JP2023119754A - Production method of cocktail dendritic cell vaccine - Google Patents

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 The present invention relates to a method for producing a vaccine that activates immunity and enhances anticancer effects, which is produced based on the patient's own cells. “Cancer vaccine” is a vaccine that suppresses the progress of cancer by enhancing the “immunity against cancer” of the patient, and also reduces or eliminates cancer cells remaining in the body. Many of the "cancer vaccines" currently in use are not for the prevention of cancer, but for the purpose of treatment, and are used for "immunotherapy against cancer."

In recent years, cancer immunotherapy has been recognized as a practical therapeutic method along with other surgical therapies, chemotherapy, and radiotherapy. Immunotherapy against cancer is a treatment method that enhances the aggressiveness of the immune system against cancer by strengthening the body's immunity or making immune cells recognize cancer as an attack target. So far, BRM therapy, cytokine therapy, cellular immunotherapy, vaccine therapy, etc. have been performed.
In particular, since the beginning of the 2000s, vaccine therapy using dendritic cells has been performed, and high effects have been observed.
Here, each immunotherapy is briefly described.

BRM (biological response modifier) therapy is a therapeutic method that has been performed in the early stage represented by Maruyama's vaccine, and is a therapeutic method using a substance that modifies a patient's biological response to tumor cells and the like. Known BRMs include PSK, bestatin, OK-432, and the like. Although this treatment method has been shown to be effective for some cancers, it is an adjunctive therapy that is effective when used in combination with other treatments that weaken the immune system, such as surgical therapy and chemotherapy. There is a strong aspect of physical therapy. In addition, there is a problem that the anti-cancer effect etc. by itself is weak, because the immunity is not always strengthened.

Cytokine therapy is a therapeutic method that kills cancer cells and virus-infected cells by directly in vivo administering cytokines that proliferate or activate lymphocytes such as T cells and NK cells. For example, cytokine therapy by administration of interleukin 2 (hereinafter referred to as "IL-2") is applicable. However, the clinical results of this therapy were not as effective as expected. It had the problem of producing serious side effects.

Cellular immunotherapy is a therapeutic method that enhances the immune system of a patient by ex vivo activation, proliferation, etc. of immune cells collected from the patient and then returning them to the body of the patient. It is also called therapy (broadly defined adoptive immunotherapy). Narrowly defined adoptive immunotherapy is classified into activated lymphocyte therapy and NK cell therapy depending on the type of immune cells treated in vitro.

Activated lymphocyte therapy is a narrowly defined activated lymphocyte therapy (activated T lymphocyte therapy) that activates and proliferates T cells in vitro, and activation that activates and proliferates NK cells. It is further classified into NK cell therapy.

Vaccine therapy is currently divided into peptide vaccines and dendritic cell vaccines. Peptide vaccines are a method of directly administering peptides, which serve as cancer markers, into the body as antigens. Immune cells respond to these antigens, recognizing cancer as a target of attack, and exhibiting a cancer suppressing effect. It is what is done. On the other hand, dendritic cell vaccines activate killer T cells and helper T cells to fight cancer by injecting antigen-presenting cells called dendritic cells with antigens that serve as cancer markers into the body. It increases aggression.
According to current research results, it is known that dendritic cell vaccines presenting antigens by dendritic cells are more effective than peptide vaccine therapy in which antigens are directly administered.
In the method for producing a cocktail dendritic cell vaccine of the present invention, monocytes of equivalent conditions (with little variation in monocyte quality) collected in one course of component blood collection are divided to obtain a plurality of types of equivalent conditions. to generate a dendritic cell vaccine.

In the dendritic cell vaccine, monocytes collected from a patient are cultured into immature dendritic cells, a ligand (an example of an antigen) is added (sometimes called a pulse) and cultured to form mature dendritic cells. As a result, it becomes a dendritic cell vaccine that presents a specific antigen.
This dendritic cell vaccine therapy is classified into what is called specific immunotherapy and what is called non-specific immunotherapy, depending on the antigen that is also used for this dendritic cell vaccine therapy.

<Specific immunotherapy>
Specific immunotherapy against cancer is the production of an appropriate amount of dendritic cells in vitro, with cancer marker antigens as ligands originating from the patient's cells, and using these as vaccines to produce killer T cells, It is a treatment method that makes helper T cells recognize cancer cells as targets and attack them. As a representative ligand, WT1 (Double T-One) peptide is used and is effective against a wide variety of cancers. This is supported by the confirmation that the WT1 gene is expressed as an antigen in various solid malignant tumors such as leukemia, lung cancer, colon cancer, pancreatic cancer, bone and soft tissue sarcoma, and malignant glioma. . Therefore, the use of a vaccine with the WT1 peptide as a ligand that allows dendritic cells to retain this means that it is possible to treat a wide variety of cancers.

<Non-specific immunotherapy>
Non-specific immunotherapy is a therapeutic method aimed at strengthening the overall immunity.
BRM therapy and cytokine therapy are also classified as non-specific immunotherapy, but in dendritic cell vaccines, NKT cells are activated by using α-galactosylceramide, an NKT cell activating ligand, resulting in high Immunostimulatory effects have been confirmed.

NKT cells are called the fourth lymphocytes, and are a type of T cells that have the characteristics of NK cells. NKT cells are activated by presentation of glycolipid antigens and produce various cytokines, and thus have the effect of inducing both immunostimulatory and suppressive reactions. Immunostimulation activates the immune system and strengthens defenses against external enemies, and immunosuppression suppresses immune overreaction and inflammation. can be maintained. In addition to being able to induce cell death of cancer cells, it has various anti-cancer effects, but its direct attack effect on cancer is low. Therefore, the anticancer action of NKT cells is expected to have secondary effects mediated by other immune cells.

Six anticancer effects of NKT cells are described below.

(1) Maturation of dendritic cells As dendritic cells mature, they present cancer antigens to immune cells to promote cancer attacks. cells cannot mature, and the suppressive effect on cancer is reduced. Cytokines produced by NKT cells mature dendritic cells to enhance the immune function against cancer.

(2) Adjuvant action It produces a substance called cytokine (IFN-γ) and has the action of activating and proliferating various immune cells. It has a suppressive effect on cancer by activating killer T cells, NK cells and macrophages.

(3) Direct Killing Effect on Cancer Cells Activated NKT cells have the effect of inducing cell death by apoptosis of cancer cells.

(4) Checkpoint inhibitory action Immune cells have receptors called immune checkpoints that suppress immune overreactions. Cancer cells use this effect to produce immunosuppressive cells (cells with immune checkpoint inhibitory function that produce immune checkpoint molecules or their ligands) to avoid attacks from immune cells. There is an action to kill this immunosuppressive cell, and the immunosuppression is released. Therefore, the attack on cancer cells by killer T cells is promoted to induce anticancer effects.

(5) Inhibition of angiogenesis Suppresses the formation of new blood vessels that supply nutrients and oxygen for cancer tissue growth.
As a result, cancer cells are unable to acquire nutrients, so it has the effect of suppressing the growth of cancer cells.

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

As described above, immunotherapy targeting NKT cells is a relatively new therapeutic method among immunotherapies for cancer, and is expected to enhance overall immunity.

Thus, various cancer immunotherapies have been developed and improved so far, and there are many methods for producing vaccines. For example, in Patent Document 1, dendritic cells and zinc at a concentration of 200 ng/mL to 400 ng/mL are included, and the cell viability of the dendritic cells is higher than when washed with saline. A 10% increase in cell viability has produced a high-quality dendritic cell vaccine. Furthermore, this dendritic cell is matured by adding a cancer antigen containing the WT1 peptide, and enhances the anticancer effect as a dendritic cell vaccine corresponding to specific immunotherapy.

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 the dendritic cells. This is a method for producing a dendritic cell vaccine, which is a non-specific immunotherapy that activates a wide range of immune cells.

JP 2018-83849 Patent No. 6447947

However, both of the dendritic cell vaccines disclosed in Patent Documents 1 and 2 are undergoing clinical studies, and are being confirmed to be more effective than conventional immunotherapy. Expected effects have not been obtained in cancer patients. This means that dendritic cell vaccines can be vaccines for various cancers and diseases depending on the antigens used. However, it can be said that a sufficient effect cannot be obtained unless the vaccine has an antigen that matches the symptoms of the patient.

For example, when specific immunotherapy is performed using the dendritic cell vaccine of Patent Document 1, even if the ability of lymphocytes to recognize cancer increases, the overall immunity of the patient itself is reduced, which is sufficient. In some cases, the therapeutic effect does not appear, such as no significant effect is obtained, or the patient dies due to other diseases due to complications.

In addition, when non-specific immunotherapy is performed using the dendritic cell vaccine of Patent Document 2, the patient's immune function is improved, but the anticancer effect is not necessarily exhibited. If the patient's immunity does not recognize cancer as an attack target, a sufficient anticancer effect cannot be obtained, and as a result, it may not appear as a cancer treatment effect.

In other words, it activates and proliferates various immune cells that are responsible for immunity in the body, and then makes the specific immune system clearly recognize the cancer cells that are targeted for attack. is important.
In addition, reducing the burden on cancer patients is also important for activating the immune system as a result, but many conventional cancer therapies impose a heavy physical burden on patients. It should also be noted that cancer patients who use immunotherapy often have advanced cancer stages and often have limited time and physical strength. be. In order to generate a dendritic cell vaccine, it is necessary to collect and differentiate the patient's own monocytes, so it is necessary to collect blood components. Monocytes are separated by a blood collection device and returned to the patient's body, which takes a long time and may cause side effects due to the anticoagulant mixed in the blood. For this reason, it is desired that the blood collected in one blood collection exhibits the maximum effect and recovers to the extent that the next therapeutic action can be performed.

In view of such circumstances, the present invention activates and proliferates (this point is important) various immune cells responsible for immunity in the body by nonspecific immunotherapy, and then, by specific immunotherapy, It has a synergistic therapeutic effect on various cancer patients by making the cancer cells to be attacked clearly recognized by the specific immune system. It is intended to provide a cocktail dendritic cell vaccine that can be obtained.

