JP4852702B2 - Dendritic cells obtained by antigen pulsing - Google Patents

Description

  The present invention relates to dendritic cells that can be used for the prevention or treatment of diseases.

(Dendritic cell therapy and its problems)
In the field of cancer treatment and the like, dendritic cell therapy, which is a kind of cell immunotherapy, has attracted attention.

  Dendritic cells are antigen-presenting cells (APCs) that have the strongest antigen-presenting ability in the living body and activate T cells by presenting incorporated antigen epitopes to T cells, thereby enhancing the immune response. be able to.

  Dendritic cell therapy is a treatment method in which the dendritic cells are pulsed in vitro using a substance extracted from cancer cells or a cancer-specific antigen, and the antigen-presenting ability is enhanced and returned to the living body.

  A substance extracted from cancer cells is generally a mixture of many kinds of substances. Therefore, dendritic cells pulsed using substances extracted from cancer cells are thought to have been pulsed by various types of cancer antigens in the cell extract substances, and thus a strong immune response against cancer is induced. It is expected to be. However, when pulsed with a mixture of many kinds of substances in this way, the antigen presented by the dendritic cells cannot be specified. Therefore, it is allowed to be used for cancer patients, but is not allowed to be used for healthy people.

  In addition, dendritic cells pulsed with cancer-specific antigens can identify antigens presented by dendritic cells, but the number of currently identified cancer-specific antigens is not yet large. There is a point.

  As another embodiment for presenting an antigen to a dendritic cell, for example, a method is known in which a gene encoding a peptide derived from the antigen is introduced into the dendritic cell and expressed. However, even though dendritic cells prepared in this way can trigger a strong immune response against a specific antigen, at present, dendritic cells using genetic engineering are It is not allowed to be administered to healthy individuals for prevention.

(About hepatitis)
Hepatitis is inflammation that occurs in the liver and is mainly caused by viruses, alcohol, and drugs. The most common hepatitis is caused by the hepatitis virus. To date, seven types of hepatitis viruses have been found. Moreover, in Japanese viral hepatitis, B type and C type account for most.

  Hepatitis can be broadly classified into acute hepatitis and chronic hepatitis, and chronic hepatitis refers to hepatitis that lasts for 6 months or more. Hepatitis A is orally infected and remains acute hepatitis and does not become chronic. On the other hand, hepatitis B and C are transmitted through blood, and there are vertical infection between mother and child and horizontal infection between family members and heterosexuals. In addition, hepatitis B and C often progress from acute hepatitis to chronic hepatitis. When chronic, hepatitis is said to have a very high transition rate to liver diseases such as cirrhosis and liver cancer.

(About hepatitis B)
Hepatitis B patients are distributed worldwide, and it is said that there are 350 million people infected with hepatitis B virus in the world. In particular, the number of infected people in China is estimated to be 120 million, and it is said that it accounts for more than 70% of the number of infected people in Southeast Asia as a whole. In addition, it is reported that about 1 million people worldwide die from this disease every year. In Japan, it is estimated that there are over 1 million people infected with hepatitis B virus, and it is estimated that about 10% are chronic hepatitis patients.

(Hepatitis B therapy and its problems)
There is no effective treatment for hepatitis B. Therefore, the treatment is aimed at reducing the number of viruses and suppressing their activity. As typical treatment methods, interferon therapy and lamivudine therapy are known.

  Interferon therapy is the first treatment for hepatitis B that was started about 20 years ago. Interferon plays a role in helping the immune system work to suppress virus growth. Since interferon therapy can be expected to be effective as the number of viruses decreases, in general, it exhibits an excellent effect in the treatment of hepatitis C in which the number of viruses is 1/1000 of that of hepatitis B. The treatment can only be effective in about 20% of patients. Moreover, when it shifts to cirrhosis, interferon becomes less effective. Furthermore, interferon used for treatment has a large cost to develop and produce a product, and there are many problems such as bringing a large economic burden of 2 to 3 million yen to a patient with one course of treatment.

  Lamivudine is a drug originally developed as a therapeutic drug for AIDS, but is known to be effective for the treatment of hepatitis B. Lamivudine has the effect of directly suppressing the synthesis of viral DNA, and has the effect of stopping the rapid deterioration and fulminating of hepatitis B. Lamivudine has been reported to be effective in patients with hepatitis B for which interferon has not been effective, but is not effective when the virus is not in a proliferative state. Furthermore, there is a problem that lamivudine-resistant virus appears by long-term administration.

  In addition, these methods for treating hepatitis B cannot be compared uniformly because the methods for evaluating the effects are different. Moreover, it is considered impossible to completely eliminate hepatitis B virus from the body using these treatment methods. Therefore, in order to prevent the increase in the number of patients with hepatitis B as described above, it is considered important to prevent hepatitis B.

(Hepatitis B prophylaxis and its problems)
From the viewpoint of prevention of hepatitis B, HB vaccine is generally effective.

  In order to prevent chronic hepatitis B, it is important to maintain and maintain anti-HBs antibodies in the body even in healthy individuals. Today's HB vaccines are made based on HBs antigen, which is the surface antigen of hepatitis B virus. Usually, by inoculating this HB vaccine three times, an anti-HBs antibody can be produced in the body of a human who is effective for the HB vaccine. Humans who have developed antibodies will not be subsequently infected with hepatitis B. However, even if the antibody level decreases several years later, once anti-HBs antibodies are made in the body, there is a possibility that they may escape infection. It is done. In addition, the antibody level can be increased again by additional inoculation.

  However, there are some problems with the HB vaccine.

  First, about 10% of healthy individuals are non-responders to the HB vaccine, and even when the HB vaccine is administered, anti-HBs antibodies cannot be produced. There are not a few HB vaccine non-responders among healthcare workers, and it is thought that there are hundreds of thousands in Japan.

  In addition, the HB vaccine cannot exert sufficient effects even in people suffering from diseases accompanied by a decrease in immune capacity, such as HIV patients, liver disease patients, and dialysis patients. Obesity and smokers also have reduced immunity, but these people are also less responsive to HB vaccines.

  Furthermore, since the HB vaccine does not work for people undergoing immunosuppressants such as liver transplant recipients, patients cannot produce anti-HBs antibodies. However, in hepatitis B-related liver transplantation, maintaining the anti-HBs antibody level has a significant effect on therapeutic results, and therefore, patients need to repeatedly administer anti-HBs human immunoglobulin after liver transplantation. The administration is necessary once every two weeks, and this brings about a great economic burden of 2.5 million yen per year. From now on, especially in Southeast Asia where there are many patients, hepatitis B-related liver transplantation is expected to increase dramatically, and so there is a global need to solve this economic problem.

  An object of the present invention is to provide dendritic cells effective for the prevention or treatment of diseases, and in particular, to provide dendritic cells that can be suitably used for the prevention or treatment of hepatitis B.

The human dendritic cells having the ability to present HBs antigens of the present invention include a step of obtaining human dendritic cells using monocytes isolated from human peripheral blood , and culturing the human dendritic cells in a medium containing HBs antigen. It was obtained by a method comprising the step of pulsing includes a HBs antigen-presenting ability, and a human dendritic cells does not induce hepatic cytotoxicity CTL.

Method for producing human dendritic cells with HBs antigen presenting ability of the present invention comprises the steps of obtaining a human dendritic cells using monocytes isolated from human peripheral blood, the human dendritic cells, including HBs antigen characterized in that it comprises a step of pulsing cultured in a medium.

The composition containing human dendritic cells having the ability to present HBs antigens of the present invention is characterized by comprising human dendritic cells having the ability to present HBs antigens of the present invention.

Vaccines pulses human dendritic cells of the present invention includes the steps of obtaining a human dendritic cells using monocytes isolated from human peripheral blood, the human dendritic cells, cultured in a medium containing a HB vaccine containing the HBs antigen was obtained by a method comprising the step of pulsing a vaccine pulsed human dendritic cells does not induce hepatic cytotoxicity CTL.

Method for producing a vaccine pulsed human dendritic cells of the present invention comprises the steps of obtaining a human dendritic cells using monocytes isolated from human peripheral blood, the human dendritic cells, including HB vaccine containing the HBs antigen characterized in that it comprises a step of pulsing cultured in a medium.

Compositions comprising a vaccine pulsed human dendritic cells of the present invention is characterized by comprising a vaccine pulsed human dendritic cells of the present invention.

The human vaccine pulse dendritic cell of the present invention is a vaccine pulse dendritic cell for the prevention or treatment of human hepatitis B, has the ability to present HBs antigen, and does not induce hepatotoxic CTL. The vaccine pulse dendritic cell is obtained by using a monocyte isolated from peripheral blood derived from a hepatitis B patient or a hepatitis B virus carrier to obtain a dendritic cell and a vaccine for the dendritic cell. A vaccine pulse dendritic cell obtained by a production method including a pulse step of culturing and pulsing in a medium containing the vaccine, and the vaccine used for the pulse is an HB vaccine containing HBs for prevention or treatment of hepatitis B It is characterized by being.

  The medicament for treatment and prevention for human hepatitis B of the present invention comprises the human vaccine pulse dendritic cell of the present invention.

The dendritic cells of the present invention are effective for the prevention or treatment of diseases, and can be particularly suitably used for the prevention or treatment of hepatitis B.

FIG. 1 shows anti-HBs antibody values before administration of HB vaccine pulse dendritic cells in Example 1. FIG. 2 is a graph showing changes in HLA-DR and CD86 of dendritic cells by the HB vaccine pulse in the Example. FIG. 3 is a graph showing changes in the ability of dendritic cells to rejuvenate T cells by the HB vaccine pulse in the Example. FIG. 4 is a graph showing the effect of HB vaccine-pulsed dendritic cells on the proliferation ability of HBs memory T cells using the HBs memory T cells in the Example. FIG. 5 is a graph showing the effect of HB vaccine pulse dendritic cells on the ability to produce anti-HBs antibodies using the HBs memory T cells in the Example. FIG. 6 shows the test items of the safety test of HB vaccine pulsed dendritic cells by the blood biochemical test in the Example. FIG. 7 is a graph showing the results of the ALT test in the example. FIG. 8 is a graph showing the results of CRP inspection in the example. FIG. 9 is a graph showing the results of PT inspection in the example. FIG. 10 is a graph showing the result of the Creatline test in the example. FIG. 11 is a table showing the results of safety tests on autoimmune reactions using serum biochemical tests in the above examples. FIG. 12 is a graph showing changes in anti-HBs antibody values by administration of HB vaccine pulse dendritic cells in the Example . FIG. 13 is a graph showing the transition of the anti-HBs antibody value of the HB vaccine non-responder by administration of HB vaccine pulse dendritic cells in the Example. FIG. 14 is a graph showing the results of a safety test regarding an autoimmune reaction using a serum biochemical test in Example 2. FIG. 15 is a graph showing changes in anti-HBs antibody values by administration of HB vaccine pulse dendritic cells in Example 3.

