JP2010259373A - Method for activation treatment of antigen-presenting cells - Google Patents

Abstract

An activated antigen-presenting cell capable of efficiently inducing immunocompetent cells containing a disease antigen-specific CD8 + CTL in vivo and / or in vitro, a medicament containing the activated antigen-presenting cell, and the activated antigen presentation A method for inducing immunocompetent cells including a disease antigen-specific CTL induced using the activated antigen-presenting cells, an immunocompetent cell induced by the method, and And a therapeutic / preventive method using the immunocompetent cell.
In addition to sensitizing antigen-presenting cells with a disease antigen, by co-sensitizing with a bisphosphonate and a cell wall skeletal component, the proportion and number of cells of the disease antigen-specific CD8 + CTL are compared with those in the case of no sensitization. Can be increased.
[Selection figure] None

Description

  The present invention relates to a method for activating an antigen-presenting cell comprising a step of sensitizing with a bisphosphonate, a bacterial cell wall skeleton component and a disease antigen. The present invention also includes a medicament containing the antigen-presenting cell, a method for treatment / prevention using the antigen-presenting cell, a method for inducing an immunocompetent cell using the antigen-presenting cell, an immunocompetent cell induced by the induction method, The present invention relates to a medicament containing the immunocompetent cells, a therapeutic / preventive method using the immunocompetent cells, and an activator for antigen-presenting cells.

  Cancer treatment methods include surgical excision, radiation, and chemotherapy using anticancer drugs. In recent years, various treatment methods other than those therapies have been studied and put into practical use. One of them is immune cell therapy using immune cells.

  Immunocytotherapy includes LAK (Lymphokine Activated Killer) therapy in which lymphocytes are activated by lymphokines outside the body and returned to the body, cytotoxic T cells that specifically recognize and injure lesions (Cytotoxic T Lymphocyte), hereinafter also referred to as CTL CTL therapy using), dendritic cell therapy using dendritic cells (hereinafter also referred to as DC), and the like.

  The dendritic cell therapy uses dendritic cells that have been processed by the MHC (Major Histocompatibility Antigen Complex) after processing the disease antigen directly or intracellularly, and the pathogen is selectively attacked in the body. It is a therapy that induces and treats disease antigen-specific CTLs. The disease antigen to be presented includes, for example, a cancer antigen protein or peptide, an infectious disease antigen protein or peptide, or a part thereof (see, for example, Non-Patent Document 1 and Non-Patent Document 2).

  Disease antigen-specific CTLs can be induced in vitro (in vitro) by co-culturing dendritic cells and lymphocytes sensitized with a disease antigen and stimulating the lymphocytes with the dendritic cells. For example, when a dendritic cell sensitized with a cancer antigen protein or peptide or an infectious disease antigen protein or peptide is used, the increase in the disease antigen CTL induced by a single mixed culture does not use the dendritic cell. It is 5 to 20 times the case.

  In dendritic cell therapy using a dendritic cell sensitized with a cancer antigen protein or peptide or an infectious disease antigen protein or peptide, in vivo (in vivo) disease antigen-specific CTL induction efficiency, that is, to all lymphocytes It is known that the proportion of occupied CTL increases to 2 to 14 times.

  Various methods have been used to improve the effect of dendritic cell therapy by increasing the induction efficiency of disease antigen-specific CTLs by dendritic cells.

  First, in addition to sensitizing dendritic cells with disease antigens, drugs that react with Toll-like receptors (hereinafter referred to as TLR) on dendritic cells are also reacted to dendritic cells. This is a method for directly increasing the induction efficiency of disease antigen-specific CTL by enhancing the antigen presentation ability of dendritic cells (adjuvant effect via TLR, for example, Non-Patent Document 3, Non-Patent Document 4). reference). For example, BCG-CWS (Bacillus of Calmette and Guerin-Cell Wall Skelton) is a component extracted and purified from Mycobacterium bovis (M. tuberculosis) Calmetogeran, and the main component is arabinogalactan mycolate -Consists of peptidoglycan. Therefore, BCG-CWS stimulates through TLR2 / 4 which acts as a receptor for bacterial peptidoglycan, and enhances the antigen presentation ability of dendritic cells.

  As another method, in addition to sensitizing dendritic cells with disease antigens, antigen-presenting molecules other than MHC molecules on dendritic cells, such as glycolipids on CD1d (Cluster Differentiation 1d) molecules, etc. And activate immunocompetent cells including iNKT cells (invariant Natural Killer T cells) other than disease antigen-specific CTL, and indirectly through the activated immunocompetent cells, disease antigen-specific CTL This is a method for increasing the induction efficiency (adjuvant effect via immunocompetent cells other than disease antigen-specific CTL, for example, see Non-Patent Document 5 and Non-Patent Document 6).

  In addition, a method is known in which dendritic cells are activated by sensitization with bisphosphonate and a disease antigen to increase the induction efficiency of CTL (Patent Document 1, Non-Patent Document 7).

  However, the increase in disease antigen-specific CTL induction by the above-described direct adjuvant effect through TLR or the indirect adjuvant effect through immunocompetent cells other than disease antigen-specific CTL is still insufficient. There is a need to develop a technology that can induce disease antigen-specific CTL more efficiently.