The present invention is a method for producing a cocktail dendritic cell vaccine that activates a patient's immunity. a component blood collection step of collecting a predetermined amount of monocytes from the patient by one puncture (meaning that the collection of a predetermined amount of blood is completed in one course; multiple punctures are allowed); a division step of dividing monocytes collected from blood or immature dendritic cells differentiated from monocytes into a plurality of groups; A cocktail dendritic cell vaccine exclusive for the patient is produced by a pulsing step of pulsing at different timings and a storing step of storing each group pulsed with the ligand.

In addition to the first invention, the cocktail dendritic cell vaccine of the second invention includes, before the preparation step, immune strength information, which is information about the patient's immunity, individual patient's biological information, and patient's disease information. an immunity/disease information acquisition step of acquiring any one or more of them, wherein the ligand pulsed in the pulse step is selected according to the acquired immunity/disease information, and of cocktail dendritic cell vaccines. .

In addition to the second invention, the cocktail dendritic cell vaccine of the third invention further has a division ratio determination step of determining the division ratio of the division step according to the acquired immunity/disease information. to produce a cocktail dendritic cell vaccine.

Further, in the cocktail dendritic cell vaccine of the fourth invention, in addition to the second invention or the third invention, the storage period of each group in the storage step is determined according to the acquired immunity/disease information A cocktail dendritic cell vaccine is manufactured by further having a storage period determination step to determine.

Further, the cocktail dendritic cell vaccine of the fifth invention is, in addition to any one of the first to fourth inventions, an observation in which observation results are obtained by observing each group pulsed with the ligand after the pulse step A cocktail dendritic cell vaccine is produced by further having a result obtaining step.

Further, in the cocktail dendritic cell vaccine of the sixth invention, in addition to any one of the first to fifth inventions, the storage period of each group in the storage step is further acquired in the observation result acquisition step A cocktail dendritic cell vaccine is manufactured based on the observation results.

Further, in the cocktail dendritic cell vaccine of the seventh invention, in addition to any one of the first to sixth inventions, at least one or more of each group divided between the division step and the pulse step is stored frozen. Alternatively, a method for producing a cocktail dendritic cell vaccine is performed by performing a freezing/refrigerating storage step of refrigerating.

Further, the cocktail dendritic cell vaccine of the eighth invention, in addition to any one of the first to seventh inventions, further has a ligand-generating step of generating a ligand based on the patient's individual body tissue, The pulsing step is configured to pulse the ligands generated in the ligand generating step to produce a cocktail dendritic cell vaccine.

Further, in the cocktail dendritic cell vaccine of the ninth invention, in addition to any one of the first to eighth inventions, each group pulsed with the ligand stored after the storage period is grouped and the patient A cocktail dendritic cell vaccine is manufactured by having a filling step of filling an injection device (including but not limited to a syringe, the same applies hereinafter) for returning to the body of the.

Further, in the cocktail dendritic cell vaccine of the tenth invention, in addition to any one of the first to ninth inventions, the ligand pulsed in the pulse step is selected from WT1 peptide, α-galactosylceramide, and neoantigen. A cocktail dendritic cell vaccine characterized by two or more ligands is produced.

INDUSTRIAL APPLICABILITY According to the method for producing a cocktail dendritic cell vaccine of the present invention, it is possible to produce a plurality of dendritic cell vaccines that are expected to have a high therapeutic effect on cancer from limited blood components.

Overview of the method for producing a cocktail dendritic cell vaccine of the present invention A diagram showing an example of the action of a dendritic cell vaccine produced by the method for producing a cocktail dendritic cell vaccine of the present invention on immunity. 1 is a flow chart showing a method for producing a cocktail dendritic cell vaccine according to Embodiment 1. Flowchart showing the production method of the cocktail dendritic cell vaccine production method of Embodiment 2 Flowchart showing the production method of the cocktail dendritic cell vaccine production method of Embodiment 3 Flowchart showing the production method of the cocktail dendritic cell vaccine production method of Embodiment 4 Flowchart showing the production method of the cocktail dendritic cell vaccine production method of Embodiment 5 Flowchart showing the production method of the cocktail dendritic cell vaccine production method of Embodiment 7 Flowchart showing the production method of the cocktail dendritic cell vaccine production method of Embodiment 8 Flowchart showing the production method of the cocktail dendritic cell vaccine production method of Embodiment 9 A diagram showing an example of a table for determining the division ratio in the division step Diagram 1 showing an example of the storage period determined in the storage step Figure 2 showing an example of the storage period determined in the storage step A table that ranks the anticancer effects of immunization by dendritic cell vaccines

First, before describing the embodiments of the present invention, the immunity of the body will be described.
Immunity is divided into innate immunity that organisms originally have and acquired immunity that is acquired later.

Innate immunity is the immunity that the human body is naturally equipped with, and is composed mainly of phagocytic cells that eat and remove foreign substances such as bacteria and viruses. Phagocytic cells have receptors that recognize molecules and structures, and are roughly divided into types that process and eliminate foreign substances and those that play a role in transmitting signals within cells. Immune cells include eosinophils, neutrophils, basophils, macrophages, dendritic cells, and NK (natural killer) cells.

Acquired immunity, on the other hand, is the mechanism by which an infected pathogen is remembered and can be effectively eliminated when re-encountered. Although it takes longer to respond than innate immunity, it has the versatility to respond to various pathogens. Immune cells include B cells and T cells, and T cells further include helper T cells, killer T cells, and regulatory T cells. B cells change into plasma cells and memory B cells depending on the state.

Furthermore, some NKT cells have properties of both innate and acquired immunity.
Among the above-mentioned immune cells, the cells on which this cocktail dendritic cell vaccine directly acts are NKT cells, helper T cells, and killer T cells. Cells, macrophages, dendritic cells, B cells, etc. are also activated.

Immune cells involved in the action of the dendritic cell vaccine produced by the method for producing the cocktail dendritic cell vaccine and substances involved in the immune function will be briefly described below.

<Explanation of terms for immune cells and substances related to immune function>
<Term description: Antigen>
It is a foreign substance that enters the body and serves as a landmark for adaptive immune cells to recognize it as a target of attack. In the case of cancer antigens, immune cells recognize them as targets of attack by using a protein structure that does not exist in normal cells as a landmark.

<Term description: Antibody>
A general term for proteins called immunoglobulins. Cancer antibodies bind to cancer cells and attack cancer cells through the following three actions.
Neutralizing action: Action to prevent the proliferation of cancer cells ADCC activity: Action to help killer T cells and NK cells to attack cancer CDC activity: Action to activate complement and damage cancer cells

<Term description: Cytokine>
It is a protein secreted from immune cells and is a general term for physiologically active substances involved in intercellular actions. It transmits signals to target cells and induces various cellular responses such as cell activation, cell proliferation, differentiation, cell death, and functional expression. Interferons and interleukins are classified as cytokines.

<Term description: Interleukin>
At present, more than 30 types of interleukins, which are a type of cytokine, have been confirmed. In particular, it is abundantly secreted by lymphocytes, a type of white blood cell that fights against foreign substances in the body, and phagocytic cells such as macrophages and neutrophils.

<Glossary: Interferon>
Interferon, which is a type of cytokine, activates NK cells and macrophages that attack cancer cells and virus-infected cells, and suppresses the proliferation of viruses and tumor cells. It is also nationally approved as an antiviral and anticancer agent, and is used to treat multiple myeloma, brain tumors, and kidney cancer.

<Term description: NK cells>
NK cells have various receptors that catch abnormal cells, and have the effect of damaging cells recognized as abnormal, such as virus-infected cells and cancer cells.

<Terminology: Macrophage>
Macrophages are a type of leukocyte that are activated by signals from biological tissues and environmental stress, and phagocytize foreign substances such as bacteria. They are also antigen-presenting cells that present phagocytosed foreign substances as antigens to helper T cells. It is also activated by cytokines (IFN-γ) produced by helper T cells.

<Terminology: Dendritic cells>
They are antigen-presenting cells that take up foreign substances and present the antigens to T cells, which have the role of recognizing them as targets of attack. Early dendritic cells are called immature dendritic cells that have the ability to take up antigen (phagocytosis).

<Explanation of terms: helper T cells>
When presented with an antigen, it recognizes it as an attack target and instructs killer T cells to attack. It also produces substances called cytokines to activate B cells, and works with B cells to determine whether a foreign substance is dangerous or to assist in the production of antibodies.

<Explanation of terms: killer T cells>
Killer T cells recognize antigens presented by MHC class I molecules and damage the cells. In this way, the response of T cells, which is acquired immunity, begins with antigen presentation of antigenic peptides by MHC molecules. At the direction of helper T cells, cells that are dangerous or unnecessary for the body, such as virus-infected cells and cancer cells, are killed. kill and remove.

<Explanation of terms: B cells>
They are stimulated by cytokines produced by helper T cells to produce antibodies. Even if the antigen is eliminated, it becomes a memory B cell and has a role to memorize the antigen and prepare for the next invasion.

<Term description: NKT cells>
These cells have characteristics of both T cells and NK cells. When activated, it also produces cytokines (IFN-γ) and induces responses to immunostimulatory/suppressive therapy. The most expected effect of the present invention is an adjuvant effect, which is to activate various immune cells in the body and, importantly, to proliferate them.

<Overview of cocktail dendritic cell vaccine>
Next, with reference to FIGS. 1 and 2, an overview of the method for producing a cocktail dendritic cell vaccine of the present invention and a mechanism of improving immunity by the cocktail dendritic cell vaccine will be described.

FIG. 1 is a diagram showing the outline of the cocktail dendritic cell vaccine production method of the present invention.
This cocktail dendritic cell vaccine is a vaccine cultured based on the patient's own cells, and because it is exclusively for the patient, it is a highly safe vaccine that does not cause rejection.
First, monocytes are collected from a patient by blood component collection.
“Component blood collection” is a method in which blood is taken out of the body using a medical tube or the like, and the target blood components/factors are separated from the blood using a dedicated device and returned to the body again. In the present invention, about 100 cc of blood components are collected by separating monocytes as blood components. Blood components after the monocyte separation contain plasma and platelets in addition to monocytes. In collecting blood components, an anticoagulant is mixed in the blood before separating the monocytes so that the blood does not clot, and then the anticoagulant is returned to the body. It puts a lot of strain on your body.