  Hereinafter, the present invention will be described with examples. However, the present invention is not limited to the following description.

1. Human dendritic cell having HBs antigen-presenting ability The human dendritic cell having HBs antigen-presenting ability of the present invention is obtained by using human monocytes isolated from human peripheral blood to obtain human dendritic cells, dendritic cells were obtained by a method comprising the step of pulsing by culturing in a medium containing HBs antigens, it has HBs antigen-presenting ability, and a human dendritic cells does not induce hepatic cytotoxicity CTL.

The human dendritic cell having the ability to present an HBs antigen of the present invention can activate the T cell by presenting the HBs antigen epitope to the T cell and enhance the immune response to the HBs antigen. Therefore, according to the present invention, it is possible to provide a human dendritic cell having the ability to present HBs antigen, which is effective for the prevention or treatment of hepatitis B.

In the human dendritic cell having the ability to present HBs antigen of the present invention, the method includes a step of obtaining the dendritic cell from blood . According to this, it is possible to provide the human dendritic cell which has the HBs antigen presentation ability of this invention from the dendritic cell derived from blood. If blood is used, human dendritic cells can be obtained by a relatively simple operation using a conventionally known method or the like.

In the human dendritic cell having the ability to present HBs antigen of the present invention, the method preferably further comprises a step of collecting the blood from a human . According to this, since a human-derived dendritic cell can be obtained, the human dendritic cell having the ability to present HBs antigen according to the present invention can be administered to a human. Therefore, according to the present invention, it is possible to provide dendritic cells having the ability to present HBs antigens that are effective in preventing or treating human hepatitis B.

In the human dendritic cell having the ability to present HBs antigen of the present invention, the HBs antigen is preferably an HBs antigen contained in an HB vaccine. For example, in the case of humans, what is actually sold as an HB vaccine is one that has been approved and approved for safety and effectiveness by humans including healthy individuals. Therefore, according to the human dendritic cell having HBs antigen-presenting ability of the present invention, it is possible to provide a dendritic cell having HBs antigen-presenting ability that can be used safely and effectively for humans. In addition, since the HB vaccine can be used for healthy individuals, the human dendritic cells having the ability to present HBs antigens of the present invention can be used not only for the purpose of treating hepatitis B for hepatitis B patients and the like, but also for healthy subjects. Can also be used for the prevention of hepatitis B. In addition to humans, hepatitis B can be prevented or treated in other organisms by using dendritic cells pulsed with an HB vaccine that is safe and effective for the organism.

2. Method for producing human dendritic cells having the ability to present HBs antigen The method for producing human dendritic cells having the ability to present HBs antigen according to the present invention comprises the steps of producing human dendritic cells using monocytes isolated from human peripheral blood. and obtaining, the human dendritic cells, characterized by comprising a step of pulsing by culturing in a medium containing HBs antigen. According to this, it is possible to obtain human dendritic cells having the ability to present an HBs antigen that are effective in preventing or treating hepatitis B.

Method for producing human dendritic cells with HBs antigen presenting ability of the present invention, includes the step of obtaining said dendritic cells from human blood. According to this, it is possible to obtain human dendritic cells having the ability to present the HBs antigen of the present invention from dendritic cells derived from human blood. If human blood is used, dendritic cells can be obtained by a relatively simple operation using a conventionally known method or the like.

The method for producing a human dendritic cell having HBs antigen-presenting ability of the present invention preferably further includes a step of collecting the blood from a human . According to this, it is possible to obtain human dendritic cells having the ability to present HBs antigen that are effective in preventing or treating human hepatitis B.

In the method for producing a human dendritic cell having HBs antigen-presenting ability of the present invention, the HBs antigen is preferably an HBs antigen contained in an HB vaccine. According to this, it is possible to provide a human dendritic cell having an ability to present an HBs antigen, which is effective for the prevention or treatment of hepatitis B, which can be used safely and effectively for an organism, for example, a human including a healthy person. .

3, a composition comprising human dendritic cells with HBs antigen presenting ability of the compositions the present invention comprising human dendritic cells with HBs antigen-presenting ability, including human dendritic cells with HBs antigen presenting ability of the present invention It is characterized by that. According to this, the human dendritic cells having the ability to present HBs antigens of the present invention are used, for example, in a state mixed with another substance, suspended in another substance, or combined with another substance. be able to. Specifically, human dendritic cells having the ability to present HBs antigens of the present invention are suspended in, for example, PBS, physiological saline, RPMI1640 medium, etc., or dendritic cells having the ability to present HBs antigens of the present invention. In addition, for example, TNF-α, IL-12, HB vaccine, GM-CSF, OK432, and other immunostimulants can be used. Therefore, according to the present invention, human dendritic cells having the ability to present HBs antigens of the present invention can be used in an aspect suitable for the purpose of use or in an aspect that enhances the effects of human dendritic cells having the ability to present HBs antigens of the present invention. It becomes possible to use.

The composition containing human dendritic cells having the ability to present HBs antigens of the present invention preferably further contains an HB vaccine additive.

In the composition containing human dendritic cells having the ability to present HBs antigen of the present invention, examples of the form of the composition include an experimental reagent and an experimental kit. According to this, the human dendritic cell which has the HBs antigen presentation ability of this invention can be used in the aspect suitable for research, for example, immunology research.

In the composition containing human dendritic cells having the ability to present HBs antigens of the present invention, the composition is preferably a pharmaceutical composition. According to this, the human dendritic cell having the ability to present HBs antigen of the present invention can be used in a mode suitable for medical treatment, for example, prevention or treatment of hepatitis B.

In the composition containing human dendritic cells having the ability to present HBs antigens of the present invention, the composition is preferably an immune response activation preparation. According to this, the human dendritic cell having the ability to present HBs antigen of the present invention can be used in a dosage form suitable for activating an immune response against HBs antigen.

In the composition containing human dendritic cells having the ability to present HBs antigens of the present invention, the composition is preferably a cellular vaccine. According to this, it becomes possible to administer the dendritic cell which has HBs antigen presentation ability of this invention directly in the living body, and can activate the immune response with respect to HBs antigen.

In the composition containing human dendritic cells having the ability to present HBs antigen of the present invention, the cell vaccine is preferably a preventive or therapeutic vaccine for hepatitis B. According to this, dendritic cells having the ability to present HBs antigens of the present invention contained in the cellular vaccine can prevent or treat hepatitis B by activating an immune response against HBs antigens in vivo. It becomes possible.

4, HBs method for preventing or treating Hepatitis B using human dendritic cells with HBs antigen presenting ability of antigen prevention of hepatitis B using human dendritic cells with presenting ability or treatment method of the present invention, the present The human dendritic cells having the ability to present the HBs antigen of the invention are used. According to this, the dendritic cell which has the HBs antigen presentation ability of this invention can prevent or treat hepatitis B by activating the immune response with respect to an HBs antigen. For example, in the case of humans, conventional HB vaccines have been effective, such as healthy individuals who have been unsuccessful with conventional HB vaccines, those who are suffering from diseases associated with decreased immune capacity, those who are being administered immunosuppressants, etc. A person who has not been able to produce hepatitis B can produce anti-HBs antibodies by the method for preventing or treating hepatitis B of the present invention, and hepatitis B can be prevented or treated.

The method for preventing or treating hepatitis B using the human dendritic cells having the ability to present HBs antigens of the present invention includes the step of administering the human dendritic cells having the ability to present HBs antigens of the present invention to an organism. preferable. According to this, the dendritic cell which has the HBs antigen presentation ability of this invention can prevent or treat hepatitis B by activating the immune response with respect to HBs antigen in the living body. In addition, the method for preventing or treating hepatitis B according to the present invention can exhibit a higher effect than conventional methods for preventing or treating hepatitis B in which an HB vaccine is directly administered in vivo. .

In the method for preventing or treating hepatitis B using human dendritic cells having the ability to present HBs antigens of the present invention, the organism is preferably a human. According to this, it is possible to prevent or treat human hepatitis B.

5. Vaccine-pulsed human dendritic cell The vaccine-pulsed human dendritic cell of the present invention comprises a step of obtaining human dendritic cells using monocytes isolated from human peripheral blood, and the human dendritic cells are treated with HBs antigen. cultured in medium containing HB vaccines containing obtained by a method comprising the step of pulsing a vaccine pulsed human dendritic cells does not induce hepatic cytotoxicity CTL.

In the vaccine pulsed human dendritic cell of the present invention, the “vaccine pulsed human dendritic cell” refers to a human dendritic cell having an antigen presenting ability obtained by a method including a step of pulsing with a vaccine.

Vaccines pulses human dendritic cells of the invention, the T cells were activated by presenting antigen epitopes contained in the vaccine to T cell, it is possible to enhance the immune response to the antigen. Therefore, according to the present invention, vaccine-pulsed human dendritic cells can be provided that are effective in preventing or treating diseases.

For example, in the case of humans, what is actually sold as a vaccine is approved and approved by humans for safety and efficacy. Therefore, according to the present invention, it is possible to provide vaccine-pulsed human dendritic cells that can be used safely and effectively for humans, for example. It should be noted that not only humans but also other organisms can be prevented or treated by using vaccine-pulsed human dendritic cells pulsed with a safe and effective vaccine for that organism.

In the vaccine pulsed human dendritic cell of the present invention, the method comprises the step of obtaining the dendritic cell from blood . According to this, it is possible to provide the vaccine pulse human dendritic cell of this invention from the dendritic cell derived from blood. If blood is used, dendritic cells can be obtained by a relatively simple operation using a conventionally known method or the like.

In vaccines pulses human dendritic cells of the present invention, the method further preferably includes the step of collecting the blood from a human. According to this, since a human-derived dendritic cell can be obtained, the vaccine pulse human dendritic cell according to the present invention can be administered to a human. Therefore, according to the present invention, vaccine-pulsed human dendritic cells that are effective for the prevention or treatment of human diseases can be provided.

In the vaccine pulsed human dendritic cell of the present invention, the HB vaccine containing the HBs antigen used for the pulse is a hepatitis vaccine . Known hepatitis vaccines include hepatitis A vaccine, hepatitis B vaccine, and the like. For example, in the case of humans, what is actually sold as a hepatitis vaccine is one that has been approved and approved for safety and efficacy by humans including healthy individuals. Therefore, according to the present invention, it is possible to provide hepatitis vaccine pulsed human dendritic cells having hepatitis virus antigen presentation ability that can be used safely and effectively for humans, for example. In addition, since the hepatitis vaccine can be used for healthy persons, the hepatitis vaccine pulse dendritic cells of the present invention are not only for the purpose of treating hepatitis for hepatitis patients, but also for healthy persons. It can be used for the purpose of preventing hepatitis. It should be noted that not only humans but also other organisms can be prevented or treated by using the dendritic cells pulsed by using a safe and effective hepatitis vaccine for the organism.