WO2007 / 029689

Blood 2004, 103, 383-389 Proc Natl Acad Sci USA. 2001, 98, 8809-8814 Nat Rev Immunol. 2004, 4, 449-511 Cancer Res. 2004, 64, 5461-5470 J Clin Invest. 2004, 114, 1800-1811 J Exp Med. 2002, 195, 617-624 Blood 2007, 110, 921-927

  The present invention has been made in view of the above circumstances, and a method for activating antigen-presenting cells (for example, dendritic cells) for efficiently inducing disease antigen-specific CD8 + CTL in vivo and / or in vitro. , A medicament containing the activated antigen-presenting cell, a method for treatment / prevention using the activated antigen-presenting cell, a method for inducing a disease antigen-specific CD8 + CTL using the activated antigen-presenting cell, and immunity induced by the method Provided are a responsible cell, a medicament containing the immunocompetent cell, a treatment / prevention method using the immunocompetent cell, and an activator for antigen-presenting cells.

  The present inventors have studied to solve the above problems. As a result, in addition to sensitizing dendritic cells with disease antigens and co-sensitizing with bisphosphonate and bacterial cell wall skeletal components, total lymphocytes of disease antigen-specific CTLs are compared to when bisphosphonate and bacterial cell wall skeletal components are not added. It was found that the ratio and the number of cells occupy significantly increased, and the present invention was completed. For example, by adding bisphosphonate and bacterial cell wall skeletal components, the proportion of disease antigen-specific CTL in the total lymphocytes is about 6.0 times higher than when not added, and the disease antigen-specific CTL is about It can be 19 times. However, the present invention is not limited to these numerical values. Both bisphosphonates and bacterial cell wall skeletal components are known to activate dendritic cells, but using both together is not just an added effect, but a synergistic effect beyond what is expected Was confirmed. According to the present invention, further development of immune cell therapy for inducing disease antigen-specific CD8 + CTL to treat / prevent cancer and / or infection is promoted.

  That is, the present invention is as follows.

上記式（Ｉ）中、Ｒは、水素原子又は低級アルキル基であり、Ｒ_１とＲ_２は、それぞれ独立して、水素原子、ハロゲン原子、ヒドロキシル基、アミノ基、チオール基、アリール基又は置換されたアリール基、アルキル基又は置換されたアルキル基、低級アルキルアミノ基、アルアルキル基、シクロアルキル基及び複素環式基からなる群から選択され、又はＲ_１とＲ_２は同じ環状構造の一部を形成し、
上記Ｒ_１とＲ_２における置換基としては、ハロゲン原子、低級アルキル基、ヒドロキシル基、チオール基、アミノ基、アルコキシ基、アリール基、アリールチオ基、アリールオキシ基、アルキルチオ基、シクロアルキル基、複素環式基等からなる群から選択される（１）又は（２）に記載の抗原提示細胞の活性化処理方法；
（４）前記ビスホスホネートが、ゾレドロン酸、パミドロン酸、アレンドロン酸、リセドロン酸、イバンドロン酸、インカドロン酸、エチドロン酸、それらの塩、及び、それらの水和物からなる群から選択される（１）から（３）のいずれか一つに記載の抗原提示細胞の活性化処理方法；
（５）前記共感作における前記ビスホスホネートの濃度が、０．００１〜２０μＭである（１）から（４）のいずれかに一つに記載の抗原提示細胞の活性化処理方法；
（６）前記共感作における前記ビスホスホネートの濃度が、０．００１〜５μＭである（５）に記載の抗原提示細胞の活性化処理方法；
（７）前記細菌細胞壁骨格成分がＢＣＧ−ＣＷＳであることを特徴とする（１）から（６）に記載の抗原提示細胞の活性化処理方法；
（８）前記疾病抗原が、がん抗原又は感染症抗原蛋白質、がん抗原又は感染症抗原ペプチド、がん細胞又は感染症細胞の細胞融解物、アポトーシス細胞、ネクローシス細胞、及び、それらの熱処理物からなる群から選択される（１）から（７）のいずれか一つに記載の抗原提示細胞の活性化処理方法；
（９）前記共感作における前記疾病抗原の濃度が、０．０１〜２０μｇ／ｍＬである（１）から（８）のいずれか一つに記載の抗原提示細胞の活性化処理方法；
（１０）前記共感作における前記疾病抗原の濃度が、０．１〜２μｇ／ｍＬである（９）に記載の抗原提示細胞の活性化処理方法；
（１１）活性化抗原提示細胞の生産方法であって、（１）から（１０）のいずれか一つに記載の活性化処理方法で抗原提示細胞を処理することを含む活性化抗原提示細胞の生産方法；
（１２）（１１）に記載の生産方法により生産された活性化抗原提示細胞、
（１３）がん及び／又は感染症のための医薬組成物であって、（１２）に記載の活性化抗原提示細胞を含む医薬組成物；
（１４）前記抗原提示細胞が、自己由来又はＨＬＡを同じくする他家由来である（１３）に記載の医薬組成物；
（１５）がん及び／又は感染症の予防・治療方法であって、（１２）に記載の活性化抗原提示細胞を投与することを含む予防・治療方法；
（１６）前記抗原提示細胞が、自己由来又はＨＬＡを同じくする他家由来である（１５）に記載の予防・治療方法。 (1) An antigen-presenting cell activation treatment method comprising the step of co-sensitizing the antigen-presenting cell with a bisphosphonate, a bacterial cell wall skeleton component, and a disease antigen;
(2) The method for activating antigen-presenting cells according to (1), wherein the antigen-presenting cells are selected from the group consisting of dendritic cells, immature dendritic cells, and artificial antigen-presenting cells;
(3) The bisphosphonate is a compound represented by the following general formula (I), a salt thereof, and a hydrate thereof,