Next, monocytes of the collected blood components are cultured by adding cytokines or the like to induce differentiation into immature dendritic cells. Thereafter, the cultured immature dendritic cells are separated into two or more vessels, one of which is added with the ligand used for specific immunotherapy, and the other is added with the ligand used for nonspecific immunotherapy. , both are cultured to mature into dendritic cells. Thus, the dendritic cells added with the antigen and cultured are produced as a dendritic cell vaccine for a specific disease.

The quality of the cultured dendritic cell vaccine is visually confirmed with a microscope or the like.
The quality of a dendritic cell vaccine includes the survival rate of dendritic cells, the morphology of dendritic cells (whether they have many branches, etc.), and whether there is contamination due to contamination by bacteria, fungi, or the like.
A dendritic cell vaccine whose quality and safety have been confirmed is stored in a storage container. After that, it is administered according to the patient's condition.
The above is the outline of the method for producing the cocktail dendritic cell vaccine of the present invention.
As described above, according to the method for producing a cocktail dendritic cell vaccine of the present invention, a combination of multiple dendritic cell vaccines that can be expected to have a high anticancer effect from blood components collected by a single puncture treatment of a patient. can generate The term "cocktail dendritic cell vaccine" is used in the present invention, and the term "cocktail" means that the dendritic cell vaccines administered to the patient are two or more dendritic cell vaccines. As a treatment to be administered to a patient, two or more types of dendritic cell vaccines may be mixed in advance and then administered, or multiple types of dendritic cell vaccines may be individually administered one by one. In addition, when three or more types of dendritic cell vaccines are used, two or more types of dendritic cell vaccines are mixed and administered, and the remaining dendritic cell vaccines are administered one by one. It's okay.

<Action 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 different kinds of dendritic cell vaccines, one of which is a dendritic cell vaccine pulsed with the ligand α-galactosylceramide, and the other of which is WT1 as a ligand. It shows the direct action of peptide-pulsed dendritic cell vaccines on pre-existing immune cells in the body. The second thickest arrows (0202, 0203) indicate that activated NKT cells act particularly strongly compared to the effects of other activated NKT cells. Thin arrows indicate the action of immune cells in the body under the action of the dendritic cell vaccine. The ellipses represent the immune cells described within the ellipses, and the triple ellipses are the immune cells specifically boosted by the cocktail dendritic cell vaccine. , the polygons in the center indicate cancer cells. The dashed lines around cancer cells indicate the immunosuppressive effects of cancer cells, forming a barrier from immune function. The drawing of blood vessels means acquisition of nutrients to cancer cells from blood vessels due to angiogenesis of cancer cells.
Next, each action will be described with reference to the numbering.

<Action of α-galactosylceramide-pulsed dendritic cell vaccine: NKT cell activation action: 0201>
Dendritic cell vaccines pulsed with α-galactosylceramide activate NKT cells. Activated NKT cells have various effects on immune function.
Below, the main five actions among them will be described.

<Action 1 of activated NKT cells: dendritic cell maturation promoting action: 0202>
Activated NKT cells produce cytokines and promote the maturation of immature dendritic cells in the body.

<Activated NKT cell action 2: adjuvant action: 0203>
Activated NKT cells produce cytokines (IFN-γ) to activate NK cells, macrophages, and killer T cells, increasing their ability to attack cancer. It also activates various immune cells. Killer T cells and helper T cells are activated by pulsing dendritic cells with the WT1 peptide, which is a ligand that has specific immunoreactivity. B cells are simultaneously positively affected by pathways of non-specific immune activity, resulting in an effect of 1+1>2, ie, a synergistic effect. This point is one of the features of the present invention.

<Effect of activated NKT cells 3: Apoptosis-inducing effect on cancer cells: 0204>
Activated NKT cells produce a kind of serine protease to degrade DNA of cancer cells, thereby causing apoptosis of cancer cells.

<Action 4 of activated NKT cells: Checkpoint inhibitory action: 0205>
Activated NKT cells work to kill immunosuppressive cells prepared by cancer cells to protect themselves from attacks by immune cells in the body. As a result, various immunosuppressions are released, and the state of immunity within cancer tissue is greatly improved. For example, it enables killer T cells to effectively attack cancer cells. Thus, the checkpoint inhibitory action based on non-specific immune activity further effectively amplifies the cancer-attacking ability of killer T cells based on specific immune activity. This point is also one of the characteristics of the present invention that an effect of 1+1>2, that is, a synergistic effect can be obtained as described in the adjuvant action.

<Action 5 of activated NKT cells: Angiogenesis inhibitory action: 0206>
Activated NKT cells inhibit angiogenesis for cancer cells to supply nutrients, make it difficult for cancer cells to obtain nutrients, and suppress cancer cell growth.

<Action of activated macrophages: cancer cell phagocytosis by macrophages: 0224>
Activated macrophages phagocytize cancer cells and dead cancer cells.

<Action of Dendritic Cell Vaccine Pulsed with WT1 Peptide: Cancer Antigen Presenting Action: 0210>
The WT1 peptide-pulsed dendritic cell vaccine presents the WT1 peptide, which is a cancer antigen, to helper T cells and killer T cells to activate them. As a result, killer T cells recognize cancer cells as targets of attack, and helper T cells activate killer T cells and produce cytokines to activate NK cells as described later, and B cells promotes the production of antibodies.

<Action of activated helper T cells: Cancer cell attack instruction: 0211>
Activated helper T cells produce cytokines (INF-γ) and direct killer T cells to attack cancer cells based on the information contained in the cytokines. In addition, the effect of cytokines activates NK cells, B cells and macrophages, increasing the attacking action against cancer.

<Action of activated helper T cells: Antibody production instruction against cancer antigen (e.g. WT1): 0212>
Activated helper T cells produce cytokines that instruct B cells to produce antibodies against cancer antigens (eg, WT1) based on the information contained in the cytokines.

<Action of activated killer T cells: Cancer cell attack action by killer T cells: 0213>
Activated killer T cells recognize and bind to WT1 expressed in cancer cells, making holes in the cell membrane and causing necrosis of the cancer cells. Furthermore, they produce substances such as TNF-β (tumor necrosis factor, a type of cytokine) and induce apoptosis of cancer cells. Thus, the direct attack of killer T cells on cancer cells is a powerful anticancer effect that attacks cancer cells from both the outside and the inside.

<Action of activated killer T cells: Neovascular destruction action: 0214>
The WT1 gene is expressed in blood vessels generated by cancer cells, and killer T cells presented with a cancer antigen (WT1) attack not only cancer cells but also new blood vessels. This destroys new blood vessels, reduces the supply of nutrients to cancer cells, and suppresses the growth of cancer cells.
This action is also one of the factors that the cocktail dendritic cell vaccine of the present invention exhibits the effect of 1+1>2.

<Action of activated NK cells: Cancer cell attack action by NK cells: 0220>
Activated NK cells show a cytotoxic response to and attack cancer cells.

<Action by proliferating/activated macrophages: Antigen presentation action by macrophages 0222>
Activated NKT cells proliferate and activate macrophages. Proliferated and activated macrophages fragment cancer cells that have been taken in by phagocytosis and express them on the cell surface. In other words, it exerts an antigen-presenting action of presenting cancer cell antigens. This presented antigen is transferred to the dendritic cell to enable it to present further antigen.

<Cancer antigen-presenting action of dendritic cells: Cancer antigen-presenting action by dendritic cells: 0221>
Immature dendritic cells take up cancer antigens, mature, differentiate into mature dendritic cells, and present cancer antigens to helper T cells and killer T cells.
This action is an anticancer action that can normally occur in the body, but due to the action of activated NKT cells, a greater anticancer effect than usual can be obtained.
Since this cancer antigen is presented by macrophage phagocytosis of the patient's own cancer cells, it is expected to be more effective than dendritic cell vaccines that use artificial antigens as ligands.
This action is also one of the factors that the cocktail dendritic cell vaccine of the present invention exhibits the effect of 1+1>2.

<Action of activated B cells: cancer antibody production: 0223>
Activated B cells produce antibodies against cancer antigens (eg WT1) and attack cancer cells. By binding to cancer cells, these antibodies suppress the proliferation of cancer cells, and serve as markers for other immune cells (for example, NK cells and killer T cells) to promote their attack.

As described above, by combining non-specific immunotherapy and specific immunotherapy, the synergistic effects of both can be achieved, and cancer cells can be attacked by various immune functions.
Therefore, higher effects can be expected than when each therapy is performed alone.

FIG. 14 is a table empirically ranking the anticancer activity when each homogeneous dendritic cell vaccine produced according to the present invention is used. Rating A is a rating as a direct attack on cancer cells and a major anticancer effect. Rating B is a rating lower than rating A as an anticancer action and a secondary action such as suppression of cancer cell proliferation. A rating of S indicates a direct attack on cancer cells by activated immune cells, indicating an enhanced anticancer effect.