In the vaccine pulsed dendritic cell of the present invention, the hepatitis vaccine is an HB vaccine containing an HBs antigen . According to this, a vaccine-pulsed human dendritic cell having an ability to present HBs antigen, which is effective for the prevention or treatment of hepatitis B, which can be used safely and effectively for an organism, for example, a human including a healthy person, is provided. Can do.

6. Method for Producing Vaccine Pulsed Human Dendritic Cells The method for producing vaccine pulsed human dendritic cells of the present invention comprises the steps of obtaining human dendritic cells using monocytes isolated from human peripheral blood, and the human dendritic cells cells, characterized in that it comprises a step of pulse cultured in medium containing HB vaccine containing the HBs antigen. According to this, vaccine-pulsed human dendritic cells that are effective in preventing or treating diseases can be obtained.

Method for producing a vaccine pulsed human dendritic cell of the invention comprises a step of obtaining the dendritic cells from human blood. According to this, it is possible to obtain the vaccine pulse dendritic cell of this invention from the dendritic cell derived from human blood. If blood is used, dendritic cells can be obtained by a relatively simple operation using a conventionally known method or the like.

Method for producing a vaccine pulsed human dendritic cells of the present invention preferably further comprises the step of collecting the blood from a human. According to this, it is possible to obtain vaccine pulse dendritic cells effective for the prevention or treatment of human diseases.

In the vaccine pulsed human dendritic cell production method of the present invention, the vaccine used for the pulse is an HB vaccine containing an HBs antigen . According to this, a vaccine-pulsed human dendritic cell having an ability to present HBs antigen, which is effective for the prevention or treatment of hepatitis B, which can be used safely and effectively for an organism, for example, a human including a healthy person, is provided. Can do.

7. Composition containing vaccine-pulsed human dendritic cells The composition containing vaccine-pulsed human dendritic cells of the present invention is characterized by containing the vaccine-pulsed human dendritic cells of the present invention. According to this, the vaccines pulse human dendritic cells of the present invention, for example, it can be used in a state such as a combination state of being mixed with other substances, a state suspended in another substance, and other materials. Specifically, the vaccine pulse dendritic cells of the present invention are suspended in, for example, PBS, physiological saline, RPMI 1640 medium or the like, or the vaccine pulse human dendritic cells of the present invention are treated with, for example, TNF- α, IL-12, HB vaccine, GM-CSF, OK432, other immunostimulants and the like can be used. Therefore, according to the present invention, the vaccine pulsed human dendritic cell of the present invention can be used in a mode suitable for the purpose of use or in a mode of enhancing the effect of the vaccine pulsed human dendritic cell of the present invention.

Compositions comprising a vaccine pulsed human dendritic cells of the present invention preferably further contains a vaccine additives.

In a composition comprising a vaccine pulsed human dendritic cells of the present invention, the composition is preferably an experimental reagent or experimental kit. According to this, the vaccines pulse human dendritic cells of the present invention, research, for example, can be used in a manner suitable for immunization studies.

In a composition comprising a vaccine pulsed human dendritic cells of the present invention, the composition is preferably a pharmaceutical composition. According to this, the vaccines pulse human dendritic cells of the present invention, medical, for example, can be used in a manner suitable for the prevention or treatment of disease.

In a composition comprising a vaccine pulsed human dendritic cells of the present invention, the composition is preferably an immune response activation formulation. According to this, it is possible to use a vaccine pulsed human dendritic cells of the present invention, in dosage forms suitable for activating the immune response to an antigen contained in the vaccine.

In a composition comprising a vaccine pulsed human dendritic cells of the present invention, the composition is preferably a cell vaccine. According to this, the vaccines pulse human dendritic cells of the present invention, it is possible to directly administered in vivo, the immune response to an antigen contained in the vaccine can be activated.

In a composition comprising a vaccine pulsed human dendritic cells of the present invention, the cellular vaccine is preferably a prophylactic or therapeutic vaccine disease. According to this, the vaccine pulsed human dendritic cells of the present invention contained in the cell vaccines, in vivo, by activating the immune response to an antigen contained in the vaccine, to prevent or treat the disease It becomes possible.

8. Prevention or treatment method of disease using vaccine pulsed human dendritic cell The prevention or treatment method of disease using vaccine pulsed human dendritic cell of the present invention uses the vaccine pulsed human dendritic cell of the present invention. Features. According to this, the vaccine pulsed human dendritic cells of the present invention, by activating the immune response to an antigen contained in the vaccine, it is possible to prevent or treat the disease. For example, in the case of humans, healthy individuals who have been unsuccessful in vaccines that have been used to prevent or treat the above diseases, those who are suffering from diseases associated with decreased immune ability, those who are taking immunosuppressants Thus, a person who could not exert a sufficient effect of the vaccine can produce an antibody by the disease prevention or treatment method of the present invention, and the disease can be prevented or treated.

The method for preventing or treating a disease using the vaccine pulsed human dendritic cell of the present invention preferably includes a step of administering the vaccine pulsed human dendritic cell of the present invention to an organism. According to this, the vaccine pulsed human dendritic cells of the present invention, in vivo, by activating the immune response to an antigen contained in the vaccine, it is possible to prevent or treat the disease. In addition, the disease prevention or treatment method of the present invention can exhibit a higher effect than conventional disease prevention or treatment methods by directly administering a vaccine into a living body.

In a method for the prevention or treatment of diseases using vaccines pulsed human dendritic cells of the present invention, the organism is preferably a human. According to this, it is possible to prevent or treat human diseases.

9. Human vaccine pulse dendritic cell The human vaccine pulse dendritic cell of the present invention is a vaccine pulse dendritic cell for the prevention or treatment of human hepatitis B, has HBs antigen presentation ability, and Obtaining dendritic cells that do not induce hepatotoxic CTL and obtain the dendritic cells using monocytes isolated from peripheral blood derived from hepatitis B patients or hepatitis B virus carriers as the vaccine pulse dendritic cells And a vaccine pulse dendritic cell obtained by a production method comprising a pulse step of culturing and pulsing the dendritic cell in a medium containing a vaccine, and the vaccine used for the pulse is prevention or treatment of hepatitis B It is characterized by being an HB vaccine containing HBs.

  In the vaccine pulse dendritic cell for human of the present invention, for example, the vaccine pulse dendritic cell induces Th2, preferably does not induce Th1, and more preferably does not induce CTL.

The human vaccine pulse dendritic cell of the present invention has the following advantages. In dendritic cell therapy performed for cancer treatment or the like, the purpose is to induce cytotoxic T cells (CTL). Therefore, the induction of Th1 is promoted and the dendritic cells are activated not to induce Th2. I am letting. In contrast, the human vaccine pulse dendritic cells of the present invention, for example, do not induce CTL, and therefore have a completely different mechanism from that of dendritic cell therapies that have been performed in cancer treatment and the like. That is, according vaccine pulsed dendritic cells in the present invention, for example, the production of antibodies capable and will without inducing hepatic cytotoxicity CTL, liver disorders associated with liver cell attack by hepatocytes cytotoxic CTL (e.g., drama Hepatitis etc.). Therefore, the human vaccine pulse dendritic cells of the present invention are extremely useful for hepatitis B patients and hepatitis B virus carriers. The mechanism that the human vaccine pulse dendritic cell of the present invention does not induce Th1 or CTL is unknown.

  In the human vaccine pulse dendritic cell of the present invention, in the step of pulsing the dendritic cell with the vaccine, the pulse time is preferably, for example, 8 hours to 24 hours. In the dendritic cell acquisition step, it is preferable to co-culture isolated monocytes with GM-CSF and IL-4.

10. Method for producing human vaccine pulse dendritic cells The method for producing human vaccine pulse dendritic cells of the present invention comprises, for example, collecting peripheral blood from a hepatitis B patient or a hepatitis B virus carrier, and from the peripheral blood. A dendritic cell obtaining step for obtaining dendritic cells from human-derived blood to obtain dendritic cells using isolated monocytes, and prevention of human hepatitis B by pulsing the dendritic cells with a vaccine or Vaccine for treatment A production method including a pulse step for obtaining dendritic cells, wherein the vaccine used for the pulse in the pulse step may be a vaccine for prevention or treatment of hepatitis B. According to the human vaccine pulse dendritic cell obtained by the production method of the present invention, for example, Th2 is preferably induced in humans and Th1 is not induced, and CTL is preferably not induced.

  In the method for producing human vaccine pulse dendritic cells of the present invention, the pulse time in the vaccine pulse step is preferably 8 to 24 hours.

  In the dendritic cell acquisition step of the method for producing human vaccine pulse dendritic cells of the present invention, it is preferable to co-culture isolated monocytes with GM-CSF and IL-4.

11. Pharmaceutical for treatment and prevention for human hepatitis B The pharmaceutical for treatment and prevention for human hepatitis B of the present invention comprises the human vaccine pulse dendritic cell of the present invention. Features. In the pharmaceutical product of the present invention, the vaccine pulse dendritic cell of the present invention preferably induces, for example, Th2, does not induce Th1, and further preferably does not induce CTL. Thus, since CTL is not induced, according to the pharmaceutical of the present invention, fulminant hepatitis is not caused by, for example, hepatocyte attack by CTL. Furthermore, according to the pharmaceutical product of the present invention, not only hepatitis B patients, but also healthy persons who have been unsuccessful in the vaccines used so far for preventing or treating hepatitis B, the vaccines are sufficient. A person who has failed to produce an effect can produce antibodies, and hepatitis B can be prevented or treated. Examples of the hepatitis B include chronic hepatitis B. The administration form of the pharmaceutical agent for the treatment and prevention of human hepatitis B of the present invention is not particularly limited, but for example, it is preferably administered subcutaneously.

12. Method for preventing or treating human hepatitis B using vaccine pulse dendritic cells The method for preventing or treating human hepatitis B using human vaccine pulse dendritic cells of the present invention is, for example, self-tree. A preventive or therapeutic method comprising administering a vaccine pulse dendritic cell derived from a dendritic cell to a hepatitis B patient or a hepatitis B virus carrier, wherein the vaccine pulse dendritic cell is a hepatitis B patient or hepatitis B Obtained by a production method comprising collecting a peripheral blood from a virus carrier, obtaining a dendritic cell using monocytes isolated from the peripheral blood, and a pulsing step of pulsing the dendritic cell with a vaccine. The human vaccine pulse dendritic cell, and the vaccine used for the pulse may be a vaccine for the prevention or treatment of hepatitis B.

  In the method for preventing or treating human hepatitis B using the human vaccine pulse dendritic cell of the present invention, the human vaccine pulse dendritic cell of the present invention may induce, for example, Th2 but not Th1. Preferably, it is further preferable not to induce CTL. According to the preventive or therapeutic method of the present invention, not only hepatitis B patients but also healthy persons who have been unsuccessful in the vaccines used so far to prevent or treat hepatitis B, the vaccines are sufficient. Those who have not been able to exert such effects can produce antibodies, and hepatitis B can be prevented or treated. Furthermore, since the prevention or treatment method of the present invention uses a dendritic cell of a hepatitis B patient or a hepatitis B virus carrier itself, the safety is extremely high. Examples of the hepatitis B include chronic hepatitis B. In the method for preventing or treating human hepatitis B using the human vaccine pulse dendritic cells of the present invention, the dosage form of the human vaccine pulse dendritic cells is not particularly limited, but for example, subcutaneous administration is preferable.

