ligand corresponding to non-specific immunotherapy. Furthermore, as ligands applicable to specific immunotherapy, multiple ligands may be used depending on factors such as the type and stage of the patient's cancer, and whether artificial cancer peptides or patient-derived cancer peptides are used. Furthermore, if the patient also has a non-cancer disease treatable by a dendritic cell vaccine, a ligand corresponding to that treatment may also be used. This step involves determining the division ratio of immature dendritic cells pulsed with these ligands. This division ratio, which is the ratio at which immature dendritic cells divide, is the ratio of dendritic cells contained in the dendritic cell vaccine produced by the method for producing a cocktail dendritic cell vaccine of the present invention. Therefore, this ratio may significantly affect the therapeutic effect of patients administered with the dendritic cell vaccine produced by the method for producing a cocktail dendritic cell vaccine of the present invention.

[0127] For example, if the information regarding immunity obtained in the immunity / disease information acquisition step indicates that the patient's immunity is high, it may be possible to reduce the amount of non-specific immune dendritic cell vaccine, for example, to 3-6%, and increase the amount of specific immune dendritic cell vaccine, for example, to 94-97%.

[0128] In addition, if the information on the patient's immune strength indicates a low level of immunity, and the cancer has been surgically removed or the cancer is still in its early stages, it may be possible to reduce the proportion of specific immune dendritic cell vaccine and set the non-specific immune dendritic cell vaccine at 30% so that it can be administered over a long period of time. Although the above are extreme cases, it is desirable to produce each dendritic cell vaccine in an optimal ratio as appropriate, depending on the immune strength and disease information of each individual patient. The division ratio here affects the number of dendritic cells, but does not correspond to the amount of vaccine related to the number of administrations. In other words, the number of dendritic cells contained per unit amount in the liquid of a nonspecific immune dendritic cell vaccine and a specific immune dendritic cell vaccine may be different. This is because the amount of ligand that can be pulsed onto dendritic cells differs depending on the ligand.

[0129] FIG. 11 is an example of a table for determining the ratio in the division step. This ratio is the division ratio of immature dendritic cells and does not correspond to the amount of dendritic cell vaccine produced. The rows are evaluation values ​​of immune strength, which are divided into three levels here: normal indicates the average value of a person with healthy immune strength, weak indicates an even weaker immune strength, and strong indicates a state in which the immune strength is more active than normal. One possible method for evaluating immunity is to obtain interferon levels and rate them on a three-point scale. For example, if the patient's immune system is weak, it is necessary to strengthen the immune system, so the non-specific immune dendritic cell vaccine is increased to a level not exceeding 20%, and the remainder is produced as a specific immune dendritic cell vaccine.If the immune system evaluation value is strong and relatively high, the focus is on specific immunotherapy, so the non-specific immune dendritic cell vaccine is reduced to around 5%, and the remainder is produced as a specific immune dendritic cell vaccine. <Embodiment 3: Explanation of the flowchart, mainly corresponding to claim 3>

[0130] 5 is a flowchart showing the procedure of embodiment 3. Explanation of steps that overlap with other embodiments will be omitted, and only the different steps will be explained. In division ratio determination step 0508, the number of divisions and division ratios for dividing the immature dendritic cells in division step 0503 are determined based on the immunity / disease information acquired in immunity / disease information acquisition step 0506, at the latest before division step 0503. In division step 0503, the immature dendritic cells are divided into a plurality of groups based on the number of divisions and division ratios determined in division ratio determination step. <Effects of Embodiment 3, mainly corresponding to claim 3>

[0131] The patient's condition is assessed through testing, and the production ratio of the dendritic cell vaccine that is optimal for the patient's condition is determined and manufactured, allowing for the production of an effective cocktail dendritic cell vaccine with stronger anti-cancer effects. <Embodiment 4: Overview, mainly corresponding to claim 4>