In the above formula (I), R is a hydrogen atom or a lower alkyl group, and R ₁ and R ₂ are each independently a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, a thiol group, an aryl group, or a substituent. Selected from the group consisting of a substituted aryl group, an alkyl group or a substituted alkyl group, a lower alkylamino group, an aralkyl group, a cycloalkyl group and a heterocyclic group, or R ₁ and R ₂ are one of the same cyclic structure Forming part,
Examples of the substituent in R ₁ and R ₂ include halogen atoms, lower alkyl groups, hydroxyl groups, thiol groups, amino groups, alkoxy groups, aryl groups, arylthio groups, aryloxy groups, alkylthio groups, cycloalkyl groups, and heterocyclic rings. The method for activating an antigen-presenting cell according to (1) or (2) selected from the group consisting of a formula group and the like;
(4) The bisphosphonate is selected from the group consisting of zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, salts thereof, and hydrates thereof (1) To the activation treatment method of an antigen-presenting cell according to any one of (3);
(5) The method for activating antigen-presenting cells according to any one of (1) to (4), wherein the concentration of the bisphosphonate in the sensitization is 0.001 to 20 μM;
(6) The activation treatment method for antigen-presenting cells according to (5), wherein the concentration of the bisphosphonate in the sensitization is 0.001 to 5 μM;
(7) The method for activating antigen-presenting cells according to (1) to (6), wherein the bacterial cell wall skeleton component is BCG-CWS;
(8) The disease antigen is cancer antigen or infectious disease antigen protein, cancer antigen or infectious disease antigen peptide, cell lysate of cancer cell or infectious disease cell, apoptotic cell, necrotic cell, and heat-treated product thereof. The method for activating an antigen-presenting cell according to any one of (1) to (7) selected from the group consisting of:
(9) The method for activating an antigen-presenting cell according to any one of (1) to (8), wherein the concentration of the disease antigen in the sensitization is 0.01 to 20 μg / mL;
(10) The method for activating antigen-presenting cells according to (9), wherein the concentration of the disease antigen in the sensitization is 0.1 to 2 μg / mL;
(11) A method for producing an activated antigen-presenting cell, comprising: treating an antigen-presenting cell with the activation treatment method according to any one of (1) to (10); Production method;
(12) an activated antigen-presenting cell produced by the production method according to (11),
(13) A pharmaceutical composition for cancer and / or infection, comprising the activated antigen-presenting cell according to (12);
(14) The pharmaceutical composition according to (13), wherein the antigen-presenting cell is autologous or derived from another family having the same HLA;
(15) A preventive / treating method for cancer and / or infectious disease, comprising administering the activated antigen-presenting cell according to (12);
(16) The prevention / treatment method according to (15), wherein the antigen-presenting cell is derived from an autologous or another family having the same HLA.

上記式（Ｉ）中、Ｒは、水素原子又は低級アルキル基であり、Ｒ_１とＲ_２は、それぞれ独立して、水素原子、ハロゲン原子、ヒドロキシル基、アミノ基、チオール基、アリール基又は置換されたアルキル基、アルキル基又は置換されたアルキル基、低級アルキルアミノ基、アルアルキル基、シクロアルキル基及び複素環式基からなる群から選択され、又はＲ_１とＲ_２は同じ環状構造の一部を形成し、上記Ｒ_１とＲ_２における置換基としては、ハロゲン原子、低級アルキル基、ヒドロキシル基、チオール基、アミノ基、アルコキシ基、アリール基、アリールチオ基、アリールオキシ基、アルキルチオ基、シクロアルキル基、複素環式基からなる群から選択される（１７）から（１９）いずれか１つに記載の免疫担当細胞の誘導方法；
（２１）前記ビスホスホネートが、ゾレドロン酸、パミドロン酸、アレンドロン酸、リセドロン酸、イバンドロン酸、インカドロン酸、エチドロン酸、それらの塩、及び、それらの水和物からなる群から選択される（１７）から（２０）のいずれか一つに記載の免疫担当細胞の誘導方法；
（２２）前記共感作における前記ビスホスホネートの濃度が、０．００１〜２０μＭである（１７）から（２１）のいずれか一つに記載の免疫担当細胞の誘導方法；
（２３）前記共感作における前記ビスホスホネートの濃度が、０．００１〜５μＭである（２２）に記載の免疫担当細胞の誘導方法；
（２４）前記細菌細胞壁骨格成分がＢＣＧ−ＣＷＳである（１７）から（２３）のいずれか一つに記載の免疫担当細胞の誘導方法；
（２５）前記疾病抗原が、がん抗原又は感染症抗原蛋白質、がん抗原又は感染症抗原ペプチド、がん細胞又は感染症細胞の細胞融解物、アポトーシス細胞、ネクローシス細胞、及び、それらの熱処理物からなる群から選択される（１７）から（２４）のいずれか一つに記載の免疫担当細胞の誘導方法；
（２６）前記共感作における前記疾病抗原の濃度が、０．０１〜２０μｇ／ｍＬである（１７）から（２５）のいずれか一つに記載の免疫担当細胞の誘導方法；
（２７）前記共感作における前記疾病抗原の濃度が、０．１〜２μｇ／ｍＬである（２６）に記載の免疫担当細胞の誘導方法。 (17) A method for inducing immunocompetent cells, comprising the following steps (i) and (ii):
(I) sensitizing the antigen-presenting cell with a bisphosphonate, a bacterial cell wall skeletal component and a disease antigen;
(Ii) a step of co-culturing the antigen-presenting cells and lymphocytes simultaneously with or after the sensitization;
(18) The method for inducing immunocompetent cells according to (17), wherein the induced immunocompetent cells include disease antigen-specific CD8 + CTL;
(19) The method for inducing immunocompetent cells according to (17) or (18), wherein the antigen-presenting cells are selected from the group consisting of dendritic cells, immature dendritic cells, and artificial antigen-presenting cells;
(20) The bisphosphonate is a compound represented by the following general formula (I), a salt thereof, and a hydrate thereof,