13. Use of human vaccine pulse dendritic cells for the treatment or prevention of human hepatitis B The use of human vaccine pulse dendritic cells for the treatment or prevention of human hepatitis B of the present invention includes, for example, A vaccine pulse dendritic cell is obtained by collecting a peripheral blood from a hepatitis B patient or a hepatitis B virus carrier and obtaining a dendritic cell using monocytes isolated from the peripheral blood, and the dendritic cell A human vaccine pulse dendritic cell obtained by a production method including a pulse step of pulsing a cell with a vaccine, wherein the vaccine used for the pulse is a vaccine for the prevention or treatment of hepatitis B, and the vaccine the pulsed dendritic cells, may be including Ndei administering to hepatitis B patients or hepatitis B virus carriers peripheral blood was collected.

  If the human vaccine pulse dendritic cells of the present invention are used for treatment or prevention of human hepatitis B, vaccines that have been used so far to prevent or treat hepatitis B as well as hepatitis B patients. Those who have not been able to demonstrate sufficient effects, such as healthy individuals who have not been successful, can produce antibodies, and hepatitis B can be prevented or treated. Furthermore, since the dendritic cells of hepatitis B patients or hepatitis B virus carriers themselves are used, the safety is extremely high. The human vaccine pulse dendritic cell of the present invention preferably induces, for example, Th2 and does not induce Th1, and further preferably does not induce CTL. Examples of the hepatitis B include chronic hepatitis B.

[Best Mode for Carrying Out the Invention]
The best mode of the present invention will be described below. The present invention is not limited to the following embodiments, and various modifications can be made without departing from the gist of the present invention.

1. Human dendritic cells having the ability to present HBs antigens The human dendritic cells having the ability to present HBs antigens according to the present embodiment include a step of obtaining human dendritic cells using monocytes isolated from human peripheral blood. , the human dendritic cells, obtained by a method comprising the step of pulsing by culturing in a medium containing HBs antigen. According to the human dendritic cell having the ability to present HBs antigen according to the present embodiment, it is possible to provide a human dendritic cell having the ability to present HBs antigen, which is effective in preventing or treating hepatitis B.

In the present embodiment, the "human dendritic cells pulsed with antigen", human dendritic cells, capture antigens decomposed in said human dendritic cells, may present the degraded antigen as a peptide Say to put it in a state.

In the present embodiment, a specific method for pulsing human dendritic cells with an antigen includes, for example, a method of culturing an antigen and human dendritic cells in the presence of an appropriate cytokine or the like. .

The method for obtaining human dendritic cells is not particularly limited as long as human dendritic cells that can take up an antigen and present this antigen are finally obtained, and various techniques can be used.

For example, it is possible to obtain a human dendritic cells from human blood. Specifically, for example, by using methods known to those skilled in the art, direct separation of human dendritic cells from the methods or human blood to induce differentiation of human dendritic cells from human blood containing progenitor cells of human dendritic cells Can be used. More specifically, for example, a method derived from CD34 positive progenitor cells of bone marrow cells, umbilical cord blood or peripheral blood mainly using GM-CSF and TNF-α, from umbilical cord blood mononuclear cells or peripheral blood mononuclear cells Mainly using GM-CSF and IL-4, using mainly GM-CSF and IFN-α from peripheral blood mononuclear cells, directly separating from peripheral blood mononuclear cells, etc. Human dendritic cells can be obtained.

Moreover, the method of obtaining these human dendritic cells can further include a step of collecting blood from a human as necessary . In particular, since the number of deaths due to hepatitis B is increasing worldwide, the effect of using the present invention in humans is great.

  Examples of the method for obtaining dendritic cells without using blood include a method for obtaining dendritic cells by inducing differentiation of ES cells. Specifically, for example, differentiation can be induced into dendritic cells by culturing ES cells established from an inner cell mass present inside the blastocyst together with appropriate feeder cells and cytokines.

In the present embodiment, for a medium for culturing human dendritic cells, inducing differentiation from progenitor cells of human dendritic cells into human dendritic cells, survival of human dendritic cells, pulsed, etc. of human dendritic cells by antigen However, RPMI 1640 medium or the like can be used, for example. Further, RPMI 1640 medium is preferable.

In the present embodiment, cytokines can be used for induction of human dendritic cells. Cytokines used in the induction of human dendritic cells, induction of differentiation into human dendritic cells, survival of human dendritic cells is not particularly limited as long as it does not inhibit the pulses or the like of human dendritic cells by antigen, for example, GM- CSF, TNF-α, IL-4, IFN-α and the like can be used.

In the present embodiment, the aspect of the HBs antigen is not particularly limited as long as it can be finally presented by human dendritic cells, and may be a complete protein antigen or a peptide antigen.

Moreover, the HBs antigen which concerns on this Embodiment can be used in the aspect combined with various substances and liquids, for example. Specifically, for example, HBs antigen is introduced into human dendritic cells using a phospholipid bilayer membrane such as a liposome, or HBs antigen is suspended in a buffer solution such as PBS or a medium such as RPMI 1640 medium. Cloudy and can be introduced into human dendritic cells.

Further, the timing of adding the HBs antigen is not particularly limited as long as the HBs antigen can be presented by human dendritic cells as a result. For example, the HBs antigen can be added after inducing differentiation of human dendritic cells. In addition, HBs antigen can be added at the time of differentiation induction and cultured together. Further, the pulse time of the HBs antigen is not particularly limited as long as the HBs antigen can be presented by human dendritic cells as a result. For example, it is preferably 8 to 24 hours. The HBs antigen is preferably an HBs antigen contained in an HB vaccine.

  There are several types of vaccines, such as live vaccines, inactivated vaccines, toxoids and the like. A live vaccine refers to a vaccine that is inoculated alive with, for example, viruses or bacteria that have weakened pathogenicity. An inactivated vaccine refers to a vaccine produced by purifying virus particles, bacterial cells, etc. and treating them with a drug such as heating or formalin to eliminate or detoxify pathogenicity. Toxoid refers to, for example, a product obtained by removing and purifying only bacterial toxins and detoxifying them by adding formalin without losing immunogenicity.

  The type of HB vaccine in the present embodiment is not limited as long as it contains HBs antigens. However, in Japan, HB vaccines that are licensed for human use and sold in Japan are inactivated vaccines. being classified. In the present embodiment, when an HB vaccine that is an inactivated vaccine is used, for example, a recombinant precipitated hepatitis B vaccine or a precipitated hepatitis B vaccine can be used.

  As a recombinant precipitated hepatitis B vaccine, for example, an HBs antigen that is an active ingredient of a vaccine is obtained by inserting a portion corresponding to the HBs antigen of hepatitis B virus DNA into DNA of yeast, animal cells, etc. A vaccine prepared by adding aluminum gel as an immune enhancer can be used. As animal cells, for example, vaccines prepared using CHO cells can be used.

  As the precipitated hepatitis B vaccine, for example, a vaccine prepared by purifying HBs antigen produced by animal cells or the like and then adding an aluminum gel can be used. As animal cells, for example, vaccines prepared using huGK-14 cells can be used.

2. Method for producing human dendritic cells having the ability to present HBs antigens The method for producing human dendritic cells having the ability to present HBs antigens according to the present embodiment uses human monocytes isolated from human peripheral blood. and a step of obtaining a Jo cells, the human dendritic cells, and a step of pulse cultured in medium containing HBs antigen. According to this, it is possible to obtain human dendritic cells having the ability to present an HBs antigen that are effective in preventing or treating hepatitis B.

The production method includes a step of obtaining the human dendritic cells from human blood . According to this, it is possible to obtain human dendritic cells having the ability to present the HBs antigen of the present invention from human blood-derived human dendritic cells. If blood is used, dendritic cells can be obtained by a relatively simple operation using a conventionally known method or the like.

Moreover, it is preferable that the manufacturing method further includes a step of collecting the blood from a human . According to this, it is possible to obtain dendritic cells having the ability to present HBs antigens that are effective in preventing or treating human hepatitis B.

The HBs antigen is an HBs antigen contained in an HB vaccine . According to this, it is possible to provide a human dendritic cell having an ability to present an HBs antigen, which is effective for the prevention or treatment of hepatitis B, which can be used safely and effectively for an organism, for example, a human including a healthy person. .

3. Composition containing human dendritic cells having HBs antigen-presenting ability The composition according to the present embodiment preferably contains human dendritic cells having the HBs antigen-presenting ability according to the present embodiment.

  Moreover, it is preferable that the composition which concerns on this Embodiment contains an HB vaccine additive further.

In the present embodiment, “the composition according to the present embodiment further includes an HB vaccine additive” means that the HB vaccine additive is included anywhere in the entire composition according to the present embodiment. Say you are in a state. That is, the place where the HB vaccine additive is contained may be anywhere outside, inside, on the cell membrane, etc. of the human dendritic cells in the composition according to the present embodiment, and is not particularly limited. By including the vaccine additive in the composition according to the present embodiment, human dendritic cells having the ability to present HBs antigen according to the present embodiment were obtained using the HBs antigen contained in the HB vaccine as the HBs antigen. Can be determined.

  Examples of HB vaccine additives include preservatives, antibiotics, stabilizers, preservatives, immunostimulants, buffers, isotonic agents, and the like. Specific examples include thimerosal, potassium aluminum sulfate, sodium hydroxide, aluminum chloride, acetic acid, sodium acetate, sodium chloride and the like. The HB vaccine additive is not limited to these.

  As a method for detecting the HB vaccine additive, for example, a known analysis method can be used. Specifically, for example, separation analysis using various chromatographs, quantitative analysis, infrared spectroscopic analysis, nuclear magnetic resonance analysis, mass analysis, structural analysis by elemental analysis, and the like can be given.

  The composition according to the present embodiment is preferably an experimental reagent or an experimental kit. Thereby, the dendritic cell which has the HBs antigen presentation ability concerning this Embodiment can be used in the aspect suitable for various research. Examples of research fields that can be used include research on signal transduction mechanisms related to immune response, immune tolerance, and cytokine production, and research on immunotherapy using dendritic cells. Moreover, it can also be used for the purpose of, for example, research related to the prevention or treatment of hepatitis B, for example, screening of genes, RNA, peptides, proteins, compounds, pharmaceuticals, cytokines and the like.

  Moreover, it is preferable that the composition which concerns on this Embodiment is a pharmaceutical composition. The pharmaceutical composition according to the present embodiment can be used, for example, in an aspect where it is directly administered in vivo, or in an aspect where it is mixed with other substances or cells in vitro.