[0132] In embodiment 4, based on the immune and disease information acquired in the immune and disease information acquisition step, a plan for administering the dendritic cell vaccine to a patient produced by the cocktail dendritic cell vaccine manufacturing method of the present invention is created, and a storage period is determined for each storage container stored in the storage step. <Configuration of Embodiment 4, mainly corresponding to claim 4>

[0133] In addition to embodiment 2 and embodiments including embodiment 2, embodiment 4 further includes a storage period determination step in which the storage period for each group in the storage step is determined based on the acquired immune status and disease information, thereby producing a cocktail dendritic cell vaccine. <Embodiment 4: Mainly corresponds to claim 4 Configuration: Storage period determination step>

[0134] The "storage period determination step" determines the storage period for each storage container of the dendritic cell vaccine, which has been divided into individual storage containers and stored frozen, based on the immunity and disease information acquired in the immunity and disease information acquisition step. For example, non-specific immune dendritic cell vaccines are administered at intervals of 2 to 8 weeks, but for patients who are determined to have a weakened immune system based on immune and disease information, they are administered at shorter intervals, closer to 2 weeks, and for patients who are determined not to have a weakened immune system, they are administered at intervals closer to 8 weeks, with a longer treatment course planned. Specific immune dendritic cell vaccines are administered at intervals of 2 to 4 weeks, but for patients whose cancer is judged to be imminently progressing based on immune status and disease information, the administration interval should be shorter, closer to 2 weeks, and the dose should be set higher. For patients whose cancer is not imminently progressing, such as those who have had the cancer area surgically removed, the vaccine can be administered at intervals closer to 4 weeks, or the dose can be gradually increased from a small amount to determine the appropriate amount.

[0135] As described above, based on the immune and disease information acquired in the immune and disease information acquisition step, an administration plan is created that includes the administration interval, administration period, and dosage of the dendritic cell vaccine produced by the cocktail dendritic cell vaccine manufacturing method of the present invention.Based on the created administration plan, in the storage step, the dendritic cell vaccine is stored in storage containers in an amount equal to or less than a single dose, and the number of divisions into which the vaccine is divided, the amount of dendritic cell vaccine to be stored in one storage container, and the storage period for each storage container are determined.

[0136] 12 and 13 show an example in which the storage period of each storage container of a dendritic cell vaccine is determined based on the administration schedule. The black triangle indicates the timing of administration and the line indicates the storage period, with each square representing two weeks. Figure 12 shows the administration plan created for the patient in Figure 11 whose immune status was assessed as weak. As shown in the figure, the storage period for each storage container has been determined so that the specific immune dendritic cell vaccine can be administered every two weeks. The storage period for the non-specific immune dendritic cell vaccine has been determined so that it can be administered twice, at the beginning and end of the treatment period, with one course planned to last three months. Figure 13 shows an example of administration after surgical treatment for cancer to prevent metastasis and recurrence. Because the severity of the cancer symptoms is relatively low, the plan is for long-term, careful treatment. As shown in the figure, the specific immune dendritic cell vaccine is initially administered every three weeks, followed by three every four weeks. The non-specific immune dendritic cell vaccine is scheduled to be administered approximately 12 weeks apart. It is preferable that the non-specific immune dendritic cell vaccine is administered in the range of one to three times during one course, regardless of the amount divided in the division step. <Embodiment 4: Explanation of the flowchart, mainly corresponding to claim 4>

[0137] 6 is a flowchart showing the procedure of embodiment 4. Explanation of steps that overlap with other embodiments will be omitted, and only the steps that are different will be explained. In the storage period determination step, a patient administration plan for the cocktail dendritic cell vaccine is created based on the immune status and disease information acquired in the immune status and disease information acquisition step. Based on this information, a storage period is determined for each storage container containing the dendritic cell vaccine, which was separated into single doses and stored in storage containers in the storage step. <Embodiment 4: Effect mainly corresponding to claim 4>