In the above formula (I), R is a hydrogen atom or a lower alkyl group, and R ₁ and R ₂ are each independently a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, a thiol group, an aryl group, or a substituent. Selected from the group consisting of a substituted alkyl group, an alkyl group or a substituted alkyl group, a lower alkylamino group, an aralkyl group, a cycloalkyl group and a heterocyclic group, or R ₁ and R ₂ are one of the same cyclic structure The substituents in R ₁ and R ₂ are halogen atoms, lower alkyl groups, hydroxyl groups, thiol groups, amino groups, alkoxy groups, aryl groups, arylthio groups, aryloxy groups, alkylthio groups, cyclo The method for inducing immunocompetent cells according to any one of (17) to (19) selected from the group consisting of an alkyl group and a heterocyclic group;
(21) The bisphosphonate is selected from the group consisting of zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, salts thereof, and hydrates thereof (17) To the method of inducing immunocompetent cells according to any one of (20);
(22) The method for inducing immunocompetent cells according to any one of (17) to (21), wherein the concentration of the bisphosphonate in the sensitization is 0.001 to 20 μM;
(23) The immunocompetent cell induction method according to (22), wherein the concentration of the bisphosphonate in the sensitization is 0.001 to 5 μM;
(24) The method for inducing immunocompetent cells according to any one of (17) to (23), wherein the bacterial cell wall skeleton component is BCG-CWS;
(25) The disease antigen is cancer antigen or infectious disease antigen protein, cancer antigen or infectious disease antigen peptide, cell lysate of cancer cell or infectious disease cell, apoptotic cell, necrotic cell, and heat-treated product thereof. The method for inducing immunocompetent cells according to any one of (17) to (24) selected from the group consisting of:
(26) The method for inducing immunocompetent cells according to any one of (17) to (25), wherein the concentration of the disease antigen in the sensitization is 0.01 to 20 μg / mL;
(27) The method for inducing immunocompetent cells according to (26), wherein the concentration of the disease antigen in the sensitization is 0.1 to 2 μg / mL.

(28) A method for producing immunocompetent cells, which comprises inducing immunocompetent cells by the induction method according to any one of (17) to (27);
(29) Immunocompetent cells produced by the production method according to (28);
(30) A pharmaceutical composition for cancer and / or infection, comprising the immunocompetent cell according to (29);
(31) The pharmaceutical composition for cancer and / or infectious disease according to (30), wherein the immunocompetent cell is autologous or derived from another family having the same HLA.
(32) A method for preventing / treating cancer and / or infectious diseases, comprising administering the immunocompetent cell according to (29);
(33) The method for preventing and / or treating cancer and / or infectious diseases according to (32), wherein the immunocompetent cells are derived from autologous or another family having the same HLA.

上記式（Ｉ）中、Ｒは、水素原子又は低級アルキル基であり、Ｒ_１とＲ_２は、それぞれ独立して、水素原子、ハロゲン原子、ヒドロキシル基、アミノ基、チオール基、アリール基又は置換されたアリール基、アルキル基又は置換されたアルキル基、低級アルキルアミノ基、アルアルキル基、シクロアルキル基及び複素環式基からなる群から選択され、又はＲ_１とＲ_２は同じ環状構造の一部を形成し、
上記Ｒ_１とＲ_２における置換基としては、ハロゲン原子、低級アルキル基、ヒドロキシル基、チオール基、アミノ基、アルコキシ基、アリール基、アリールチオ基、アリールオキシ基、アルキルチオ基、シクロアルキル基、複素環式基からなる群から選択される（３４）又は（３５）記載の抗原提示細胞の活性化剤；
（３７）前記ビスホスホネートが、ゾレドロン酸、パミドロン酸、アレンドロン酸、リセドロン酸、イバンドロン酸、インカドロン酸、エチドロン酸、それらの塩、及びそれらの水和物からなる群から選択される（３４）から（３６）のいずれか一つに記載の抗原提示細胞の活性化剤；
（３８）前記細菌細胞壁骨格成分がＢＣＧ−ＣＷＳであることを特徴とする（３４）から（３７）のいずれか一つに記載の抗原提示細胞の活性化剤。 (34) an activator of an antigen-presenting cell comprising a bisphosphonate and a bacterial cell wall skeleton component;
(35) The antigen-presenting cell activator according to (34), wherein the antigen-presenting cell is selected from the group consisting of dendritic cells, immature dendritic cells, and artificial antigen-presenting cells;
(36) The bisphosphonate is a compound represented by the following general formula (I), a salt thereof, and a hydrate thereof,