  Moreover, it is preferable that the composition which concerns on this Embodiment is an immune response activation formulation. The immune response activating preparation according to the present embodiment can be used, for example, in an aspect in which it is directly administered in vivo, or in an aspect where it is mixed with other substances or cells in vitro.

  As an aspect of using the composition according to the present embodiment in vitro, for example, by mixing the immune response activation preparation according to the present embodiment and a T cell and presenting the HBs antigen epitope to the T cell, T The usage method which activates a cell is mentioned. Further, these T cells can be used by being administered in vivo.

  Moreover, as an aspect which administers the composition which concerns on this Embodiment directly in the living body, the injection and infusion as a cell vaccine are mentioned, for example. The cell vaccine is preferably a preventive or therapeutic vaccine for hepatitis B. In addition, as a place where the composition according to the present embodiment is directly administered into a living body, for example, intravenous administration, intraarterial administration, intrathoracic administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intramuscular (for example, Intrahepatic) administration, lymph node administration, and the like. Subcutaneous injection is preferred because of the simplicity of administration.

4. Prevention or treatment of hepatitis B using dendritic cells having HBs antigen-presenting ability The dendritic cells having HBs antigen-presenting ability according to the present embodiment are used for, for example, prevention or treatment of hepatitis B. Can do.

  Prevention or treatment of hepatitis B using dendritic cells having the ability to present HBs antigens can be used in combination with, for example, conventional methods for preventing or treating hepatitis B.

Moreover, the prevention or treatment of hepatitis B using the human dendritic cells having the ability to present HBs antigen according to the present embodiment includes a step of administering human dendritic cells having the ability to present HBs antigen to an organism. Is preferred. The organism is not particularly limited as long as it is an organism that can be infected with hepatitis B. Examples of organisms that can be infected with hepatitis B include organisms that can naturally infect hepatitis B and organisms that can be artificially infected with hepatitis B. Specifically, organisms that can naturally infect the hepatitis B include artificially infecting the hepatitis B, such as primates such as humans and chimpanzees, and ducks such as woodchuck and pekindak. Examples of organisms that can be used include squirrels, rats, ducks, geese, herons, vines, mice, and the like. In addition, if the rejection reaction can be suppressed, the organism derived from the dendritic cell and the organism to be administered may be the same species or different species. In the case of allogeneic organisms, the dendritic cells according to the present embodiment can be administered into the removed self body, or can be administered into a non-self body that matches the degree to which the histocompatibility antigen can be administered. It is also possible.

For example, the organism to be administered is preferably a human. In this case, it is desirable that the HLA types match as much as possible. Accordingly, the human dendritic cells are preferably derived from human donors. If autologous cells are used, it is not necessary to consider the suitability of the HLA type, and thus human dendritic cells derived from the subject person are more preferable.

As described above, the human dendritic cells having the ability to present HBs antigen according to the present embodiment are used in a mode of being directly administered in vivo, or mixed with T cells in vitro to activate T cells, This T cell can be used in a mode of being administered in vivo. However, since T cells are effector cells, once they function, they die and disappear in a short time. Therefore, in order to prevent and treat hepatitis B, it is necessary to administer T cells repeatedly at a high frequency. On the other hand, dendritic cells also act on naive T cells, HBs memory T cells, and B cells, and repeatedly give stimuli, thus providing a long and powerful effect. From the above, it is considered that it is more preferable to use the human dendritic cells having the ability to present HBs antigen according to the present embodiment in a mode of directly administering in vivo.

5, the following use examples and its effect of human dendritic cells with HBs antigen presenting functions of the present embodiment, examples and its effect use of human dendritic cells with HBs antigen presenting functions of the present embodiment Show. In this use example, it is preferable to include a step of administering human dendritic cells having the ability to present HBs antigen according to the present embodiment to humans. This use example can be applied to the prevention or treatment of hepatitis B. In addition, this use example is not limited to the following cases, The use aspect of the human dendritic cell which concerns on this use example can be variously changed in the range which does not exceed the summary of this invention.

5-1, Application to prevention of hepatitis B About 10% of healthy people are non-responders to HB vaccine, and anti-HBs antibody even if HB vaccine is administered Is known to be unable to produce Since hepatitis B is transmitted through the blood, medical professionals and the like are considered to have a particularly high need for anti-HBs antibodies, but there are many HB vaccine non-responders in these people.

Therefore, by administering the human dendritic cells having the ability to present HBs antigens according to the present embodiment to these HB vaccine non-responders, it becomes possible for these people to produce anti-HBs antibodies. In order to prevent infection with hepatitis B, it is said that it is necessary to maintain the anti-HBs antibody value at least 10 mIU / mL. According to the results of Experimental Example 11 to be described later, the average of the anti-HBs antibody value of the HB vaccine non-responder is 30 mIU / mL by administering the human dendritic cells having the ability to present HBs antigen according to the present embodiment once. It was found that a value of 10 mIU / mL or more can be maintained for about 3 to 6 months. Therefore, the human dendritic cells having the ability to present HBs antigen according to the present embodiment enable the current HB vaccine non-responder to produce anti-HBs antibodies and prevent infection with hepatitis B. it can.

Moreover, the HB vaccine currently used for the prevention of hepatitis B needs to be inoculated usually three times at regular intervals. According to the results of Experimental Example 11 to be described later, not only the HB vaccine non-responder but also the HB vaccine is successful by administering the human dendritic cell having the ability to present HBs antigen according to the present embodiment once. It has been found that responder anti-HBs antibody levels can also be increased. Therefore, the human dendritic cells having the ability to present HBs antigens according to the present embodiment can be used not only for the HB vaccine non-responder but also for the HB vaccine responder as a new preventive measure for hepatitis B. , May reduce the number of inoculations and the economic burden.

In addition, it is known that conventional HB vaccines are not able to exert sufficient effects on people who have reduced immune functions, such as HIV patients, liver disease patients, dialysis patients, obese people, and smokers. It is considered that such people can also produce anti-HBs antibodies by using human dendritic cells having the ability to present HBs antigens according to the present embodiment.

5-2, Application to Treatment of Hepatitis B Antiviral agents have been mainly used so far for the treatment of hepatitis B. However, it is known that mutant viruses that do not work with antiviral agents appear frequently by administering antiviral agents for a long period of time. As a result, the amount of hepatitis B virus increased again, and there was a risk of hate for hepatitis.

Thus, it is expected that the frequency of appearance of such mutant viruses can be reduced by using human dendritic cells having the ability to present HBs antigens according to the present embodiment, which is a great advantage in the treatment of hepatitis B. it is conceivable that.

  In addition, those who are administered immunosuppressants such as liver transplant recipients are in an immunosuppressed state, and thus the normal HB vaccine is not effective and anti-HBs antibodies cannot be produced. However, in hepatitis B-related liver transplantation, it is known that maintaining anti-HBs antibody levels has a significant positive effect on treatment outcome, so patients should receive anti-HBs human immunoglobulins for 2 weeks after transplantation. It is necessary to administer at a single frequency. This causes a burden of about 2.5 million yen per year on the patient, and there is a global need to solve this economic problem.

Thus, it is considered that the patient can produce an anti-HBs antibody by administering to the patient the human dendritic cell having the ability to present HBs antigen according to the present embodiment. Furthermore, from the results of Experimental Example 11 described later, the human dendritic cells having the ability to present HBs antigens according to the present embodiment are administered at a frequency of, for example, once every 3 to 6 months, so that 10 mIU / mL or more. It is expected that anti-HBs antibody values can be maintained. From this, the use of human dendritic cells having the ability to present HBs antigens according to the present embodiment makes it possible to significantly reduce the administration frequency compared to the use of anti-HBs human immunoglobulins. The economic burden can be greatly reduced.

  In addition, the existing HB vaccine is a preventive vaccine for hepatitis B, and the therapeutic vaccine for hepatitis B has been used only experimentally.

On the other hand, as described above, human dendritic cells having the ability to present HBs antigens produced using the HB vaccine according to the present embodiment are not limited to the prevention of hepatitis B. It can be used for treatment. This expands the usage of HB vaccines that are currently being used and are recognized for safety and effectiveness, and has a new mode of use that enhances the effectiveness of existing HB vaccines. It can be said that it was found.

From the above, the human dendritic cells having the ability to present HBs antigens according to the present embodiment have a wide use and great advantages.

6. Vaccine-pulsed human dendritic cell The vaccine-pulsed human dendritic cell according to the present embodiment comprises a step of obtaining a human dendritic cell using monocytes isolated from human peripheral blood, and the human dendritic cell , cultured in a medium containing a HB vaccine containing the HBs antigen obtained by a process comprising the step of pulses. According to this, a vaccine pulse dendritic cell effective in prevention or treatment of a disease can be provided.

The method comprises obtaining a human dendritic cells from human blood.

The method preferably further includes the step of collecting the blood from a human .

In the method, it is preferable that peripheral blood is collected from a patient with chronic hepatitis B or a carrier, and human dendritic cells are obtained using monocytes isolated from the peripheral blood. This is because in the case of humans, peripheral blood can be collected repeatedly safely and easily.

In addition, the addition timing of the vaccine is not particularly limited as long as the antigen contained in the vaccine can be presented by human dendritic cells as a result. For example, the vaccine may be added after inducing differentiation of human dendritic cells. it can. In addition, a vaccine can be added at the time of differentiation induction and cultured together. Further, the pulse time by the vaccine is not particularly limited as long as the antigen contained in the vaccine can be presented by human dendritic cells as a result. For example, it is preferably 8 to 24 hours.

As a result, the vaccine is not particularly limited as long as the antigen contained in the vaccine can be presented by human dendritic cells. For example, a live vaccine, an inactivated vaccine, a toxoid, an antitoxin, or the like may be used. it can. Further, the form of the vaccine is not particularly limited as long as the antigen contained in the vaccine can be presented by human dendritic cells as a result. For example, a liquid vaccine, a precipitated vaccine, a freeze-dried vaccine, etc. Can be used.

The vaccine used for the pulse is an HB vaccine containing HBs antigen .

7. Manufacturing method of vaccine pulse human dendritic cell The manufacturing method of the vaccine pulse human dendritic cell which concerns on this Embodiment is the process of obtaining a human dendritic cell using the monocyte isolated from the human peripheral blood, human dendritic cells, and a step of pulsing by culturing in a medium containing HB vaccine containing the HBs antigen. According to this, a vaccine pulse dendritic cell effective in the prevention or treatment of a disease can be obtained.

The production method further includes the step of obtaining the human dendritic cells from human blood . According to this, it is possible to obtain the vaccine pulse dendritic cell of this invention from the human dendritic cell derived from human blood. If blood is used, dendritic cells can be obtained by a relatively simple operation using a conventionally known method or the like.

Moreover, it is preferable that the manufacturing method further includes a step of collecting the blood from a human . According to this, it is possible to obtain vaccine pulsed human dendritic cells effective for the prevention or treatment of human diseases.