[0138] The storage period of each storage container is determined based on an administration plan appropriate to the patient's condition, including the number of aliquots of the dendritic cell vaccine produced by the cocktail dendritic cell vaccine manufacturing method of the present invention into storage containers, the amount of aliquots, the administration interval, the administration period, etc. Therefore, the cocktail dendritic cell vaccine can be used effectively in accordance with the patient's condition and can be kept in a stable state until administration. <Embodiment 5: Mainly corresponds to claim 5 Overview>

[0139] In embodiment 5, a dendritic cell vaccine composed of mature dendritic cells that have been cultured after pulsing with a ligand in the pulse step is observed to check the viability of the dendritic cells, the presence of foreign matter, etc., and to confirm that the vaccine is of a quality that is acceptable for administration to patients. <Embodiment 5: Configuration mainly corresponding to claim 5>

[0140] In addition to the configuration of any one of embodiments 1 to 4, embodiment 5 further includes an observation result acquisition step of observing each group pulsed with a ligand after the pulsing step and obtaining observation results, thereby producing a cocktail dendritic cell vaccine. <Embodiment 5: Mainly corresponds to claim 5 Configuration: Observation result acquisition step>

[0141] The "observation result acquisition step" is a step in which the mature dendritic cells obtained by culturing in the pulse step are observed under a microscope or the like to confirm whether the dendritic cell vaccines of each group have been produced with the specified quality, such as the maturation rate and survival rate of the dendritic cells and whether there is any contamination due to the inclusion of foreign matter.

[0142] The maturation rate of dendritic cells is determined by the amount of ligand taken up and maturation, which typically requires a culture period of 2 to 3 days. Dendritic cells only acquire the ability to present antigens once they have matured, so if they are not fully matured, sufficient effects will not be obtained. Therefore, if the maturation rate is determined to be low, it is desirable to increase the maturation rate by extending the culture period, etc.

[0143] Regarding survival rates, some cells die during the process of differentiating from monocytes to immature dendritic cells and then to mature dendritic cells. In addition, some cells die after reaching the end of their lifespan after maturation. If a large number of cells die, the effectiveness decreases, and measures such as increasing the dosage may be necessary.

[0144] Contamination due to foreign matter involves checking for fungi that have been mixed in during the production process of this cocktail dendritic cell vaccine, or for the proliferation of bacteria that were originally present in the blood during culture.If contamination due to the proliferation of fungi or bacteria is found, it is dangerous to administer this cocktail dendritic cell vaccine to patients, so production of this cocktail dendritic cell vaccine must be stopped and the preparation steps must be started again.

[0145] The number of mature dendritic cells per unit area is also counted to estimate the total number of mature dendritic cells. This makes it possible to know how many doses of dendritic cell vaccine have been secured. <Embodiment 5: Explanation of the flowchart, mainly corresponding to claim 5>

[0146] 7 is a flowchart showing the procedure of embodiment 5. Explanation of steps that overlap with other embodiments will be omitted, and only the steps that are different will be explained. In the observation result acquisition step 0710, the state inside the dendritic cell vaccines that have been divided into multiple groups and cultured in the pulse step 0704 is observed using a microscope or the like to obtain information related to the quality of the dendritic cell vaccine, such as the maturity of the dendritic cells, the survival rate of the dendritic cells, the number of dendritic cells, and whether or not there is contamination due to foreign matter. If there are no problems with the dendritic cell vaccines in each group, the process proceeds to the storage step 0705, where they are stored until administration. <Embodiment 5: Effect mainly corresponding to claim 5>

[0147] By checking the condition of dendritic cells in dendritic cell vaccines and whether they are contaminated with foreign matter, the quality of the cocktail dendritic cell vaccine can be guaranteed and medical problems can be prevented in advance. In addition, by checking the activity level and cell quantity of dendritic cells, it is possible to create a dosing plan and estimate the post-administration effects in advance. <Embodiment 6: Mainly corresponds to claim 6 Overview>