In the above formula (I), R is a hydrogen atom or a lower alkyl group, and R ₁ and R ₂ are each independently a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, a thiol group, an aryl group, or a substituent. Selected from the group consisting of a substituted aryl group, an alkyl group or a substituted alkyl group, a lower alkylamino group, an aralkyl group, a cycloalkyl group and a heterocyclic group, or R ₁ and R ₂ are one of the same cyclic structure Forming part,
Examples of the substituent in R ₁ and R ₂ include halogen atoms, lower alkyl groups, hydroxyl groups, thiol groups, amino groups, alkoxy groups, aryl groups, arylthio groups, aryloxy groups, alkylthio groups, cycloalkyl groups, and heterocyclic rings. (34) or (35) an antigen-presenting cell activator selected from the group consisting of formula groups;
(37) The bisphosphonate is selected from the group consisting of zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, salts thereof, and hydrates thereof (34) (36) The activator of an antigen-presenting cell according to any one of
(38) The activator for an antigen-presenting cell according to any one of (34) to (37), wherein the bacterial cell wall skeleton component is BCG-CWS.

First Embodiment: Preparation of Activated Antigen Presenting Cells First, the activated antigen presenting cells of the present invention will be described in detail.

  The activated antigen-presenting cell of the present invention is an antigen-presenting cell co-sensitized with bisphosphonate, a bacterial cell wall skeleton component and a disease antigen.

  The antigen-presenting cells used in the present invention are not particularly limited, and for example, any of immature dendritic cells, mature dendritic cells, other antigen-presenting cells, artificial antigen-presenting cells, and mixtures thereof may be used. Among these, immature dendritic cells and mature dendritic cells are preferable, and immature dendritic cells are more preferable. This is because immature dendritic cells can induce disease antigen-specific CTLs and / or γδT cells more preferably when sensitized with bisphosphonates, bacterial cell wall skeleton components and disease antigens. The artificial antigen-presenting cell is an artificially-presented antigen-presenting cell that expresses, for example, at least major histocompatibility antigen (MHC) class I and costimulatory molecules (for example, CD80, CD86, etc.). Examples include cells prepared by genetic engineering. Furthermore, the artificial antigen-presenting cells may be those obtained by modifying a tumor-derived cell line to express MHC class I and costimulatory molecules as described above. Examples of the cell lines include breast cancer-derived MDA-MB-231 (class I antigen HLA-A * 0201), renal cancer-derived TUHR10TKB (class I antigen HLA-A * 0201 / A * 2402), gastric cancer-derived JR-st strain (class I antigen HLA-A * 2402) and the like. These cell lines can be obtained from, for example, ATCC, RIKEN BioResource Center, and the like. The production of the artificial antigen-presenting cell is disclosed in US Publication No. US-2005-0048646-A1, the entire contents of which are incorporated herein by reference.

  In the present invention, the bisphosphonate is not particularly limited and refers to a compound that is an analog of pyrophosphate, in which O (oxygen atom) of P—O—P of the pyrophosphate skeleton is substituted with C (carbon atom). Examples of the bisphosphonate used in the present invention include a compound represented by the following general formula (I), a salt thereof, and a hydrate thereof.

In the above formula (I), R is a hydrogen atom or a lower alkyl group, and R ₁ and R ₂ are each independently a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, a thiol group, or a substituted group. Selected from the group consisting of an aryl group which may be substituted, an alkyl group which may be substituted, a lower alkylamino group, an aralkyl group, a cycloalkyl group and a heterocyclic group, and R ₁ and R ₂ are ones of the same cyclic structure. A part may be formed.
Examples of the substituent in R ₁ and R ₂ include a halogen atom, a lower alkyl group, a hydroxyl group, a thiol group, an amino group, an alkoxy group, an aryl group, an arylthio group, an aryloxy group, an alkylthio group, a cycloalkyl group, Selected from the group consisting of heterocyclic groups and the like.

In the present specification, examples of the halogen atom include a fluoro atom, a chloro atom, and a bromine atom; examples of the alkyl group include a direct group such as a methyl, ethyl, propyl, isopropyl, butyl, pentyl, heptyl, octyl, and pentadecanyl group. chain or branched _C 1 _{-C 30} alkyl group or the like; examples of the lower alkyl group, for example, straight-chain or branched _C 1 _{-C 10} alkyl group or the like; the aryl group, for example, phenyl, naphthyl group or the like; the aralkyl group includes, for example, aryl - lower alkyl group or the like; the cycloalkyl group, for example, cyclooctyl, C ₁ -C ₁₀ cycloalkyl groups such adamantyl; heterocyclic groups include pyridyl, furyl, Pyrrolidinyl, imidazolyl, quinolyl, isoquinolyl group and the like are shown respectively.