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

FIG. 14 compares the sum of the ratings of the action of the non-specific immune cell vaccine and the specific immune cell vaccine and the rating of the action of the cocktail dendritic cell vaccine of the present invention.
Among the anti-cancer effects of the non-specific immune cell vaccine and the specific immune cell vaccine, killer T cells overlap, so if one rating is included in the total, the action of rating A is 5 and the action of rating B is 5. becomes 5.
On the other hand, the anti-cancer activity of the cocktail dendritic cell vaccine is graded as 6 for grade S, 1 for grade A, and 1 for grade B. and the sum of the effects of specific immune cell vaccines.
This is due to enhancement of the activity of killer T cells and NK cells by adjuvant action, and effective anticancer action of killer T cells by checkpoint inhibitory action.
<Embodiment 1 Mainly corresponding to claim 1 Overview>

In this embodiment, monocytes are obtained from a patient by component blood collection, differentiated into immature dendritic cells, and then divided into a plurality of groups to generate different types of vaccines. Provided is a method for producing a cocktail dendritic cell vaccine that has a synergistic effect on cancer cell attack compared to the case where multiple routes act alone by utilizing their activities.
<Embodiment 1 Mainly Corresponding to Claim 1 Configuration>

Embodiment 1 of the present invention includes a preparation step for preparing to collect blood components from a patient, and a predetermined amount of monocytes is punctured once from the patient who is ready to collect blood components (collecting a predetermined amount of blood is completed in one cycle). a blood component collection step of collecting blood components in a step of collecting blood components in a step of collecting blood components in a step of collecting blood components in a step of collecting blood components in a step of collecting blood components in the step of collecting blood components in a plurality of groups of monocytes or immature dendritic cells differentiated from monocytes collected only from the patient; a dividing step of dividing into groups, a pulse step of optionally pulsing different types of ligands in each group divided by the dividing step at different timings starting from the time of division, and a storing step of storing each group pulsed with the ligand and a method for producing the patient-specific cocktail dendritic cell vaccine.
<Embodiment 1 Mainly corresponding to claim 1, configuration, preparation steps>

A "preparation step" is a step of preparing for blood component collection from a patient.
In blood collection, "monocytes", which are the origin of dendritic cells, are collected, but at this time, it is necessary to prepare enough monocytes to collect. In addition, we confirm whether there is any risk due to the disease and determine whether the patient is in a condition to withstand component blood collection.

“Monocyte” is a type of white blood cell, which is a blood cell produced in the bone marrow like other white blood cells, and accounts for 3 to 6% of white blood cells. Once inside the tissues of the body, monocytes transform into macrophages and dendritic cells. Therefore, the more monocytes exist, the more dendritic cells can be cultured.
<White blood cell count>

Therefore, the white blood cell count is examined to confirm whether or not there is a sufficient white blood cell count, and if the white blood cell count does not meet a predetermined standard, the predetermined white blood cell count is raised by medication. The white blood cell count is at least 3000/μl or more, preferably 4000/μl or more.
<Coping with bleeding risk>

In blood component collection, blood is collected outside the body, and after the components are separated, it is returned to the body, which is repeated.
For this reason, an anticoagulant is mixed into the blood so that the blood does not clot in the component blood collector. If your platelet count is lower than that of a healthy person, or if you have blood coagulation problems or bleeding from the affected area due to cancer, bleeding may not stop. Therefore, it is necessary to treat possible bleeding sites and secure platelet counts.
<Securing physical strength>

Blood component collection involves a heavy burden, so it is necessary to secure physical strength to withstand component blood collection. Patients with advanced cancer stage are likely to have weakened physical strength.
<Embodiment 1 Mainly Corresponding to Claim 1 Configuration Component Blood Collection Step>

The “component blood collection step” means that a predetermined amount of monocytes is punctured once from a patient who is ready for component blood collection (predetermined amount of blood collection is completed in one course. do not interfere) is the step of collecting blood components.

“Component blood collection” is a task of extracting blood from a patient's body (bleeding), separating target components with a component blood collecting machine, and returning the separated remaining blood to the body (blood feeding). This is repeated until the desired amount of the desired component is obtained. Therefore, one puncture includes puncturing with a needle connected to a tube for withdrawing blood and puncturing with a needle connected to a tube for returning blood to the body.

Monocytes are obtained in component blood collection involved in the production of the cocktail dendritic cell vaccine of the present invention. In a general method, a blood collection needle is inserted into the patient's left upper extremity to collect blood, and a blood collection needle is also inserted into the right upper extremity of the patient. Prick and return the treated blood. The collected blood is mixed with an anticoagulant such as sodium citrate, separated by a centrifuge, and separated into individual blood components. A layer containing monocytes is extracted from this and collected in a culture bag. The remaining ingredients are remixed and returned to the body.
It takes about 3 hours to repeat this process and process about 4 L of whole blood in the body. Therefore, although the time required for blood component collection varies depending on the amount of monocytes required, it takes approximately 1 hour and 30 minutes or longer.

In the component blood collection step, monocytes are separated as a target component, and approximately 100 cc of blood containing monocytes as the main component (which may contain plasma, platelets, etc.) is collected. This blood component preferably contains 200×10 ⁷ or more monocytic cells.

Separate blood collection tubes and needles can be used for blood withdrawal and blood delivery in a shorter time, but blood withdrawal and blood return are combined, and are performed with a single puncture. Also good. In this case, blood removal, separation, blood feeding, and operation are performed sequentially. Thus, the method is not particularly limited as long as it is a method of collecting monocytes from the patient's blood.

In addition, if it becomes possible to generate monocytes from stem cells or iPS cells in the future, stem cells or iPS cells obtained from a patient may be cultured by this method. In the present invention, in consideration of the patient's physical strength, etc., all of them are included as long as a single puncture can obtain monocytes necessary for subsequent treatments.
<Embodiment 1 Mainly corresponding to claim 1 Configuration Division step>

The "dividing step" is a step of dividing monocytes collected from the patient's own blood or immature dendritic cells differentiated from monocytes into a plurality of groups. Immature dendritic cells differentiated from monocytes are divided into separate culture vessels (for example, petri dishes, beakers, graduated cylinders, etc.) at a predetermined ratio. This ratio is divided based on what ratio ultimately produces the dendritic cell vaccine pulsed with the ligand of interest. Based on this ratio, the ligand is pulsed in the pulse step described later, and at least one of the culture vessels containing the immature dendritic cells divided into a plurality of groups is pulsed with the NKT activating ligand. , which is not less than 3% of the entire ratio and does not exceed 30%. In addition, when dividing at the monocyte stage, first divide into equal parts, mature to immature dendritic cells under the same conditions for a certain period of time, and at that stage, combine the two again and divide again. may This has the effect of homogenizing the divided immature dendritic cells. The advantage of splitting at the monocyte stage is that in the unlikely event of contamination, the contaminated split portion cannot be used, but the remaining split portion can be utilized. In addition, the conditions for maturing monocytes to immature dendritic cells may differ slightly depending on the patient's immunity, and the conditions for immature dendritic cells are good for immature dendritic cells after trial and error of multiple types of conditions. can be produced.

NKT cells are said to account for about 0.1% of T cells in peripheral blood, and are very few cells. Therefore, vaccines, which are non-specific immunotherapy for the purpose of activating NKT cells, may be wasted even if they are administered in large doses. In other words, NKT-activating ligand-pulsed dendritic cell vaccines tend to plateau with dose. Therefore, it is desirable to allocate more to specific immunotherapy.

In addition, the division in the "division step" may be performed by dividing the cells in the state of monocytes to differentiate them into immature dendritic cells. It is desirable that the container to be divided is divided into containers such as petri dishes, in which culture can be performed as it is. The division ratio at this time is also the same as in the case of division in the state of immature dendritic cells.
However, it is preferable to divide at the stage of immature dendritic cells. This is because it is possible to suppress variations in the quality of cells contained in a plurality of divided groups. This is likely because cells are subject to variability in the rate at which they mature within their culture vessel, and the effect of ligand pulsing may vary if the monocyte divides at an early stage.
<Embodiment 1 Mainly Corresponding to Claim 1 Configuration Division Step First Culture Substep>

The splitting step includes a "first culturing substep". The first culturing substep is a step of culturing monocytes collected from the patient's own blood in an incubator for about 5 to 7 days to differentiate them into immature dendritic cells. Monocytes are placed in a medium (serum-free medium optimized for dendritic cells), stimulating factor cytokines (e.g., interleukin, etc., IL4, GM-CSF, etc.) are added and cultured to generate immature dendritic cells. induces differentiation into cytoplasmic cells. A feature of live immature dendritic cells is that after 2 days in culture, downstream regulation of CD14 surface expression, increased dextran reuptake, and increased response to MIP-1a can be detected. The state of differentiation of immature dendritic cells can be observed. Regarding the culture method here, various studies and proposals have been made for efficiency and stabilization, so that method may be used. Any method can be used as long as immature dendritic cells can be efficiently and stably generated from monocytes. Note that the first culture step is preferably performed in one culture vessel. This is because variations occur in culture. However, when dividing in the monocyte state and culturing after division, each group is cultured. In addition, it is also possible to vary the conditions for culturing divided monocytes in order to control the quality of immature dendritic cells. Culture conditions can also be determined according to the type of ligand so that the ligand to be pulsed later will be effective. Regarding the effectiveness of the ligand, it is necessary to comprehensively consider the antigen-presenting ability, the rate of maturation, and the control of the properties of the immature dendritic cells that are part of the cells when they are reintroduced into the patient's body. . In addition, culture conditions include temperature, type of medium, nutrients to be given, time, and the like.
<Embodiment 1 Mainly corresponding to claim 1 Configuration Pulse step>

The “pulsing step” is a step of pulsing different types of ligands to each group divided in the division step, possibly at different timings starting from the time of division.
A "ligand" refers to a substance that forms a protein complex with a biological substance to serve a biological purpose. Here, it induces activation of immunity by specifically binding to various receptors on immune cell membranes. Ligands are composed of amino acids, peptides, proteins, glycolipids, and the like.
"Pulse" as used herein refers to the addition of an antigen-containing chemical to a culture subject to promote culture. Specifically, it is a step of adding ligands such as peptides or α-galactosylceramide to immature dendritic cells and culturing them to mature to generate mature dendritic cells. Note that the rate at which immature dendritic cells mature may differ depending on the type of pulsed ligand. Therefore, it is necessary to pulse at different timings starting from the division time. This is because specific immunotherapy and non-specific immunotherapy should be started almost at the same time in order to expect a synergistic effect. In addition, it is preferable to shorten the storage period (frozen storage period) as much as possible in order to minimize the amount of highly active mature dendritic cells or dysfunctional mature dendritic cells.

Here, there are two types of pulsing ligands, one of which is an NKT cell ligand classified as non-specific immunotherapy, which is also called NKT cell target therapy.
The other is classified as specific immunotherapy and uses cancer antigens as ligands.