[0148] In the sixth embodiment, an administration plan for the cocktail dendritic cell vaccine is created based on the observation results obtained in the observation result obtaining step, and a storage period is determined in the storage step. The effectiveness of dendritic cell vaccines varies depending on the number, activity, and maturity of dendritic cells, but the quantity and quality of dendritic cells obtained by culture largely depend on the properties of the patient's monocytes. Therefore, a feature of this embodiment is that, based on the actual culture results, an administration plan is created in the storage step, and the number of aliquots to be divided into storage containers and the amount of dendritic cell vaccine to be stored in the storage containers are determined and stored accordingly. <Configuration of Embodiment 6, mainly corresponding to claim 6>

[0149] The sixth embodiment has the same configuration as the fifth embodiment, but differs in the effect of the storage step. <Embodiment 6: Mainly corresponds to claim 6 Configuration Storage step>

[0150] In the storage step of this embodiment, the dosage per administration, the number of administrations, etc. are determined based on the observation results obtained in the observation result obtaining step, such as the number of mature dendritic cells and the activity of dendritic cells, and the number of divisions to be divided in the storage container dividing step described above is then determined. Furthermore, in the storage period determination step, the dosage and number of administrations may be determined based on the administration interval and administration period determined based on the patient's immune status and disease information. <Embodiment 6: Processing flow mainly corresponding to claim 6> The processing flow of the sixth embodiment is the same as that of the fifth embodiment, but the action of the storage step is different. Details of the action of the storage step have been explained in the configuration, so they will be omitted here. <Embodiment 6: Effect mainly corresponding to claim 6>

[0151] The quality of each dendritic cell vaccine is confirmed by observation, and the dosage and number of administrations are determined, so that dendritic cell vaccines with guaranteed effectiveness can be divided and stored according to the quality of the immunostimulatory vaccine. <Embodiment 7: Overview, mainly corresponding to claim 7>

[0152] In embodiment 7, at least one of the multiple groups of immature dendritic cells divided in the division step is cryopreserved, and the subsequent pulsing step and storage step are carried out at a different time from the other groups. <Embodiment 7 Configuration mainly corresponding to claim 7>

[0153] In addition to the configuration of any one of embodiments 1 to 6, embodiment 7 further includes a frozen / refrigerated storage step in which at least one of each group divided between the division step and the pulping step is frozen or refrigerated, thereby producing a cocktail dendritic cell vaccine. <Embodiment 7: Mainly corresponds to claim 7 Configuration: Freezing / refrigerating storage step>

[0154] In the "freezing / refrigerating storage step," a portion of the immature dendritic cells divided into multiple groups is frozen and stored. These are then stored for a period of approximately one to three months. After this, the ligand to be used in the pulsing step is determined, and the pulsing and storage steps are carried out to create one or more dendritic cell vaccines that make up the cocktail dendritic cell vaccine. Of course, storage for longer than three months is not prohibited.

[0155] This storage period is intended to ensure time to identify and generate the composition of proteins (including peptides) to be used as ligands from patient-derived tissues, such as neoantigens. Patient-derived tissues are the patient's own cells, and are used to search for genetic mutations that are beginning to transform into cancer cells and identify their composition, which requires a long time to generate. For this reason, until the patient-derived ligand is produced, dendritic cell vaccines are produced using existing ligands from immature dendritic cells that were not preserved in the freezing or refrigeration steps, and then administered. Once the patient-derived ligand is produced, a dendritic cell vaccine using this ligand is produced and administered instead.