Moreover, it is preferable that the vaccine used for the pulse is a disease prevention or treatment vaccine. According to this, effective in the prevention or treatment of such diseases, it is possible to provide a vaccine pulsed human dendritic cells.

The vaccine used for the pulse is preferably a hepatitis vaccine. According to this, a hepatitis vaccine pulsed human dendritic cell having the ability to present hepatitis virus antigens, which is effective for the prevention or treatment of hepatitis, which can be used safely and effectively for living organisms, for example, humans including healthy individuals, is provided. Can do.

The hepatitis vaccine is an HB vaccine containing HBs antigen . According to this, a hepatitis vaccine pulsed human dendritic cell having an ability to present an HBs antigen, which is effective for the prevention or treatment of hepatitis B, which can be used safely and effectively for an organism, for example, a human including a healthy person, is provided. be able to.

8. Composition Containing Vaccine Pulsed Human Dendritic Cells The composition according to the present embodiment preferably includes the vaccine pulsed human dendritic cell according to the present embodiment.

  Moreover, it is preferable that the composition which concerns on this Embodiment contains a vaccine additive further.

  In the present embodiment, “the composition according to the present embodiment further includes a vaccine additive” means that the vaccine additive is included somewhere in the entire composition according to the present embodiment. Say the state. That is, the place where the vaccine additive is contained may be anywhere outside the dendritic cell in the composition according to the present embodiment, inside, on the cell membrane, etc., and is not particularly limited. By including the vaccine additive in the composition according to this embodiment, it is possible to determine that the vaccine pulse dendritic cell according to this embodiment has been obtained using the antigen contained in the vaccine as the antigen. .

  Examples of vaccine additives include preservatives, antibiotics, stabilizers, preservatives, immunostimulants, buffers, isotonic agents, coloring agents, and the like. Specific examples include thimerosal and formalin as preservatives, erythromycin, erythromycin lactobionate, kanamycin sulfate, and other additives as antibiotics. Examples of other additives include purified sucrose, sodium L-glutamate, gelatin, lactose, potassium glutamate, D-sorbitol, human serum albumin, polysorbate 80, glycine, glucose, sodium hydroxide, potassium aluminum sulfate, aluminum chloride, Acetic acid, sodium acetate, sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride, phenol red, L-arginine hydrochloride, disodium phosphate, monopotassium phosphate, L-arginic acid, lysine hydrochloride Etc. These other additives can be added as stabilizers, preservatives, immunostimulants, buffers, isotonic agents, colorants. In addition, a vaccine additive is not limited to these.

  The composition according to the present embodiment is preferably an experimental reagent or an experimental kit. Thus, the vaccine pulse dendritic cells according to the present embodiment can be used in a mode suitable for various studies. Examples of research fields that can be used include research on signal transduction mechanisms related to the disease and research on immunotherapy using dendritic cells. Moreover, it can also be used for the purpose of, for example, research related to prevention or treatment of the disease, for example, screening of genes, RNA, peptides, proteins, compounds, pharmaceuticals, cytokines and the like.

  Moreover, it is preferable that the composition which concerns on this Embodiment is a pharmaceutical composition. The pharmaceutical composition according to the present embodiment can be used, for example, in an aspect where it is directly administered in vivo, or in an aspect where it is mixed with other substances or cells in vitro.

  Moreover, it is preferable that the composition which concerns on this Embodiment is an immune response activation formulation. The immune response activating preparation according to the present embodiment can be used, for example, in an aspect in which it is directly administered in vivo, or in an aspect where it is mixed with other substances or cells in vitro.

  As an aspect of using the composition according to the present embodiment in vitro, for example, the immune response activation preparation according to the present embodiment and T cells are mixed, and the antigen epitope contained in the vaccine is converted into T cells. The usage method which activates T cell by showing is mentioned. Further, these T cells can be used by being administered in vivo.

  Moreover, as an aspect which administers the composition which concerns on this Embodiment directly in the living body, the injection and infusion as a cell vaccine are mentioned, for example. The cell vaccine is preferably a disease prevention or treatment vaccine. In addition, as a place where the composition according to the present embodiment is directly administered into a living body, for example, intravenous administration, intraarterial administration, intrathoracic administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, intramuscular (for example, Liver etc.) administration, lymph node administration and the like. Subcutaneous injection is preferred because of the simplicity of administration.

9, the vaccine pulsed human dendritic cells according to the prevention or treatment embodiment of disease using vaccines pulsed human dendritic cells can be used in the prevention or treatment of disease. Examples of the disease include hepatitis B and chronic hepatitis B.

Vaccines pulses human dendritic cells according to the present embodiment, for example, can be used in combination with a method for the prevention or treatment of conventional the disease.

Moreover, it is preferable that the prevention or treatment of the disease according to the present embodiment includes a step of administering vaccine-pulsed human dendritic cells to an organism. Examples of the organism include organisms that can be infected with the disease. In addition, if the rejection reaction can be suppressed, the organism derived from the dendritic cell and the organism to be administered may be the same species or different species. In the case of allogeneic organisms, the dendritic cells according to the present embodiment can be administered into the removed self body, or can be administered into a non-self body that matches the degree to which the histocompatibility antigen can be administered. It is also possible.

For example, the organism to be administered is preferably a human. In this case, it is desirable that the HLA types match as much as possible. Accordingly, the human dendritic cells are preferably derived from human donors. If autologous cells are used, it is not necessary to consider the suitability of the HLA type, and thus human dendritic cells derived from the subject person are more preferable.

As described above, the vaccine pulsed human dendritic cell according to the present embodiment is used in a mode of being administered directly into a living body, or mixed with a T cell in vitro to activate the T cell. It can be used in a mode of administration in vivo. However, since T cells are effector cells, once they function, they die and disappear in a short time. Therefore, in order to prevent and treat diseases, it is necessary to administer T cells repeatedly at a high frequency. On the other hand, dendritic cells act on naive T cells, memory T cells, and B cells and repeatedly give stimuli, and thus have a long and powerful effect. From the above, it is considered that it is more preferable to use the vaccine pulse dendritic cell according to the present embodiment in a mode in which it is directly administered into a living body.

10, the following vaccine pulses example of the use of human dendritic cells and their effects of the present embodiment, illustrating the use example and its effect of the vaccine pulsed human dendritic cells according to the present embodiment. In this use example, it is preferable to include a step of administering the vaccine-pulsed human dendritic cell according to the present embodiment to a human. This use example can be applied to the prevention or treatment of diseases. In addition, this use example is not limited to the following cases, The use aspect of the human dendritic cell which concerns on this use example can be variously changed in the range which does not exceed the summary of this invention.

  Until now, in the dendritic cell therapy which has been performed in cancer treatment or the like, the dendritic cells are pulsed using a substance extracted from the cancer cells. As described above in the background section, the substance extracted from the cell is generally a mixture of many kinds of substances. Therefore, it is considered that the dendritic cells pulsed using the cell extract material were pulsed by many kinds of antigens in the cell extract material. The dendritic cells treated in such a manner are considered to be a group of many types of dendritic cells having antigen-presenting ability including antigens that cannot be identified or present antigens not related to diseases. The dendritic cells thus prepared are allowed to be used for the treatment of patients already suffering from diseases in the field of cancer treatment and the like, but are not allowed to be administered to healthy individuals. Therefore, it is impossible to use dendritic cells pulsed with a cell extract for healthy people for the prevention of diseases.

In contrast, vaccines pulse human dendritic cells in the present embodiment can be produced by pulsing human dendritic cells with a vaccine. What is actually sold as a vaccine is one that has been approved and approved for safety and effectiveness by humans including healthy individuals. Thus, human dendritic cells pulsed with a vaccine according to this embodiment can be used not only for patients for the treatment of diseases but also for healthy people for the prevention of diseases.

  In addition, for example, tuberculosis has been steadily decreasing after the war in Japan, but the rate of decrease has slowed since around 1980, and the number of patients has increased for three consecutive years since 1997. In addition, long-term administration of anti-tuberculosis drugs is indispensable for the treatment of tuberculosis, but it is very likely that drug-resistant bacteria will be generated, and in order to prevent this, the combined use of two or more types of drugs is a rule.

Therefore, the vaccine pulsed human dendritic cells according to the present embodiment can be used in the treatment of tuberculosis. Here, vaccine-pulsed human dendritic cells obtained by a method including a step of pulsing with a tuberculosis vaccine such as BCG vaccine can be used as the vaccine-pulsed human dendritic cells according to the present embodiment. According to this, it is expected that the appearance frequency of the drug-resistant bacteria as described above can be lowered, and this is considered to be a great advantage in the treatment of tuberculosis. In addition, for other diseases, the same effect may be expected by using vaccine-pulsed human dendritic cells for treatment, and it is expected to be a great advantage for treatment of the disease.

  In addition, as another method for presenting antigens to dendritic cells, the gene encoding the antigen is incorporated into a plasmid vector or the like for transfection, or incorporated into a virus vector or the like for infection. Methods for expressing antigens are known. Today, however, dendritic cells that have been genetically engineered in this way may be used for the treatment of diseased patients, although they may be used to treat patients with the disease. It has not been.

In contrast, vaccines pulse human dendritic cells in the present embodiment, as described above, can be used not only as a therapeutic vaccine for a disease to the patient, it is possible to use as a prophylactic vaccine disease to healthy subjects.

  In addition, one of the causes of patients for whom the vaccine is not effective is considered to be a decrease in the function of dendritic cells. That is, it is conceivable that dendritic cells cannot be sufficiently activated only by administration of a vaccine to a living body as in the past.

Therefore, for example, a mode in which the vaccine-pulsed human dendritic cells according to the present embodiment are directly administered in vivo, or mixed with T cells outside the living body to activate T cells and administer the T cells in vivo. By using in an aspect, it becomes possible to produce an antibody against the target antigen even for a healthy person who has not been able to produce an antibody due to a vaccine so far.

From the above, the vaccine-pulsed human dendritic cells according to the present embodiment are recognized for wide use and great advantages. In addition, it is considered that the prevention or treatment of diseases using vaccine-pulsed human dendritic cells according to the present embodiment is completely distinguished from conventional dendritic cell therapy.

Note that in the best mode section for carrying out the invention, 6, 10, in the definition of the pulse, a specific method of the pulse, a method for obtaining human dendritic cells, for human dendritic cell cultures Unless otherwise stated, the culture medium, cytokines, vaccine additive detection methods, and the like can use the same interpretations, methods, and substances as those described in 1 to 5 of the same paragraph.

  Hereinafter, the present invention will be specifically described based on experimental examples, but the present invention is not limited to the examples.

(Experimental Example 1) Preparation of human dendritic cells (1) Using a syringe (Terumo Syringe SS-50ESZ: Terumo) with a lumen wetted with heparin (Novo-Heparin injection 1000: manufactured by Aventis), the subject ( The subjects were 5 healthy volunteers who had already received HB vaccine repeatedly.) 40-60 mL of blood was collected per person. The obtained blood and the same amount of phosphate-buffered saline (PBS) as the blood were thoroughly mixed in a dedicated clean bench. Next, mononuclear cells were separated by specific gravity centrifugation using Ficoll-Conray solution (specific gravity 1.077). Centrifugation was performed at room temperature, 1800 rpm to 3000 rpm for 30 minutes, and the intermediate layer after centrifugation was taken out as mononuclear cells.