  In the present invention, the bisphosphonate is preferably pharmaceutically acceptable, and examples thereof include those having a bone resorption inhibitory action and generally used as a therapeutic agent for osteoporosis. Examples thereof include zoledronic acid, its salts and / or their hydrates (eg sodium zoledronic acid hydrate (ZOMETA®, Novartis Pharma)), pamidronic acid, its salts and / or their hydrates (E.g. disodium pamidronate pentahydrate (AREDIA (R), Novartis Pharma)), alendronic acid, its salts and / or hydrates thereof (e.g. sodium alendronate trihydrate (ONCLAST ( Registered trademark), Arisu Pharmaceutical Co., Ltd.), risedronic acid, a salt thereof and / or a hydrate thereof (eg, risedronate sodium hydrate), ibandronic acid, a salt thereof and / or a hydrate thereof (eg, ibandronic acid) Sodium), incadronic acid, a salt thereof and / or a hydrate thereof (for example, disodium incadronate) Etidronate, their salts and / or their hydrates (e.g. etidronate disodium) and the like. Among these, zoledronic acid, pamidronic acid, alendronic acid, salts thereof and / or hydrates thereof are particularly preferable.

  The bacterial cell wall skeleton component used in the present invention is a component constituting the bacterial cell wall skeleton. Examples of the bacterium include Mycobacterium, Nocardia, and Corynebacterium. Bacterial cell wall skeletal components are insoluble residues obtained by physically crushing these bacteria and purifying them by denuclearization, deproteinization, degreasing, etc. is there. Although it does not restrict | limit in particular, For example, BCG-CWS is mentioned. BCG-CWS is a component extracted and purified from Mycobacterium tuberculosis. The main component is composed of arabinogalactan mycolate and peptidoglycan.

  In the disease antigen used in the present invention, the “disease” is not particularly limited, and examples thereof include cancer and infectious diseases. The cancer is not particularly limited, and includes any cancer, such as cancer that is difficult to treat (including precancerous conditions). The infectious disease is not particularly limited, and examples thereof include viral infections such as AIDS and hepatitis B / C, cell infections, bacterial infections, fungal infections, and protozoal infections. The form of the disease antigen used in the present invention is not particularly limited, and examples thereof include cancer antigens or infectious disease antigen proteins and peptides thereof. Examples of the disease antigen include cancer cell or infectious cell lysates, apoptotic cells, necrotic cells, and heat-treated products thereof.

  When the disease antigen is a cancer antigen, any antigen can be used regardless of the type of cancer of the cancer antigen. For example, any cancer antigen such as prostate cancer, liver cancer, pancreatic cancer and the like can be used. Examples of the cancer antigen include those encoded by the MAGE gene family such as MAGE1, MAGE3, GAGE, BAGE, and RAGE. Other cancer antigens include, for example, cancer antigens caused by mutations in p53, K-ras, CDK4 and bcl-c-abl gene products, and overexpressed in cancer cells such as c-erb2 (neu) protein. Cancer antigens, and oncogenic virus antigens such as the E7 protein of HPV-16. Furthermore, tumor fetal antigens such as carcinoembryonic antigen (CEA) and α-fetoprotein (AFP), as well as differentiation antigens such as prostate-specific antigen and CD-10 (CALLA antigen) expressed on leukemia and lymphoma B cells Can be used.

  In addition, the type of infectious disease of the infectious disease antigen is not particularly limited, and for example, intractable diseases among viral infectious diseases such as AIDS, hepatitis B / C, Epstein-Barr virus (EBV) infection, HPV infection, etc. Also mentioned. Parasitic antigens such as malaria parasite sporozoide proteins can also be used.

  In the present invention, a synthetic peptide can be used as the disease antigen peptide. This makes it possible to reduce the burden on the patient as compared with the case of using a cancer antigen collected from his own cancer tissue or the like. As the cancer antigen protein or peptide, for example, those listed in Tables 1 to 3 below can be used, and these can be easily obtained or synthesized by those skilled in the art.

  In the present invention, sensitizing an antigen-presenting cell with a substance refers to reacting the antigen-presenting cell with the substance, and preferably presenting the substance directly or indirectly on the surface of the antigen-presenting cell. It means to make it. In addition, co-sensitization of antigen-presenting cells refers to sensitizing antigen-presenting cells with two or more substances simultaneously, continuously, or intermittently. The substance is preferably a bisphosphonate, a bacterial cell wall skeleton component and / or a disease antigen.

  Next, the method for activating the antigen-presenting cell of the present invention and the method for producing the activated antigen-presenting cell of the present invention will be described in detail based on the following example using dendritic cells as antigen-presenting cells. In the present invention, the antigen-presenting cell activation treatment refers to a treatment including co-sensitization of an antigen-presenting cell with a bisphosphonate, a bacterial cell wall skeleton component, and a disease antigen as described above.

  First, preparation of antigen-presenting cells (in this example, dendritic cells) will be described. Dendritic cell preparation begins with obtaining a sample to obtain dendritic cell progenitor cells. As the sample, peripheral blood, bone marrow fluid, umbilical cord blood and the like can be used. Peripheral blood is preferably used in consideration of availability and low burden on patients. It is preferable to collect an amount of blood that does not burden the provider. As a blood collection method, whole blood collection using a vacuum blood collection tube, a blood collection bag, or the like can be used. Further, when it is necessary to secure a large amount of cells, it is possible to directly obtain peripheral blood mononuclear cells by using a method of collecting mononuclear cell components using a component blood collection device. Heparin or citric acid may be added to the collected blood so that coagulation does not occur.