[0156] Furthermore, the effectiveness of a dendritic cell vaccine with the ligand used cannot be determined until it is actually administered. This is due to the compatibility between the patient's cancer and the antigen used as the ligand, and if the effect is low, changing to a different ligand may be more effective. For this reason, we administer a dendritic cell vaccine produced with immature dendritic cells that were not stored in the freezing and refrigeration steps, confirm the effect, and then decide on the ligand to be used for immature dendritic cells that were stored in the freezing and refrigeration steps. To determine whether the vaccine is effective, after administering the dendritic cell vaccine to the patient, immune and disease information is acquired in the immune and disease information acquisition step, and a ligand is selected in the ligand selection step based on that immune and disease information. <Embodiment 7: Processing flow mainly corresponding to claim 7>

[0157] 8 is a flowchart showing the procedure of embodiment 7. Explanation of steps that overlap with other embodiments will be omitted, and only the steps that are different will be explained. In the freezing / refrigerating preservation step 0811, at least one of the multiple groups that were differentiated into immature dendritic cells in the division step 0803 is frozen / refrigerated for preservation. The remaining groups are directly transferred to the pulse step 0804 and the storage step 0805. The group preserved in the freezing / refrigerating preservation step undergoes the ligand step and storage step at a later timing than the other groups. <Embodiment 7: Effect mainly corresponding to claim 7>

[0158] By storing the immature dendritic cells divided in the division step under freezing or refrigeration, treatment can be carried out using a portion of the dendritic cell vaccine produced using existing ligands by the method for producing a cocktail dendritic cell vaccine of the present invention, while identifying and producing cancer antigens derived from the patient's biological tissue, and once completed, producing a dendritic cell vaccine using the frozen and stored immature dendritic cells. This allows the production of a dendritic cell vaccine with a stronger anti-cancer effect.

[0159] Furthermore, it is not possible to know whether the ligand is sufficiently effective in the patient until it is administered. If the effect of administering the dendritic cell vaccine from the previously generated group is not sufficient, a dendritic cell vaccine can be generated using a different ligand. Whether or not it is effective can be determined by examining the rate of killer T cell induction. If a sufficient effect is observed, the same ligand as the previously generated dendritic cell vaccine can be used again. This increases the chances of achieving a greater anti-cancer effect. <Embodiment 8: Mainly corresponds to claim 8 Overview>

[0160] In embodiment 8, a cancer antigen based on the patient's individual biological tissue is generated and used as a ligand. <Embodiment 8: Configuration mainly corresponding to claim 8>

[0161] In addition to the configuration of any one of embodiments 1 to 7, embodiment 8 further includes a ligand generation step of generating a ligand based on the patient's individual body tissue, and the pulse step is configured to pulse the ligand generated in the ligand generation step to produce a cocktail dendritic cell vaccine. <Embodiment 8 (mainly corresponding to claim 8) Configuration: Ligand generation step>

[0162] The "ligand generation step" is a step in which cancer antigens to be used as ligands are generated from patient-derived tissue. Patient-derived tissue refers to tissue obtained from the patient's cancer cells or cells beginning to transform into cancer cells, and the identified composition of proteins (including peptides) is generated. For example, cancer cells or cells transforming into cancer cells have mutated gene structures not found in other normal cells, and this can be used to identify them.

[0163] In this way, cancer antigens produced from patient-derived tissues are, unlike artificial antigens, ideal markers for the patient's cancer, and are therefore expected to be highly effective. <Embodiment 8: Processing flow mainly corresponding to claim 8>

[0164] 9 is a flowchart showing the procedure of embodiment 8. Explanation of steps that overlap with other embodiments will be omitted, and only the steps that are different will be explained. The ligand generation step 0912 is performed in parallel with the preparation step 0901, the blood component collection step 0902, and the division step 0903, and must be completed at least before the pulsing step 0904. In this ligand generation step, a ligand is generated based on the patient's own biological information, and the ligand is used in the pulsing step on at least one of the immature dendritic cells divided into multiple groups. In this way, one or more of the multiple dendritic cell vaccines that make up the cocktail dendritic cell vaccine are generated based on the patient's own biological information. <Embodiment 8: Effect mainly corresponding to claim 8>

[0165] According to this embodiment, one of the dendritic cell vaccines that make up the cocktail dendritic cell vaccine uses a cancer antigen generated based on the patient's own biological information (tissue derived from the patient), so reliable effectiveness can be expected. <Embodiment 9: Mainly corresponds to claim 9 Overview>