An “NKT cell activating ligand” is a glycolipid that activates NKT cells.
NKT cells are activated by presentation of NKT cell activating ligands by antigen-presenting cells such as dendritic cells. Activated NKT cells can produce a large amount of interferon (IFN-γ) and activate various immune cells. In addition, it does not target cancer cells, and since the receptors of NKT cells are common without individual differences, a certain effect can be expected for everyone. In recent years, α-galactosylceramide has been attracting attention as an NKT cell activating ligand, and ligands with even higher effects than α-galactosylceramide are being researched and developed. Therefore, if a new NKT cell activating ligand is developed in the future, it may be used.

Cancer antigens are proteins (including peptides) that have a unique structure that cancer cells have, and that do not exist in normal cells. By presenting an antigen to T cells by antigen-presenting cells such as dendritic cells, the T cells are activated and recognize cancer cells as targets of attack, using the protein structure of cancer antigens as a marker. is. Therefore, if the amino acid structure of the artificial cancer antigen presented by dendritic cells matches the amino acid structure of the cancer cells invading the patient, it is effective, but if it does not match, it is ineffective. . Under these circumstances, a cancer antigen called WT1 peptide has shown relatively high efficacy in recent years.

“WT1 peptide” is an artificial cancer antigen with a protein structure that is commonly seen in various cancers, so it can be used for most cancer patients, effect can be expected. Therefore, it is very efficient to use WT1 peptide as a ligand for non-specific cancers.

In addition, there are many artificial cancer antigens other than WT1, but unlike this, proteins extracted from the patient's own cancer cells or body tissues are cultured or identified and artificially used as antigens. There is also a way to generate In this case, since it is based on the patient's own cancer cells or similar, the amino acid structure of the antigen matches, so a higher effect can be expected. As such a ligand, what is called a neoantigen is also being put to practical use.

A "neoantigen" is called a neoantigen, and is produced by artificially identifying the structure of a protein expressed in cells that have undergone genetic changes that are not found in normal cells. In order to carry out this identification, it is necessary to analyze new cancer tissue, which is the stage prior to gene changes, and can be extracted mainly from blood. Neoantigens vary from patient to patient and may change with cancer progression. Therefore, it is effective to use newly identified neoantigens as much as possible.

As described above, research on cancer antigens that can be used as ligands is progressing day by day, and any newly discovered ligands belonging to specific immunotherapy can be used in the method for producing a cocktail dendritic cell vaccine of the present invention. can be used as a ligand for

As described above, various ligands can be used in the pulse step, but at least one of the groups of immature dendritic cells divided in the division step corresponds to nonspecific immunotherapy. For the remaining groups, ligands that are relevant for specific immunotherapy are used. In other words, immature dendritic cells divided in a division step are used so that both ligands applicable to non-specific immunotherapy and ligands applicable to specific immunotherapy are included.
<Embodiment 1 Mainly corresponding to claim 1 Configuration Pulse step Second culture sub-step>

The pulsing step includes a second culturing substep in which immature dendritic cells are pulsed with a ligand and cultured to mature into mature dendritic cells. When the immature dendritic cells take in the added ligand as an antigen, they are matured by culturing for about 1 to 3 days. The ratio of mature dendritic cells to the whole after culture may be 70% or more, preferably 80% or more, but the schedule may be such that immature dendritic cells partially remain. Such immature dendritic cells act as an immune system according to the state of the body after being reintroduced into the patient's body, and can be used to strengthen the immune system that was not fully prepared by diagnosis and treatment planning.
<Embodiment 1 Mainly corresponding to Claim 1 Configuration Storage step>

The "storage step" is the step of storing each ligand-pulsed group.
In the pulse step, the liquid produced as a dendritic cell vaccine by maturation into mature dendritic cells is injected into a storage container such as a vial for each group and stored.
That is, a storage container filled with the dendritic cell vaccine for the number of administrations to the patient or more is produced.
This is stored in a frozen state at -80°C or below until administration, but the storage is not limited to frozen storage, and if the vaccine is suitable for refrigeration, it may be stored in refrigeration. In addition, if the dendritic cell vaccine is to be administered to a patient immediately after production, it may be stored under refrigeration or at room temperature.
The storage step does not necessarily have to be performed for all divided groups, and may be configured to be performed only for some groups. Alternatively, each group of matured dendritic cells that have been divided may be reintegrated and stored in a state in which a plurality of types of matured dendritic cells are mixed. In particular, when the storage step is performed by low-temperature freezing, the entire mature dendritic cell group should be integrated before storage so that minute variations in quality deterioration of mature dendritic cells due to storage do not occur among multiple mature dendritic cell groups. can be considered.
<Embodiment 1 Mainly Corresponding to Claim 1 Configuration Storage Step Storage Container Division Sub-Step>

The storage step may include a "storage container splitting 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 why the dose is set to one dose or less is that the optimal dose of the dendritic cell vaccine differs depending on the patient. For example, in some patients, an effect was observed even with an amount equivalent to 1×10 ⁶ cells, but in some patients, no effect was observed unless an amount equivalent to 1×10 ⁷ cells was administered. In addition, once thawed, it is used up. For this reason, one storage container should be the minimum amount assumed, and the number of storage containers required to secure one 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 Mainly corresponding to claim 1 Description of flow>

FIG. 3 is a flow chart showing the flow of the method for producing the cocktail dendritic cell vaccine of the present invention.
The details of each step are the same as those described in the configuration, and the general flow will be described in the description of this flowchart.
First, in a preparation step 0301, preparations are made so that blood components can be collected from a patient to be treated. Specifically, the number of white blood cells is examined, and if the number is below a predetermined value, a drug that increases white blood cells is administered and the progress is observed. In addition, nutritional supplements, hydration, etc. are provided, and blood components are collected without problems.
After that, in blood component collection step 0302, blood component collection is performed. The components to be collected in component blood collection are monocytes, and about 100 cc of blood containing monocytes as the main component is collected.
Next, in a division step 0303, the immature dendritic cells differentiated from the monocytes collected from the patient are divided into a plurality of groups at a predetermined ratio. Differentiation of monocytes into immature dendritic cells is carried out by placing monocytes in a culture medium, adding cytokine as a stimulating factor, and culturing for about 5 days.
Next, in the ligand pulse step 0304, different ligands are added to the culture medium containing the immature dendritic cells, which are divided into a plurality of groups, and the cells are cultured. Here, immature dendritic cells take up ligands and mature into mature dendritic cells over a period of about 2 to 3 days.
Finally, in the storage step 0305, the matured dendritic cells that have been divided into a plurality of groups and cultured are divided into single doses, transferred to storage containers, and frozen at −80° C. or lower.
The above is the flow of production of the cocktail dendritic cell vaccine of the present invention.
<Embodiment 1 Mainly corresponding to claim 1 Effect>

A cocktail dendritic cell vaccine composed of multiple dendritic cell vaccines, including a non-specific immune dendritic cell vaccine and a specific immune dendritic cell vaccine, can be produced from a single blood collection. The homogeneity of the dendritic cells of multiple dendritic cell vaccines is ensured by culturing from a single blood collection, and a definite synergistic effect is achieved. In particular, homogeneity is further enhanced when a cocktail dendritic cell vaccine is produced by dividing the isolated monocytes into a plurality of groups after growing them to some extent and pulsing them with different ligands. This is because the variation in the speed of cells becomes large in the initial stage of growth, and the variation gradually decreases as the growth progresses.
Furthermore, since component blood collection is a heavy burden on patients and cannot be performed continuously in a short period of time, by generating multiple dendritic cell vaccines from a single blood collection, synchronism of administration timing can be ensured without wasting time. can.
Therefore, the patient can obtain a high anticancer effect due to the synergistic effect of the improvement of immunity and the improvement of anticancer activity.
<Embodiment 2 Mainly corresponding to claim 2 Overview>

In Embodiment 2, in addition to the procedure of Embodiment 1, before the preparation step, the immune status of the patient and information related to the disease are acquired, and the optimal ligand is selected according to the patient's condition. is.
<Embodiment 2 Configuration mainly corresponding to claim 2>

Embodiment 2, in addition to the procedure of Embodiment 1, acquires any one or more of immunity information, which is information about the immunity of the patient, biometric information of the individual patient, and disease information of the patient before the preparation step. a step of acquiring information on immunity/disease, wherein the ligand pulsed in the pulsing step has a ligand selection step of selecting according to the information on immunity/disease acquired, and manufacturing a cocktail dendritic cell vaccine. . Regarding the details of each configuration, the description of the configuration that overlaps with the first embodiment will be omitted, and the additional steps will be described.
<Embodiment 2 Mainly corresponding to claim 2 Configuration Immunity/disease information acquisition step>

The "immunity/disease information acquisition step" is a step of acquiring any one or more of immunity information, which is information relating to the patient's immunity, individual patient biometric information, and patient's disease information, before the preparation step.
"Immunity/disease information" refers to information related to a patient's immunity, including information related to immunity obtained from blood tests, etc., as well as information related to cancer, complications associated with cancer, and diseases other than cancer. It consists of disease information obtained from various test information such as blood tests, tumor markers, cancer tissue biopsies, X-rays, CT scans, and MRIs.

The blood test can check the white blood cell count, the amount of immunoglobulin, the presence or absence of immunodeficiency, the amount of complement, and the presence or absence of autoimmune disease.
The white blood cell count can measure the ratio of lymphocytes and monocytes, and is an important item in the production of this immunologically active vaccine. Immunoglobulins include IgG, IgA, and IgM, and if there is an abnormality in the value, a disease other than cancer may be suspected, so prior treatment may be considered. For immunodeficiency, HIV1 and HIV2 antibodies are tested, but if there is a problem, T cells and B cells, which have major immune functions, do not function normally, so it is necessary to take measures to improve immunodeficiency.
A tumor marker is used to test the value of a substance such as a characteristic protein produced by the type of cancer. Typical examples include CEA, AFP, BCA225, BFP, CA15-3, CA19-9, etc., and it is possible to know whether cancer metastasis is suspected. If the type of cancer is known, the ligand to be pulsed can be selected according to the type. Alternatively, it is conceivable that the ligand to be pulsed in the first place is custom-made according to the patient's cancer site. Biopsies of cancer tissues are similar and involve selection of ligands.
X-rays, CTs, MRIs, etc. are used to confirm the degree of progression of the original cancer disease site, and to confirm the site suspected of metastasis using tumor markers, etc., using actual images.