  Next, mononuclear cells including dendritic cell precursor cells are separated from the collected blood. As a separation method, any method for separating nucleated cells from erythrocytes can be used. For example, methods that utilize Ficoll fractionation or Ficoll-Paque density gradients or elution are commonly used. The collected cells are preferably washed several times with a medium, physiological saline, phosphate buffered saline (hereinafter referred to as PBS), etc., in order to remove platelets and the like.

Next, monocytes (CD14 positive cells) that are dendritic cell precursor cells are separated from the recovered mononuclear cells. CD14 is known as a marker expressed in monocytes that are precursor cells of dendritic cells. Therefore, monocytes can be isolated and recovered using Magnetic Cell Sorting (Miltenyi Biotec, hereinafter referred to as MACS) using anti-CD14 antibody magnetic beads. This method is preferable because it is simple and the recovery rate of monocyte cells is high. Alternatively, the recovered mononuclear cells are transferred to a culture flask and cultured for 1 hour or longer under conditions of 34 ° C. to 38 ° C., more preferably 37 ° C., 2% to 10%, more preferably 5% CO _2. Or the like may be used as a precursor cell of a dendritic cell.

  Thereafter, differentiation induction from the precursor cells of the obtained dendritic cells to immature dendritic cells or mature dendritic cells is performed. AIM-V medium (Invitrogen) is used as a culture medium. In addition to AIM-V medium, RPMI-1640 medium (Invitrogen), Dulbecco's modified Eagle medium (Invitrogen, hereinafter referred to as DMEM), TIL (Immuno-Biological Laboratories, Inc.), epidermal keratinocyte medium (Kohjin Bio Inc.) Commercially available media used for cell culture such as company, hereinafter referred to as KBM), Iskov media (Invitrogen, hereinafter referred to as IMEM), and the like can be used. Moreover, 0.5-20% of bovine serum, fetal bovine serum (hereinafter referred to as FBS), human serum, human plasma, etc. can be added as necessary.

When obtaining an immature dendritic cell, an immature dendritic cell is obtained by adding a differentiation-inducing factor to a culture medium and culturing a precursor cell of the dendritic cell. Any of cytokines can be used as the differentiation inducing factor. For example, granulocyte-macrophage colony-stimulating factor (hereinafter referred to as GM-CSF), interleukin 4 (hereinafter referred to as IL-4. The same applies to other interleukins), stem cell factor (hereinafter referred to as SCF). IL-13, tumor necrosis factor α (hereinafter referred to as TNF-α) and the like can efficiently induce immature dendritic cells. Moreover, it is preferable to add IL-1, IL-2, IL-3 etc. as needed. More preferably, it is possible to induce efficiently when a combination of GM-CSF and IL-4 is used. The culture is performed at 34 ° C. to 38 ° C., preferably 37 ° C., 2% to 10%, preferably 5% CO ₂ , and the culture period is preferably 5 to 7 days.

Moreover, when obtaining a mature dendritic cell, the further differentiation-inducing factor is added and culture | cultivated 5 to 7 days after culture | cultivation start. Any of cytokines can be used as the differentiation-inducing factor. For example, GM-CSF, IL-4, SCF, IL-1β, IL-6, IL-13, TNF-α, prostaglandin E ₂ It is preferable to induce mature dendritic cells efficiently (hereinafter referred to as PGE ₂ ). It is preferable to add IL-1, IL-2, IL-3, etc. as necessary. More preferably it is capable of inducing good GM-CSF, IL-4, IL-6, IL-1β, the use of a combination of PGE ₂ and TNF-alpha efficiency. The culture is performed at 34 ° C. to 38 ° C., preferably 37 ° C., 2% to 10%, preferably 5% CO ₂ , and the culture time is preferably 24 to 48 hours.

  In addition, hematopoietic stem cells (CD34 positive cells) are collected as dendritic cell precursor cells, and GM-CSF, TNF-α and flt-3 ligand (FL), c-kit ligand (SCF), or thrombopoietin (TPO) are used. A method of obtaining immature dendritic cells or mature dendritic cells by adding them alone or in combination, or by directly using a specific gravity solution such as Percoll from blood or peripheral blood mononuclear cells. A method of collecting the image can also be used.

  Next, the obtained antigen-presenting cells (in this example, the immature dendritic cells or mature dendritic cells) are co-sensitized with bisphosphonate, a bacterial cell wall skeleton component, and a disease antigen.

  The concentration of bisphosphonate is not particularly limited as long as it is a concentration that normally sensitizes cells with bisphosphonate. For example, the literature [The Journal of Immunology, 2001, Vol. 166, 5508-5514, or Blood, 2001, Vol. 98, no. 5, 1616-1618] is preferably 0.001 μM to 20 μM, more preferably 0.001 μM to 5 μM.

  The concentration of the bacterial cell wall skeleton component is not particularly limited as long as it is a concentration at which cells are usually sensitized by the bacterial cell wall skeleton component. For example, in the case of BCG-CWS, it is 20 μg / mL.

  The concentration of the disease antigen is not particularly limited as long as it is usually a concentration that sensitizes dendritic cells with the disease antigen. For example, in the case of a disease antigen protein or peptide as described above, the literature [Cancer Research, 1999, Vol. 59, 2167-2173, The Journal of Immunology, 1995, Vol. 154, 2257-2265, or The Journal of Immunology, 1994, 153, 996-1003], preferably 0.01 to 20 μg / mL, more preferably 0.1 to 2 μg / mL. It is preferably mL.