  (2) Simultaneously, autologous serum was obtained from each subject according to the following procedure. First, about 10 mL of blood was collected per subject using another syringe that does not use heparin (Terumo Syringe SS-10ESZ: manufactured by Terumo Corporation). Next, the obtained blood was centrifuged at 3000 rpm using a sterilized vacutainer blood collection tube (manufactured by Becton Dickinson) to separate the serum.

  (3) PBS was added to the mononuclear cells obtained in (1) and mixed well. Next, this sample was centrifuged at 1800 rpm to 2700 rpm for 10 minutes at 4 ° C., and mononuclear cells were again collected by the same procedure as (1). By repeating this operation at least twice, the mononuclear cells were thoroughly washed.

(4) The mononuclear cells obtained in (3) were counted, and 2 mL each was added to a 35 mm dish at a cell density of 2 × 10 ⁶ to 2 × 10 ⁷ / mL. As the medium, RPMI 1640 medium (manufactured by Nipro Co., Ltd.) to which the autologous serum of each subject obtained in (2) was added at a concentration of 1 to 5% was used. The culture was performed in a dedicated culture room under conditions of 37 ° C., 5% CO ₂ concentration, and 75% air.

  (5) After culturing the mononuclear cells of (4) for 90 minutes, adherent cells were separated according to the following procedure. First, the dish was removed from the culture chamber and allowed to stand for 5 minutes in a clean bench. Thereafter, the dish was tilted diagonally, and the culture was slowly applied to the entire adherent cells for washing to remove lymphocytes.

  (6) With respect to the adherent cells (monocytes) obtained in (5), GM-CSF (manufactured by Peprotech EC) is 800 U / mL and IL-4 (manufactured by Peprotech EC) is 500 U / mL per dish. The cells were cultured for 4 days in 2 mL of RPMI 1640 medium supplemented with 1 mL 5 mL of autologous serum.

  (7) On the fourth day of culture, 1 mL of the culture supernatant was discarded, and 1 mL of a medium having the composition described in (6) was newly added, followed by further culturing for 4 days.

  (8) On day 8 of culture, dendritic cells were collected from the dish and the number of dendritic cells was measured.

(Experimental example 2) Human dendritic cell pulse by HB vaccine (1) The human dendritic cell obtained in Experimental example 1 was divided into two. On one dendritic cell, HB vaccine (r-HB vaccine “Mitsubishi”: manufactured by Mitsubishi Pharma) (composition: HBs antigen 20 μg, thimerosal 0.01 w / v%, aluminum potassium sulfate 5.2 g (each per 1 mL)) Was added to a final concentration of 2.5-5 μg / mL and cultured for 8-24 hours to prepare dendritic cells pulsed with HB vaccine containing HBs antigen. (Hereinafter, this cell is referred to as “HB vaccine pulse dendritic cell”.) Cultivation was carried out in a dedicated culture room under conditions of 37 ° C., 5% CO ₂ concentration, and 75% air. The other dendritic cell was cultured as a negative control without the addition of HB vaccine.

  (2) After culture, HB vaccine pulse dendritic cells were collected from the dish. The collected cells and PBS were mixed and centrifuged at 4 ° C. and 1500 rpm for 5 minutes, and then the supernatant was discarded. By repeating this operation 5 times, the dendritic cells were sufficiently washed. The obtained dendritic cells were added to 500 μL of PBS. The same operation was performed for dendritic cells of negative control.

(Experimental Example 3) Administration of HB vaccine pulsed human dendritic cells (1) Anti-HBs antibodies before administration of HB vaccine pulsed human dendritic cells were measured. First, blood was collected from five subjects (healthy volunteers who had already received HB vaccine administration repeatedly), and serum was obtained from each subject using the same method as in Experimental Example 1 (2). Using the serum, anti-HBs antibody values were measured by the CLIA method. The results are shown in FIG. As shown in FIG. 1, 2 out of 5 subjects can produce anti-HBs antibody (anti-HBs antibody positive, responder), 3 cannot produce anti-HBs antibody (anti-HBs antibody negative, non -Responder).

(2) Administration of HB vaccine pulsed human dendritic cells to subjects. First, a prick test for each dendritic cell was performed on each subject using 50 μL of 500 μL of HB vaccine pulsed human dendritic cells obtained in Experimental Example 2. As a result, the prick test was negative in all subjects. Next, 450 μL of each HB vaccine pulsed human dendritic cell was subcutaneously administered to each subject. In the process of preparation and administration of HB vaccine pulse dendritic cells shown in Experimental Examples 1 to 3, the operator works by wearing a sterilized gown, sterilizing mask, and sterilizing cap, and the operation is as clean as possible. I went on the bench. As the reagent used, a sterilized reagent was purchased and used (PBS: manufactured by Invitrogen, RPMI 1640: manufactured by Nipro, etc.), and it was confirmed that the reagent was not turbid with the naked eye and a microscope at the time of use. Moreover, it was confirmed that there was no contamination by microorganisms during the culture process by measuring endotoxin in the culture supernatant.

(Experimental example 4) Surface antigen analysis of HB vaccine pulsed human dendritic cell The cell surface antigen was analyzed using flow cytometry.

(1) obtained in Experimental Example 2, HB vaccine pulsed human dendritic cells, and, human dendritic cells not pulsed, 5 ° C., was recovered by centrifugation for 5 minutes at 1500 rpm. The collected cells were washed with FCM PBS (pH 7.22: manufactured by Gibco) and collected again by centrifugation at 5 ° C. and 1500 rpm for 5 minutes.

  (2) Each dendritic cell obtained in (1) was divided into four Spitz tubes, and (a) 10 μL of FITC-labeled mouse IgG (Cat. No. 349041: Becton Dickinson), (b) 2 μL of PE-labeled mouse IgG (Cat. No. 349043: manufactured by Becton Dickinson), (c) 10 μL of FITC-labeled mouse anti-human HLA-DR monoclonal antibody (PM-32414X: manufactured by Becton Dickinson), (d) A sample to which 10 μL of PE-labeled anti-human CD86 (B70 / B7-2) (Mat. No. 555658, manufactured by Pharmingen) was added was prepared. These were placed on ice for 30 minutes, 2 mL of lysing solution (Becton Dickinson) was added and mixed, and centrifuged at 5 ° C. and 2000 rpm for 5 minutes.

  (3) The collected dendritic cells were sufficiently washed with FCM PBS (pH 7.22), and then surface antigens were analyzed using FACScalibur (Becton Dickinson).

  (4) The results of the analysis are shown in FIG. In FIG. 2, the horizontal axis represents the analyzed surface antigen, and the vertical axis represents the relative concentration of the surface antigen. From this result, HB vaccine-pulsed dendritic cells are found to have increased HLA-DR expression by 1.2 times and CD86 expression by 1.6 times compared to non-pulsed dendritic cells. all right. From these results, it was found that HB vaccine pulsed dendritic cells were mature.

(Experimental Example 5) Functional analysis-1 of HB vaccine pulsed human dendritic cells
(1) Mononuclear cells were collected from the peripheral blood of another person who did not receive the vaccine in the same manner as in Experimental Example 1 (1).

(2) The operation of isolating T cells from mononuclear cells was performed using autoMACS (Miltenyi Biotec) and Pan T cell isolation kit (Miltenyi Biotec). First, MACS-Buffer is added to 1 × 10 ⁷ mononuclear cells to make 80 μL, and further 20 μL of hapten-labeled antibody cocktail (CD11b, CD16, CD19, CD36, CD56) is added, and the mixture is heated at 6-12 ° C. Mix for minutes. Next, MACS-Buffer was added and washed, and the operation of removing the supernatant was performed twice. Subsequently, MACS-Buffer was added to 1 × 10 ⁷ mononuclear cells to make 80 μL, and further 20 μL of MACS anti-hapten antibody-labeled microbeads were added and mixed at 6 to 12 ° C. for 15 minutes. Next, MACS-Buffer was added for washing, and the supernatant was removed. Subsequently, 500 μL of MACS-Buffer was added to 1 × 10 ⁷ mononuclear cells, and depletion was performed using the autoMACS Deploy program to collect T cells.

(3) (a) unpulsed human dendritic cells 1 × 10 ⁴ cells, (b) HB vaccine pulsed human dendritic cells 1 × 10 ⁴ cells, and the T cells obtained in (2) 2 × 10 ⁵ cells were added and cultured for 108 hours. As the medium, RPMI 1640 medium supplemented with 10% FCS (manufactured by Filtron) was used.

(4) ³ H-labeled thymidine was added to each, followed by further culturing for 12 hours, and then cells were collected from the dish.

(5) The results of evaluation of uptake ability using a γ-ray counter (LS6500: manufactured by Beckman) are shown in FIG. The vertical axis in FIG. 3 shows the thymidine uptake capacity, and the horizontal axis shows the cases (a) and (b) shown in (3) from the left. From this result, compared with non-pulsed dendritic cells, HB vaccine-pulsed human dendritic cells have a T cell blast rejuvenation ability of 1.5 to It was found that the rate was enhanced 2 times.

(Experimental example 6) Functional analysis of human dendritic cells pulsed with HB vaccine-2
(1) 60 mL of blood was collected from each subject (healthy volunteers who had been repeatedly administered HB vaccine and HB vaccine responder), and Experimental Example 1 (1) and Experimental Example 5 (2 ) Was used to collect T cells. (Hereinafter, this T cell is referred to as “HBs memory T cell”.)
(2) Dendritic cells were collected from the same subject using the method of Experimental Example 1 and divided into two. One dendritic cell was an HB vaccine pulsed dendritic cell using the method of Experimental Example 2, and the other dendritic cell was not pulsed with an HB vaccine and used as a negative control.

(3) (a) HBs memory T cells 1 × 10 ⁵ cells and HB vaccine 10 μg, (b) HBs memory T cells 1 × 10 ⁵ cells and unpulsed dendritic cells 1 × 10 ⁴ cells, (c) HBs Memory T cells 1 × 10 ⁵ cells and HB vaccine pulse dendritic cells 1 × 10 ⁴ cells were mixed and cultured.

(4) Seven days later, thymidine labeled with ³ H was administered 12 hours before the measurement, and thymidine uptake ability was measured using a γ-ray counter in the same manner as in Experimental Example 5 (5).

(5) The results are shown in Figure 4. The vertical axis in FIG. 4 represents the relative cpm ratio, and the horizontal axis shows the cases (a), (b), and (c) described in (3) from the left. From this result, the cpm mixed with the HB vaccine pulsed dendritic cell of (c) is 7 times the HBs memory T cell compared with the case of the mixed culture with the non-pulsed dendritic cell of (b). Was proliferating.