By using such information related to immunity and diseases, for example, it can be used as an index for selecting the ligand to be used, or can be used to determine the administration schedule related to the administration interval and administration period of the cocktail dendritic cell vaccine. It is conceivable that

The immunity/disease information acquisition step may be acquired after administration of the dendritic cell vaccine produced by the method for producing a cocktail dendritic cell vaccine of the present invention. For example, confirmation of the state of induction of killer T cells, confirmation of an increase in the level of IFN-γ, and the like. When a specific immune dendritic cell vaccine is administered, the induction of killer T cells can be confirmed as a confirmation index of the effect. In addition, administration of a non-specific immune dendritic cell vaccine increases the IFN-γ level. For example, with three or more divided groups, after the first two dendritic cell vaccines are administered into the patient's body, the immature dendritic cells set aside in reserve are pulsed with a new ligand based on the evaluation results to allow them to mature. , may additionally generate new dendritic cell vaccines.

On the other hand, how much dendritic cell vaccine should be administered to a patient to obtain an effect differs depending on the individual patient. Specifically, whether or not a specific immune dendritic cell vaccine induces killer T cells depends on the patient. In addition, when IFN-γ reaches a predetermined level or higher, no further administration of non-specific immune dendritic cell vaccines is necessary until the effects decrease. Under the circumstances described above, it is possible to determine the production amount of the dendritic cell vaccine while confirming the patient's immune response by administering the dendritic cell vaccine produced by the method for producing the cocktail dendritic cell vaccine of the present invention. Conceivable.
<Embodiment 2 Mainly Corresponding to Claim 2 Configuration Ligand Selection Step>

このように、リガンドとして使用するがん抗原は、がんの部位にもよるが多数あり、どのリガンドが患者にとって効果的かは、患者の検査結果（がんの発生箇所、がんの進行度、腫瘍マーカー値など）で判断する必要がある。 The "ligand selection step" is a step of selecting a ligand to be pulsed in the pulse step according to the acquired immunity/disease information.
First, specific immunotherapy uses cancer antigens as ligands, and there are many artificially generated cancer antigens.
Table 1 below lists typical artificial cancer antigens.

In this way, there are many cancer antigens that can be used as ligands, depending on the site of the cancer. , tumor marker values, etc.).

α-galactosylceramide is known as a ligand used for nonspecific immunotherapy. Research is also underway on ligands that have an effect superior to that of α-galactosylceramide. If an effective ligand appears in the future, it will be included as a ligand that can be used in the cocktail dendritic cell vaccine as long as it activates NKT cells.
<Embodiment 2 Mainly corresponding to claim 2 Description of flowchart>

FIG. 4 is a flow chart showing the procedure of the second embodiment. Descriptions of steps that overlap with the first embodiment will be omitted, and only different steps will be described.
First, in an immunity/disease information acquisition step 0406, necessary tests (for example, blood test, tumor marker, CT scan, MRI, etc.) are performed according to the patient's condition, and the patient's immunity and immunity/disease-related immunity/disease information are obtained. Get disease information.
After that, as in the first embodiment, preparation step 0401, blood component collection step 0402, and division step 0403 are performed.
In parallel with this, before the pulse step 0404, at the ligand selection step 0407, the optimal ligand is selected based on the immunity/disease information.
For example, as an example, an artificial ligand is selected according to the patient's cancer site.
After that, the pulse step 0404 and the storage step 0405 are performed in the same manner as in Embodiment 1, but the ligand to be pulsed in the pulse step 0404 is 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 corresponding to claim 2 Effect>

As described above, according to the cocktail dendritic cell vaccine of Embodiment 2, the optimal ligand can be selected and produced according to the site and type of cancer, so that the vaccine has a higher anticancer effect. In addition to cancer, when suffering from a disease such as viral or bacterial infection, the antigen of the virus or bacteria is used as a ligand for a part of divided immature dendritic cells. Then, a cocktail dendritic cell vaccine can be produced for concurrent treatment of multiple diseases.
<Embodiment 3 Mainly corresponding to claim 3 Overview>

Embodiment 3, in addition to Embodiment 2, determines the ratio of immature dendritic cells to be divided in the division step based on the immunity/disease information acquired in the immunity/disease information acquisition step. .
<Embodiment 3 Configuration mainly corresponding to claim 3>

Embodiment 3, in addition to the configuration of Embodiment 2, further has a division ratio determination step for determining a division ratio when dividing in the division step according to the obtained immunity/disease information, and a cocktail dendritic cell vaccine. is manufactured. Regarding the details of each configuration, the description of the configuration that overlaps with that of the second embodiment will be omitted, and the additional steps will be described.
<Embodiment 3 Mainly Corresponding to Claim 3 Configuration Division Ratio Determination Step>

The "dividing ratio determining step" is a step of determining the dividing ratio of immature dendritic cells in the dividing step.
The method for producing a cocktail dendritic cell vaccine of the present invention includes a dendritic cell vaccine using a ligand that corresponds to specific immunotherapy and a dendritic cell vaccine using an NKT cell activating ligand that corresponds to nonspecific immunotherapy. and
Furthermore, as ligands applicable to specific immunotherapy, multiple ligands are used depending on factors such as the patient's cancer type, stage, and whether to use artificial cancer peptides or patient-derived cancer peptides. sometimes. In addition, when a disease other than cancer that can be treated by a dendritic cell vaccine is concurrently present, a ligand corresponding to the treatment may be used. It is this step that determines at what ratio the immature dendritic cells pulsed with these ligands will be split.
This division ratio is the ratio at which immature dendritic cells are divided, and this 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 greatly 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.

For example, in the information on immunity acquired in the immunity/disease information acquisition step, if the patient's immunity indicates a high value, the non-specific immune dendritic cell vaccine is, for example, 3 to 6 % and a specific immune dendritic cell vaccine as high as 94-97%.

In addition, the information on the patient's immunity shows a low level of immunity, and if the affected part of the cancer is surgically removed or the cancer is still in the early stage, specific immune dendritic cells It is conceivable that the proportion of vaccines will be reduced and that of non-specific immune dendritic cell vaccines will be 30% so that they can be administered for a long period of time.
Although the cases described above are extreme cases, it is desirable to manufacture each dendritic cell vaccine at an appropriate and optimal ratio according to the individual patient's immunity and disease information.
Although the division ratio here affects the number of dendritic cells, it does not match the amount of vaccine related to the number of administrations. That is, the number of dendritic cells per unit amount contained in the liquid of the non-specific immune dendritic cell vaccine and the specific immune dendritic cell vaccine may be different. This is because the amount of ligand that can be pulsed into dendritic cells varies from ligand to ligand.

FIG. 11 is an example of a table for determining ratios in division steps.
This ratio is the division ratio of immature dendritic cells and does not correspond to the amount of dendritic cell vaccine produced.
The row is the evaluation value of immunity, and here it is divided into three levels, and normal indicates the average value of people with healthy immunity, and weak indicates that the immunity is lower than that. , Strong indicates a state in which the immune force is more activated than usual.
For the evaluation value of immunity, for example, a method of obtaining a numerical value of interferon and evaluating the value on a three-grade basis can be considered.
To give a specific example, if the immunity is low, it is necessary to strengthen the immunity, so the non-specific immune dendritic cell vaccine is increased to no more than 20%, and the rest is specific Generated by immune dendritic cell vaccine. In addition, if the evaluation value of immunity is strong and relatively high, focus on specific immunotherapy and reduce the non-specific immune dendritic cell vaccine to around 5%, and the rest will be specific immune dendritic cells. Produce as a vaccine.
<Embodiment 3 Mainly corresponding to claim 3 Description of flow chart>

FIG. 5 is a flow chart showing the procedure of the third embodiment. Descriptions of steps that overlap with other embodiments will be omitted, and only different steps will be described.
The division ratio determination step 0508 is based on the immunity/disease information acquired in the immunity/disease information acquisition step 0506. At the latest, the division number and division ratio for dividing the immature dendritic cells in the division step 0503 are determined by the division step. Determined before 0503. In the division step 0503, the immature dendritic cells are divided into a plurality of groups based on the division number and the division ratio determined in the division ratio determination step.
<Embodiment 3 Mainly corresponding to claim 3 Effect>

Since the patient's condition is ascertained through testing and the production ratio of the dendritic cell vaccine that is optimal for the patient's condition is determined and manufactured, an effective cocktail dendritic cell vaccine with a higher anticancer effect can be manufactured.
<Embodiment 4 Mainly Corresponding to Claim 4 Overview>

In the fourth embodiment, based on the immunity/disease information acquired in the immunity/disease information acquisition step, a regimen for administering the dendritic cell vaccine produced by the method for producing a cocktail dendritic cell vaccine of the present invention to a patient is created. and determine the storage period for each storage container stored in the storage step.
<Embodiment 4 Mainly Corresponding to Claim 4 Configuration>

In addition to the embodiments including Embodiment 2 and Embodiment 2, Embodiment 4 further has a storage period determination step of determining the storage period of each group in the storage step according to the acquired immunity/disease information. to produce a cocktail dendritic cell vaccine.
<Embodiment 4 Mainly Corresponding to Claim 4 Configuration Storage Period Determination Step>

In the "storage period determination step", based on the immunity/disease information acquired in the immunity/disease information acquisition step, the dendritic cell vaccine, which is divided into storage containers and stored frozen, is stored in each storage container. determine the duration;
For example, non-specific immune dendritic cell vaccines are administered at intervals of 2 to 8 weeks. For patients who are judged not to have weakened immune system, it is planned to administer at intervals close to 8 weeks and extend the treatment period for one course.
The specific immune dendritic cell vaccine is administered at intervals of 2 to 4 weeks, but for patients whose cancer is judged to be imminently progressing based on immunity/disease information, the administration interval is 2. Set a short interval close to a week and set a large dose, and for patients whose cancer progression is not imminent, such as surgical removal of the cancer-affected area, 4 weeks It may be planned to administer at intervals close to , or to determine the appropriate dose by gradually increasing the dose from a small amount.