[0166] In embodiment 9, a cancer antigen based on the patient's individual biological tissue is generated and used as a ligand. <Configuration of Embodiment 9, mainly corresponding to Claim 9>

[0167] In addition to the configuration of any one of embodiments 1 to 8, embodiment 9 produces a cocktail dendritic cell vaccine by further including a filling step of filling each group pulsed with a ligand that has been stored after a storage period into an injection device (including, but not limited to, a syringe; the same applies below) for introducing each group back into the patient's body on a group-by-group basis. <Embodiment 9: Mainly corresponds to claim 9 Configuration: Filling step>

[0168] A single dose may be contained in one storage container, or in some cases multiple containers may be used for one dose depending on the patient, but one syringe or other device is used for one administration, and one dose is filled into it. <Embodiment 9: Processing flow mainly corresponding to claim 9>

[0169] Figure 10 is a flowchart showing the procedure of embodiment 9. Explanations of steps that overlap with other embodiments will be omitted, and only the different steps will be described. In filling step 1013, a single dose of the dendritic cell vaccine stored and frozen in a storage container in storage step 1005 is thawed and filled into a syringe or the like. The cocktail dendritic cell vaccine filled in a syringe or the like is then administered to a patient. <Embodiment 10: Mainly corresponds to claim 10 Overview>

[0170] Embodiment 10 is a method for producing a cocktail dendritic cell vaccine, including any one of Embodiments 1 to 9, characterized in that the ligand used in the ligand pulse step is any two or more of WT1 peptide, neoantigen, and α-galactosylceramide. <Embodiment 10: Mainly corresponds to claim 10 Details>

[0171] As explained in the ligand selection step above, WT1 peptide is a protein structure expressed in almost all cancers, making it expected to be effective in various cancer sites and for a wide range of patients. On the other hand, neoantigens are expected to be highly effective because they use tissues in which the WT1 peptide is expressed in the patient's cancer. However, they require analysis of the patient's own body tissues to identify the structure, which can be time-consuming and expensive. For this reason, neoantigens are likely to be administered to patients who can afford it, and they may be administered alone or in combination with WT1 peptide. As for α-galactosylceramide, it is likely to be administered to most patients, and its use in combination with WT1 peptide or neoantigen is expected to be highly effective. <Configuration corresponding to claim 10 in embodiment 10>

[0172] The configuration of the tenth embodiment is the same as the configuration of any one of the first to ninth embodiments. <Embodiment 10: Processing flow mainly corresponding to claim 10>

[0173] The process flow of embodiment 10 is the same as the process flow of any one of embodiments 1 to 9. A cocktail dendritic cell vaccine is produced by pulsing two or more of the WT1 peptide, neoantigen, and α-galactosylceramide in the ligand pulsing step. <Embodiment 10: Mainly corresponds to claim 10 Effect>

[0174] According to this embodiment, a specific immune dendritic cell vaccine using the WT1 peptide as a ligand, which is likely to be effective against various cancers in many people, is used in combination with a non-specific immune dendritic cell vaccine using α-galactosylceramide as a ligand, which is suitable for everyone and is expected to be effective, thereby enabling high anti-cancer effects to be expected against various cancers in many people. [Explanation of symbols]

[0175] Effect of α-galactosylceramide-pulsed dendritic cell vaccine 0202 Promoting dendritic cell maturation 0203 Adjuvant action 0204 Apoptosis-inducing effect on cancer cells 0205 Checkpoint inhibitory activity 0206 Anti-angiogenic effect 0210 Cancer antigen presentation 0211 Cancer cell attack instructions 0212 Instruction for antibody production against cancer antigens 0213 Killer T cells attack cancer cells 0214 Destructive effect of new blood vessels 0220 NK cell attack on cancer cells 0221 Antigen presentation by dendritic cells 0222 Cancer antigen presentation by macrophages 0223 Antibody production in cancer 0224 Cancer cell phagocytosis by macrophages