(Experimental Example 7) Functional analysis of HB vaccine pulsed human dendritic cells-3
(1) 60 mL of blood was collected from a subject (healthy volunteer who has already received repeated administration of the HB vaccine, and the HB vaccine responder), and was simply treated using the method of Experimental Example 1 (1). Nuclear cells were collected.

(2) Mononuclear cells 6 × 10 ⁷ cells, (a) nothing mixed, (b) HB vaccine 10 μg, (c) unpulsed dendritic cells 1 × 10 ⁵ cells, (d) HB vaccine Pulsed dendritic cells 1 × 10 ⁵ cells were mixed and cultured. The culture conditions were the same as in Experimental Example 1 (4).

  (3) After 5 days, the culture supernatant was collected, and the anti-HBs antibody value was measured by the CLIA method.

(4) The results are shown in FIG. The vertical axis in FIG. 5 indicates the anti-HBs antibody value, and the horizontal axis indicates the cases (a), (b), (c), and (d) described in (2) from the left. From this result, when HB vaccine and non-pulsed human dendritic cells were mixed, anti-HBs antibody could not be produced, but when HB vaccine pulsed human dendritic cells were mixed, anti-HBs antibody It was found that 15 to 56 mIU / mL can be produced.

(Experimental example 8) Hypersensitivity reaction test (0 to 24 hours) accompanying administration of HB vaccine pulsed human dendritic cells
(1) In Experimental Example 3, after the HB vaccine pulsed human dendritic cells were subcutaneously administered to each subject, the presence or absence of a hypersensitive reaction such as inflammation accompanying the vaccine administration was confirmed at 0 to 24 hours.

(2) As a result, no acute hypersensitivity reactions such as fever, redness, and shock were observed in all 5 subjects who received HB vaccine pulsed human dendritic cells. Therefore, it was confirmed that the HB vaccine pulsed human dendritic cells are safe.

(Experimental example 9) Safety test by blood biochemical examination (Days 0, 1, 3, 14)
(1) In order to further confirm the safety of HB vaccine-pulsed human dendritic cells administered to subjects in Experimental Example 3, each subject was tested for 50 or more items regarding CBC, liver function, kidney function, etc. Went. Typical inspection items are shown in FIG.

(2) The results of ALT, CRP, PT, and Creatine test are shown in FIGS. In each figure, the horizontal axis indicates the number of days since administration, and the vertical axis indicates the test item and its value. From these results, no abnormalities were observed in blood biochemical tests in all five subjects who were administered HB vaccine pulsed human dendritic cells. Therefore, HB vaccine pulsed human dendritic cells were found to be safe.

(Experimental example 10) Safety test on autoimmune reaction using serum biochemical test (0th and 14th days)
(1) In order to examine the presence or absence of autoimmune reaction by HB vaccine pulse dendritic cells administered to subjects in Experimental Example 3, measurement of IgG amount, IgM amount, thyroid test, microsome test, antinuclear antibody Autoantibody tests such as

(2) The results are shown in FIG. From these results, HB vaccine pulsed dendritic cells did not show any autoimmune reaction in all 5 subjects who received HB vaccine pulsed human dendritic cells. Therefore, HB vaccine pulsed human dendritic cells were found to be safe.

(Experimental example 11) Evaluation of anti-HBs antibody inducing ability of HB vaccine pulsed human dendritic cells in vivo (Day 0, 2, 3, 4, 12 weeks)
(1) From each subject who received HB vaccine pulsed human dendritic cells in Experimental Example 3, serum was collected using the method of Experimental Example 1 (2), and anti-HBs antibody levels were measured by the CLIA method. .

(2) The results are shown in FIGS. 12 and 13. The horizontal axis of FIGS. 12 and 13 indicates the week after administration, and the vertical axis indicates the anti-HBs antibody value . FIG. 13 is a graph showing the transition of anti-HBs antibody values of subjects who are HB vaccine non-responders. From the results shown in FIG. 12 , it was found that two of three subjects who were HB vaccine non-responders were able to produce anti-HBs antibodies by one administration of HB vaccine pulse dendritic cells. Moreover, the anti-HBs antibody level also rose about two subjects who are HB vaccine responders. Since these 4 subjects have an anti-HBs antibody value of 10 mIU / mL or more, one administration of HB vaccine pulse dendritic cells can produce an antibody in an amount sufficient for the prevention or treatment of hepatitis B. it is conceivable that. From the results of FIG. 13 , it was found that the subject who was this HB vaccine responder could maintain an anti-HBs antibody value of 10 mIU / mL or more for at least 3 months.

  Blood was collected from subjects (two hepatitis B patients and one patient after hepatitis B liver transplantation), and dendritic cells were prepared using the blood in the same manner as in Experimental Example 1 of Example 1. . Next, HB vaccine pulse dendritic cells were prepared using the obtained dendritic cells in the same manner as in Experimental Example 2 of Example 1.

  In the same manner as in Experimental Example 1 of Example 1, 50 μL of the obtained HB vaccine pulsed dendritic cells (500 μL) were used to perform a prick test on each subject's own dendritic cells. As a result, the prick test was negative in all subjects. Next, 450 μL of each HB vaccine pulse dendritic cell was subcutaneously administered to each subject. After administration, the subject showed no change in subjective symptoms.

In order to further confirm the safety of HB vaccine-pulsed human dendritic cells administered to the subjects, 50 items on CBC, liver function, kidney function, etc. were given to each subject on days 0, 3, 14, and 28. The above inspection was performed. In all the subjects, blood chemical tests showed no change before and after administration, and no abnormality was observed. FIG. 14 shows the results of ALT, CRP, PT, and WBC tests on one hepatitis B patient among the subjects. In the figure, the horizontal axis indicates the number of days since administration, and the vertical axis indicates the test item and its value. Therefore, it was found that HB vaccine pulsed human dendritic cells are safe for hepatitis B patients.

To a subject subjected to subcutaneous administration of their HB vaccines pulse human dendritic cells in Example 2 (B hepatitis), after two weeks from the administration, and after 6 weeks, respectively, HB vaccine pulsed human dendritic Cells were administered subcutaneously at 450 μL. Serum was collected from the subject, and anti-HBs antibody levels were measured by CLIA method using the serum (0, 2, 6, 10, 14, 18 and 22 weeks). The results are shown in Figure 15. As shown in the figure, HBs antibody was produced after the end of the first administration (week 0), and the amount of HBs antibody also increased after the end of the third administration (week 6). During this measurement period, there was no increase in liver function tests and no increase in transamylase.

As a result, after the administration of the vaccine pulsed human dendritic cells resulted in no increase in the transformer amylase, according to the vaccine pulsed human dendritic cells of the present invention, without inducing hepatic cytotoxicity CTL, HBs It was found that antibodies could be produced. This is because transamylase is usually increased when hepatotoxic CTLs are induced. Although the mechanism of not inducing this hepatotoxic CTL is unknown, it is considered that the composition of the HB vaccine used for the pulse is closely related to the mechanism of not inducing this hepatotoxic CTL. Also, vaccines pulse human dendritic cells of the present invention may be a hepatitis B patient, because it can induce HBs antibody production, it has been shown to be induced mainly humoral immunity.

Claims (26)

Obtaining a human dendritic cells using monocytes isolated from human peripheral blood, the human dendritic cells, obtained by a method comprising the step of pulsing by culturing in a medium containing HBs antigen, HBs Human dendritic cells that have antigen-presenting ability and do not induce hepatotoxic CTL. The human dendritic cell according to claim 1 , wherein the HBs antigen is an HBs antigen contained in an HB vaccine. Wherein the step of obtaining a human dendritic cells using monocytes isolated from human peripheral blood, the human dendritic cells, to claim 1 or 2 including the step of pulse cultured in medium containing HBs antigen A method for producing dendritic cells. The production method according to claim 3 , wherein the HBs antigen is an HBs antigen contained in an HB vaccine. A composition comprising the human dendritic cell according to claim 1 or 2 . The composition according to claim 5 , further comprising an HB vaccine additive. The composition according to claim 5 or 6 , wherein the composition is an experimental reagent or an experimental kit. The composition according to claim 5 or 6 , wherein the composition is a pharmaceutical composition. The composition according to claim 5 or 6 , wherein the composition is an immune response activation preparation. The composition according to claim 5 or 6 , wherein the composition is a cellular vaccine. The composition according to claim 10 , wherein the cellular vaccine is a preventive or therapeutic vaccine for hepatitis B. Obtained by a method comprising the steps of obtaining a human dendritic cells using monocytes isolated from human peripheral blood, the human dendritic cells, and a step of pulse cultured in medium containing HB vaccines containing HBs antigen A vaccine-pulsed human dendritic cell that does not induce hepatotoxic CTL. Obtaining a human dendritic cells using monocytes isolated from human peripheral blood, the human dendritic cells, claim including the step of pulsing by culturing in a medium containing HB vaccines containing HBs antigen 12 A method for producing vaccine-pulsed human dendritic cells according to 1. A composition comprising the vaccine-pulsed human dendritic cell according to claim 12 . The composition according to claim 14 , further comprising a vaccine additive. The composition according to claim 14 or 15 , wherein the composition is an experimental reagent or an experimental kit. The composition according to claim 14 or 15 , wherein the composition is a pharmaceutical composition. The composition according to claim 14 or 15 , wherein the composition is an immune response activation preparation. The composition according to claim 14 or 15 , wherein the composition is a cellular vaccine. The composition according to claim 19 , wherein the cellular vaccine is a disease prevention or treatment vaccine. A vaccine pulse dendritic cell for humans, wherein the vaccine pulse dendritic cell is a vaccine pulse dendritic cell for the prevention or treatment of human hepatitis B, has an ability to present HBs antigen, and is a hepatocyte Dendritic cell acquisition step of obtaining dendritic cells using monocytes isolated from peripheral blood derived from hepatitis B patients or hepatitis B virus carriers without inducing toxic CTL and A vaccine pulse dendritic cell obtained by a production method including a pulsing step of culturing the dendritic cell in a medium containing a vaccine and pulsing, and the vaccine used for the pulse is for prevention or treatment of hepatitis B Vaccine pulse dendritic cells for humans, which is an HB vaccine containing HBs. The vaccine pulse dendritic cell for human according to claim 21 , wherein in the vaccine pulse step, a pulse time is 8 hours to 24 hours. The human vaccine pulse dendritic cell according to claim 21 or 22 , wherein in the dendritic cell obtaining step, the isolated monocyte is co-cultured with GM-CSF and IL-4. 24. The human vaccine pulse dendritic cell according to any one of claims 21 to 23 , which induces Th2 and does not induce Th1 in humans. 25. A pharmaceutical product for treatment and prevention for human hepatitis B, comprising the human vaccine pulse dendritic cell according to any one of claims 21 to 24 . 26. The pharmaceutical product according to claim 25 , wherein the hepatitis B is chronic hepatitis B.

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