As described above, based on the immunity/disease information acquired in the immunity/disease information acquisition step, the administration interval, administration period, and administration of the dendritic cell vaccine produced by the method for producing a cocktail dendritic cell vaccine of the present invention. A dose, etc., is created as an administration plan, and based on the created administration plan, in the storage step, the dendritic cell vaccine is stored in a storage container in an amount equal to or less than one dose, and the number of divisions to be divided into the storage containers, Determine the amount of dendritic cell vaccine to be stored in one storage container and the storage period for each storage container.

FIGS. 12 and 13 are examples of determining the storage period of each storage container for the dendritic cell vaccine based on the administration regimen.
The black triangle indicates the timing of administration, the line indicates the storage period, and one square indicates two weeks.
FIG. 12 shows a dosing regimen prepared for a patient with a weak evaluation value of immunity in FIG. 11 . As shown in the figure, the storage period of each storage container is 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 at the beginning and the end of the treatment period, and it is planned to perform one course over three months.
FIG. 13 shows, for example, the case of administration for the purpose of preventing metastasis and recurrence after surgical treatment of cancer, and since the urgency of cancer symptoms is relatively low, long-term treatment is recommended. It is planned. As shown, the specific immune dendritic cell vaccine is planned to be administered initially for 3 weeks and then every 3 weeks for the next 3 weeks. The non-specific immune dendritic cell vaccine is planned to be administered about 12 weeks apart.
The non-specific immune dendritic cell vaccine is preferably administered once to three times during one course, regardless of the divided dose in the dividing step.
<Embodiment 4 Mainly Corresponding to Claim 4 Description of Flowchart>

FIG. 6 is a flow chart showing the procedure of the fourth embodiment. Descriptions of steps that overlap with other embodiments will be omitted, and only different steps will be described.
The storage period determination step creates an administration plan of the present cocktail dendritic cell vaccine to the patient based on the immunity/disease information acquired in the immunity/disease information acquisition step. Based on this information, the storage period for each storage container storing the dendritic cell vaccine, which is stored in the storage container for each dose in the storage step, is determined.
<Embodiment 4 Mainly corresponding to claim 4 Effect>

Based on a dosage regimen suitable for the patient's condition, administration of the dendritic cell vaccine produced by the method for producing a cocktail dendritic cell vaccine of the present invention, including the number and amount of divisions into storage containers, administration intervals, administration periods, etc. Since the storage period of each storage container is determined based on the plan, the cocktail dendritic cell vaccine can be used effectively according to the patient's condition, and can be maintained in a stable state until administration.
<Embodiment 5 Mainly corresponding to claim 5 Overview>

In Embodiment 5, a dendritic cell vaccine composed of mature dendritic cells cultured by pulsing a ligand in a pulsing step is observed to confirm the survival state of dendritic cells, contamination of foreign matter, etc., and administered to a patient. Confirm that the quality is satisfactory even if the
<Embodiment 5 Mainly Corresponding to Claim 5 Configuration>

Embodiment 5, in addition to the configuration of any one of Embodiments 1 to 4, further has an observation result obtaining step of obtaining observation results by observing each group pulsed with the ligand after the pulsing step. It manufactures dendritic cell vaccines.
<Embodiment 5 Mainly corresponding to claim 5 Configuration Observation result acquisition step>

In the “observation result acquisition step”, the mature dendritic cells cultured in the pulse step are magnified and observed with a microscope or the like, and various factors such as the dendritic cell maturation rate, survival rate, and contamination due to foreign matter contamination are observed. This is the step of confirming whether the group's dendritic cell vaccine has been produced with a predetermined quality.

The maturation rate of dendritic cells means that dendritic cells take up the ligand and mature, but usually requires a culture period of 2 to 3 days. Since dendritic cells have the ability to present antigens only after becoming mature dendritic cells, sufficient effects cannot be obtained unless maturation has progressed. Therefore, if the maturation rate is determined to be low, it is desirable to increase the maturation rate by adding an additional culture period.

As for the survival rate, some cells die during the process of differentiating from monocytes to immature dendritic cells and mature dendritic cells. In addition, some cells die after maturity and lifespan. If the number of dead cells is large, the effect will decrease, so it may be necessary to take measures such as increasing the dosage.

Contamination by foreign matter is confirmed by confirming whether fungi contaminating in the process of producing this cocktail dendritic cell vaccine or bacteria originally present in the blood are not proliferating by culturing. If there is contamination with fungal or bacterial growth, it is dangerous to administer to the patient, and production of this cocktail dendritic cell vaccine should be stopped and the preparation steps should be repeated.

Also, the number of mature dendritic cells per unit area is counted to estimate the total number of mature dendritic cells.
As a result, it is possible to grasp how many doses of dendritic cell vaccine have been secured.
<Embodiment 5 Mainly corresponding to claim 5 Explanation of flowchart>

FIG. 7 is a flow chart showing the procedure of the fifth embodiment. Descriptions of steps that overlap with other embodiments will be omitted, and only different steps will be described.
In the observation result acquisition step 0710, the state inside the dendritic cell vaccine cultured in the plurality of groups in the pulse step 0704 is observed with a microscope or the like, and the maturity of the dendritic cells, the viability of the dendritic cells, the dendritic cell Obtain information related to the quality of dendritic cell vaccines, such as the number of cells and the presence or absence of contamination due to foreign matter. If there is no problem with the dendritic cell vaccine of each group, the process proceeds to storage step 0705 and is stored until administration.
<Embodiment 5 Mainly corresponding to claim 5 Effect>

By confirming the state of the dendritic cells in the dendritic cell vaccine and whether there is contamination with foreign substances, the quality of the cocktail dendritic cell vaccine can be guaranteed and medical problems can be prevented in advance. In addition, by confirming the degree of activity of dendritic cells and the number of cells, it is possible to prepare an administration regimen and to estimate in advance the effect after administration.
<Embodiment 6 Mainly corresponding to claim 6 Overview>

In Embodiment 6, an administration plan for the cocktail dendritic cell vaccine is created based on the observation result obtained in the observation result obtaining step, and the storage period is determined in the storage step.
The effects of dendritic cell vaccines vary depending on the number of dendritic cells, activity, and maturity, but the quantity and quality of dendritic cells obtained by culture largely depend on the properties of the patient's monocytes. Therefore, based on the results of the actual culture, it is recommended to create a dosage plan in the storage step, determine the number of divisions to be divided into storage containers, the amount of dendritic cell vaccine to be stored in the storage container, etc., and store them. It is a feature of morphology.
<Embodiment 6 Mainly Corresponding to Claim 6 Configuration>

Embodiment 6 has the same configuration as that of Embodiment 5, and the effect of the storage step is different.
<Embodiment 6 Mainly corresponding to Claim 6 Configuration Storage step>

In the storage step of the present embodiment, the number of mature dendritic cells and the activity level of dendritic cells, which are the observation results obtained in the observation result obtaining step, are used to determine the single dose, the number of times of administration, and the like, The number of divisions to be divided in the storage container division step described above is determined.
Further, in the storage period determination step, the dose per administration and the number of administrations may be determined based on the administration interval and administration period determined based on the patient's immunity/disease information.
<Embodiment 6 Mainly corresponding to Claim 6 Flow of processing>
The processing flow of Embodiment 6 is the same as that of Embodiment 5, and the effect of the storage step is different. The details of the operation of the storage step are omitted since they have been described in the configuration.
<Embodiment 6 Mainly corresponding to claim 6 Effect>

Since the performance of each dendritic cell vaccine is confirmed by observation and the dose and frequency of administration are determined, dendritic cell vaccines can be divided and stored to ensure efficacy according to the performance of the immunologically active vaccine.
<Embodiment 7 Mainly corresponding to claim 7 Overview>

In Embodiment 7, at least one of a plurality of groups of immature dendritic cells divided in the division step is cryopreserved, and the subsequent pulsing step and storage step are performed at a time different from that of the other groups. .
<Embodiment 7 Mainly Corresponding to Claim 7 Configuration>

Embodiment 7, in addition to the configuration of any one of Embodiments 1 to 6, is a frozen/refrigerated storage in which at least one or more of each group divided between the dividing step and the pulsing step is stored frozen or refrigerated. It further has a storage step to produce a cocktail dendritic cell vaccine.
<Embodiment 7 Mainly corresponding to claim 7 Configuration Freezing/refrigerating storage step>

In the "freezing/cold storage step", some of the immature dendritic cells divided into a plurality of groups are cryopreserved. As a result, it is stored for a period of 1 to 3 months as a guide, after which the ligand to be used in the pulsing step is determined, the pulsing step and the storage step are performed, and one or more dendritic cells constituting the cocktail dendritic cell vaccine are prepared. Create a cell vaccine. Of course, this does not preclude storage for three months or longer.

This storage period is, for example, to secure a period for identifying and producing the composition of proteins (including peptides) used as ligands from patient-derived tissues such as neoantigens. Patient-derived tissue is the patient's own cell, which requires a lot of time to generate, looking for mutations in genes that are beginning to transform into cancer cells and identifying their composition. For this reason, immature dendritic cells, which were not stored in a frozen/refrigerated storage step until this patient-derived ligand was produced, were used to generate and administer a dendritic cell vaccine using an existing ligand, while administering it to the patient. Once the derived ligand is generated, a dendritic cell vaccine is generated using this ligand, and administration is performed by switching to this.