Claims

1. 1. A cocktail dendritic cell vaccine for treating leukemia or solid malignant tumors, comprising a combination of a first dendritic cell vaccine and a second dendritic cell vaccine, the first dendritic cell vaccine comprises first mature dendritic cells obtained by pulsing a first group of immature dendritic cells with α-galactosylceramide and culturing the cells; the second dendritic cell vaccine comprises second mature dendritic cells obtained by pulsing a second group of immature dendritic cells with a WT1 peptide and culturing the resulting cells; the first group of immature dendritic cells and the second group of immature dendritic cells are prepared by dividing monocytes collected in a single blood component collection procedure from a patient with leukemia or solid malignant tumor who is ready for blood component collection into a plurality of groups, and then differentiating each group of monocytes into immature dendritic cells, or by dividing immature dendritic cells differentiated from monocytes collected in a single blood component collection procedure from the patient into a plurality of groups; A cocktail dendritic cell vaccine, wherein the first dendritic cell vaccine and the second dendritic cell vaccine are administered to the patient simultaneously.

2. The cocktail dendritic cell vaccine according to claim 1, wherein the first dendritic cell vaccine and the second dendritic cell vaccine are mixed in advance before administration.

3. The cocktail dendritic cell vaccine according to claim 1, wherein the first dendritic cell vaccine and the second dendritic cell vaccine are administered separately, one type at a time.

4. 2. A method for producing a cocktail dendritic cell vaccine for treating leukemia or solid malignant tumors according to claim 1, comprising: a preparation step of collecting a predetermined amount of monocytes from a patient with leukemia or a solid malignant tumor who is ready for apheresis in a single blood collection operation; a dividing step of dividing monocytes obtained by blood component collection only from the patient into a plurality of groups, and then differentiating each group of monocytes into immature dendritic cells, or dividing immature dendritic cells differentiated from monocytes obtained by blood component collection only from the patient into a plurality of groups; a pulsing step in which at least one first group of immature dendritic cells from the groups divided in the dividing step is pulsed with α-galactosylceramide, and the remaining second group of immature dendritic cells is pulsed with WT1 peptide as a ligand, and cultured to mature the cells into first and second mature dendritic cells, respectively; a storage step of storing the ligand-pulsed mature dendritic cells of each group; A manufacturing method comprising:

5. an immunity / disease information acquisition step, which acquires, before the preparation step, one or more of immunity information, which is information about the patient's immunity, biological information of the patient, and disease information of the patient; The method for producing a cocktail dendritic cell vaccine according to claim 4, further comprising a ligand selection step in which the ligand pulsed in the pulsing step is selected according to the acquired immunity / disease information.

6. The method for producing a cocktail dendritic cell vaccine according to claim 5, further comprising a step of determining a division ratio in the division step, the division ratio being determined based on the acquired immunity / disease information.

7. 7. The method for producing a cocktail dendritic cell vaccine according to claim 5 or 6, further comprising a storage period determination step in which the storage period for each group in the storage step is determined based on the acquired immunity and disease information.

8. Further comprising an observation result acquisition step of observing each group pulsed with the ligand after the pulsing step to acquire an observation result; A method for producing a cocktail dendritic cell vaccine according to any one of claims 4 to 7, wherein the storage period for each group in the storage step is further determined based on the observation results obtained in the observation result obtaining step.

9. A method for producing a cocktail dendritic cell vaccine described in any one of claims 4 to 8, further comprising a freezing / refrigerating storage step in which at least one of each divided group is frozen or refrigerated between the dividing step and the pulsing step.

10. A method for producing a cocktail dendritic cell vaccine described in any one of claims 4 to 9, further comprising a filling step of filling each group pulsed with a ligand that has been stored after a storage period into an injection device (including, but not limited to, a syringe) for introducing each group back into the patient's body on a group-by-group basis.

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