In addition, it is impossible to know how effective the dendritic cell vaccine by the ligand to be used is to the patient 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, it may be more effective to change to a different ligand. For this reason, after administering a dendritic cell vaccine generated by immature dendritic cells that were not stored in the frozen/refrigerated storage step and confirmed the effect, immature trees that were stored in the frozen/refrigerated storage step to determine the ligands to be used for the cells.
After administering the dendritic cell vaccine to the patient, the determination of whether it is effective is performed by acquiring immunity/disease information in the immunity/disease information acquisition step, and then performing the ligand selection step based on the immunity/disease information. to select the ligand.
<Embodiment 7 Mainly corresponding to Claim 7 Flow of processing>

FIG. 8 is a flow chart showing the procedure of the seventh embodiment. Descriptions of steps that overlap with other embodiments will be omitted, and only different steps will be described.
In the freezing/refrigerating storage step 0811, at least one or more groups among those differentiated into immature dendritic cells in the division step 0803 and divided into a plurality of groups are frozen/refrigerated. The remaining groups proceed directly to pulse step 0804 and storage step 0805 . The group preserved in the frozen/refrigerated storage step undergoes the ligand step and the storage step at different timings later than the other groups.
<Embodiment 7 Mainly corresponding to claim 7 Effect>

Some dendritic cell vaccines produced using existing ligands in the method for producing a cocktail dendritic cell vaccine of the present invention by freezing or refrigerating the immature dendritic cells split in the splitting step While proceeding with treatment with , a cancer antigen derived from the patient's living tissue is identified and produced, and a dendritic cell vaccine is produced using immature dendritic cells that have been cryopreserved at the time of completion. As a result, a dendritic cell vaccine with a higher anticancer effect can be generated.

Moreover, it is not known until after administration whether the ligand has sufficient effect on the patient. If the administration of the dendritic cell vaccines of the previously generated group is not sufficiently effective, a different ligand is used to generate the dendritic cell vaccine. Whether or not there was an effect can be determined by examining the rate of induction of killer T cells. If a sufficient effect is observed, the same ligand as in the previously produced dendritic cell vaccine may be used. By doing so, it is possible to increase the possibility of obtaining a higher anticancer effect.
<Embodiment 8 Mainly corresponding to claim 8 Overview>

Embodiment 8 involves generating cancer antigens based on the patient's individual anatomy and using them as ligands.
<Embodiment 8 Configuration mainly corresponding to claim 8>

Embodiment 8, in addition to the configuration of any one of Embodiments 1 to 7, further has a ligand generation step of generating a ligand based on the patient's individual body tissue, wherein the pulse step is generated in the ligand generation step A cocktail dendritic cell vaccine is produced by pulsing the ligands.
<Embodiment 8 Mainly Corresponding to Claim 8 Configuration Ligand Generation Step>

A "ligand generating step" is a step of generating cancer antigens to be used as ligands from patient-derived tissue. Patient-derived tissue is produced by identifying the composition of proteins (including peptides) obtained from the patient's cancer cells or cells beginning to transform into cancer cells. For example, cancer cells or cells that are changing into cancer cells have a mutated gene structure that is not found in other normal cells.

Unlike artificial antigens, cancer antigens produced from patient-derived tissues are optimal markers of cancer in patients, and are therefore expected to be highly effective.
<Embodiment 8 Mainly corresponding to Claim 8 Flow of processing>

FIG. 9 is a flow chart showing the procedure of the eighth embodiment. Descriptions of steps that overlap with other embodiments will be omitted, and only different steps will be described.
The ligand generation step 0912 is performed in parallel with the preparation step 0901, blood component collection step 0902, and division step 0903, and is completed at least before the pulse step 0904 is performed. In this ligand generation step, a ligand is generated based on the patient's own biological information, and the ligand is used in at least one or more of the immature dendritic cells divided into a plurality of groups in the pulse step. . By doing so, one or more of the plurality of dendritic cell vaccines constituting the cocktail dendritic cell vaccine are generated based on the patient's own biological information.
<Embodiment 8 Mainly corresponding to claim 8 Effect>

According to this embodiment, one of the dendritic cell vaccines constituting the cocktail dendritic cell vaccine uses a cancer antigen generated based on the patient's own biological information (patient-derived tissue), so that effect can be expected.
<Embodiment 9 Mainly corresponding to claim 9 Overview>

Embodiment 9 involves generating cancer antigens based on the patient's individual anatomy and using them as ligands.
<Embodiment 9 Mainly Corresponding to Claim 9 Configuration>

Embodiment 9 is, in addition to the configuration of any one of Embodiments 1 to 8, an injection device for returning the ligand-pulsed groups stored after the storage period to the body of the patient in group units. A cocktail dendritic cell vaccine is produced by further having a filling step (including, but not limited to, a syringe; the same shall apply hereinafter).
<Embodiment 9 Mainly Corresponding to Claim 9 Configuration Filling Step>

In some cases, a single dose is enough for a single storage container, and in some patients, multiple doses are used for a single dose. to fill a single dose.
<Embodiment 9 Mainly corresponding to Claim 9 Flow of processing>

FIG. 10 is a flow chart showing the procedure of the ninth embodiment. Descriptions of steps that overlap with other embodiments will be omitted, and only different steps will be described. The filling step 1013 is a step of thawing the frozen dendritic cell vaccine stored in the storage container in the storage step 1005 for a single dose and filling it into a syringe or the like. Thus, the cocktail dendritic cell vaccine filled in a syringe or the like is administered to the patient.
<Embodiment 10 Mainly corresponding to claim 10 Overview>

Embodiment 10. Any one of Embodiments 1 to 9, wherein the ligand used in the ligand pulsing step is any two or more ligands of WT1 peptide, neoantigen, and α-galactosylceramide. A method for producing a cocktail dendritic cell vaccine comprising
<Embodiment 10 Mainly corresponding to claim 10 Details>

As explained in the ligand selection step, the WT1 peptide has a protein structure that is expressed in almost all cancers, and is expected to be effective in cancer sites and a wide range of patients. On the other hand, neoantigens are expected to be highly effective because they use tissue that is expressed in the patient's cancer, but they require analysis of the patient's own body tissue and identification of the structure, which is time-consuming and costly. There is such a problem. Therefore, neoantigen is likely to be administered to patients who can afford it economically, and neoantigen alone or WT1 peptide may be used in combination. Regarding α-galactosylceramide, there is a high possibility that it will be administered to most patients, and a high effect can be expected by using it in combination with WT1 peptide or neoantigen.
<Embodiment 10 Corresponding to Claim 10 Configuration>

The configuration of the tenth embodiment is the same as the configuration of any one of the first to ninth embodiments.
<Embodiment 10 Mainly corresponding to Claim 10 Flow of processing>

The processing flow of the tenth embodiment is the same as the processing flow of any one of the first to ninth embodiments. The ligand pulsed in the ligand pulsing step produces a cocktail dendritic cell vaccine with two or more ligands selected from WT1 peptide, neoantigen, and α-galactosylceramide.
<Embodiment 10 Mainly corresponding to claim 10 Effect>

According to this embodiment, the WT1 peptide, which is highly likely to be effective against various cancers in many people, is used as a ligand for a specific immune dendritic cell vaccine, which is suitable for everyone and can be expected to be effective. A high anticancer effect can be expected against various cancers in many people by using a combination of non-specific immune dendritic cell vaccines using α-galactosylceramide as a ligand.

0201 Effect of α-galactosylceramide-pulsed dendritic cell vaccine 0202 Dendritic cell maturation promoting effect 0203 Adjuvant effect 0204 Apoptosis-inducing effect on cancer cells 0205 Checkpoint inhibiting effect 0206 Angiogenesis inhibiting effect 0210 Cancer antigen presentation Action 0211 Cancer cell attack instruction 0212 Antibody production instruction against cancer antigen 0213 Cancer cell attack action by killer T cells 0214 New blood vessel destruction action 0220 Cancer cell attack action by NK cells 0221 Antigen presentation action by dendritic cells 0222 Macrophages cancer antigen presentation activity 0223 cancer antibody production activity 0224 cancer cell phagocytosis by macrophages

Claims (10)

a preparatory step of preparing for an apheresis from the patient;
A predetermined amount of monocytes is collected from a patient who is ready for component blood collection by one-time puncture (meaning that the collection of a predetermined amount of blood is completed in one course. Multiple punctures are allowed.). a blood component collection step;
a dividing step of dividing monocytes or immature dendritic cells differentiated from monocytes collected only from the patient into a plurality of groups;
a pulse step of pulsing different types of ligands for each group divided in the division step, optionally at different timings starting from the time of division;
a storage step of storing each ligand-pulsed group;
A method for producing the patient-specific cocktail dendritic cell vaccine comprising: Before the preparation step, an immunity/disease information acquisition step of acquiring any one or more of immunity information, which is information on the immunity of the patient, biometric information of the individual patient, and disease information of the patient,
2. The method for producing a cocktail dendritic cell vaccine according to claim 1, further comprising a ligand selection step of selecting the ligand pulsed in the pulse step in accordance with acquired immunity/disease information. 3. The method for producing a cocktail dendritic cell vaccine according to claim 2, further comprising a split ratio determining step of determining the split ratio in the split step according to the acquired immunity/disease information. 4. The cocktail tree according to claim 2 or claim 3, wherein the storage period of each group in the storage step further has a storage period determination step of determining the storage period according to the acquired immunity/disease information. A method for producing a shaped cell vaccine. 5. The method for producing a cocktail dendritic cell vaccine according to any one of claims 1 to 4, further comprising an observation result acquisition step of observing each group pulsed with the ligand after the pulsing step to obtain an observation result. 6. The production of the cocktail dendritic cell vaccine according to any one of claims 1 to 5, wherein the storage period for each group in the storage step is further determined according to the observation results obtained in the observation result obtaining step. Method. 7. The cocktail dendrite according to any one of claims 1 to 6, further comprising a frozen/refrigerated storage step of freezing or refrigerating at least one of each divided group between the dividing step and the pulsing step. A method for producing a cellular vaccine. Claims 1 to 7, further comprising a ligand generating step of generating a ligand based on the patient's individual body tissue, wherein the pulsing step is configured to pulse the ligand generated in the ligand generating step. A method for producing a cocktail dendritic cell vaccine according to any one of A filling step of filling each group pulsed with the ligand stored after the storage period into an injection device (including but not limited to a syringe, the same shall apply hereinafter) for returning to the patient's body on a group-by-group basis. 9. The method for producing a cocktail dendritic cell vaccine according to any one of claims 1 to 8, further comprising: 9. The cocktail dendritic cell according to any one of claims 1 to 8, wherein the ligands pulsed in the pulsing step are WT1 peptide and two or more ligands selected from α-galactosylceramide and neoantigen. Vaccine manufacturing method.

Images