In addition, when dendritic cells are sensitized with a cell group containing apoptotic cells and / or necrotic cells as disease antigens, for example, 1) a cancer cell or a cancer cell line is cultured and spontaneously prepared. 2) The cancer cell or cancer cell line is formed by UV irradiation (1 J / cm ² · sec for 2 minutes). 3) The cancer cell or cancer cell line is 85 ° C. For example, a method of forming by heat treatment for 10 minutes may be used.

  There are no particular restrictions on the timing of sensitization with the bisphosphonate, bacterial cell wall skeleton component, and disease antigen, and solutions containing these components can be added in order, or the three components can be added in suspension in the same solution.

  The activated antigen-presenting cell of the present invention (activated dendritic cell in this example) prepared (produced) by such an activation treatment method of the present invention can efficiently induce disease antigen-specific CD8 + CTL. . For example, in both cases of in vitro and in vivo, the activated antigen-presenting cell of the present invention can be used as a medicament capable of inducing disease antigen-specific CTL. In particular, as a medicine, a therapeutic / preventive agent for cancer and / or infectious diseases is preferable. In the present invention, immunocompetent cells recognize the specificity of antigens including T cells, iNKT cells, NK cells (Natural Killer Cell), B cells, monocytes, dendritic cells, macrophages and / or the like. A cell having the ability to participate in a dynamic immune reaction, or a cell group containing one or more of these. When used in vitro, the activated antigen-presenting cell of the present invention can be used as a composition capable of inducing disease antigen-specific CD8 + CTL. In addition, when used in vivo, the activated antigen-presenting cell of the present invention is a dendritic cell capable of inducing disease antigen-specific CD8 + CTL after washing and removing free bisphosphonate, bacterial cell wall skeleton component and disease antigen. It can be used as a vaccine such as a vaccine. Further, in any case where the activated antigen-presenting cell of the present invention is used in vitro or in vivo, for example, a cytokine (for example, IL-2) or other protein (for example, albumin) is used in combination. May be.

Second Embodiment: Medicament Containing Activated Antigen Presenting Cell of the Present Invention Next, an example of using a dendritic cell as an antigen presenting cell in the same manner as described above for a medicament using the activated antigen presenting cell of the present invention. Based on

  First, the activated antigen-presenting cells (activated dendritic cells) obtained in the first embodiment described above are collected by centrifugation or the like. Next, the collected cells are washed. The liquid to be washed is isotonic, and any liquid can be used as long as it can be used as a medicine. Considering the subsequent administration to the patient, it is preferable to use physiological saline, PBS or the like. And the collect | recovered dendritic cell can be used as a pharmaceutical by suspending in the physiological saline. Further, serum components such as albumin and cytokines can be added as necessary. In particular, the medicament is preferably used as a therapeutic / preventive agent for cancer and / or infectious diseases. Accordingly, as a related embodiment, the present invention includes the use of the activated antigen presenting cells of the present invention for the manufacture of a medicament for cancer and / or infection.

Third Embodiment: Treatment / Prevention Method Using Activated Antigen Presenting Cells of the Present Invention Next, as to the treatment / prevention method using dendritic cells of the present invention, dendritic cells are presented with antigens in the same manner as described above. A description will be given based on an example used as cells.

  By administering the activated antigen-presenting cell (activated dendritic cell) of the present invention obtained in the first embodiment described above or the medicament of the present invention obtained by the second embodiment, cancer is produced. And / or treatment / prevention against infectious diseases.

The number of cells to be administered can be appropriately selected according to the administration method and the patient's condition, and is not particularly limited. Usually, the dose is preferably 10 ⁶ to 10 ⁸ per person, more preferably 10 ⁷ per person or more. The number of doses varies depending on the patient's condition, but usually 4 to 6 doses are administered as one course. The administration interval depends on the number of dendritic cells to be administered and is not particularly limited. Usually, it is preferable to be once a week to once a month. Further, when the number of dendritic cells to be administered is 5 × 10 ⁶ , it is once every 2 weeks, and if it is 2 × 10 ⁷ or more, one month It is preferable to administer once. The method of administration is not particularly limited, but it may be injected intravenously, subcutaneously, intradermally, by injection, directly into the regional lymph node, directly into the affected area, or administered systemically as an infusion Is also possible. Alternatively, it can be injected from an artery near the lesion.

  These disease antigen-specific CTLs not only directly kill cancer cells and infected cells, but also can indirectly injure these cells via cytokines such as interferon γ (hereinafter referred to as IFNγ). For example, it can be effectively used for various treatments and preventions such as cancer and infectious diseases. Advantages of using the activated antigen-presenting cell of the present invention as a vaccine include the following. That is, in a dendritic cell vaccine sensitized only with a conventional disease antigen, only the disease antigen-specific CD8 + CTL is activated. In contrast, the activated antigen-presenting cell vaccine of the present invention can activate γδT cells in addition to the activation of disease antigen-specific CD8 + CTL. Furthermore, cytokines derived from these disease antigen-specific CD8 + CTL and / or γδ T cells can activate helper T cells, NK cells, iNKT cells and B cells. As a result, the humoral and cellular immune systems throughout the living body are activated, and as a result, the therapeutic / preventive effect can be improved. Moreover, it is preferable that the antigen-presenting cell used for the activation treatment is derived from the patient's self or from another family with the same HLA. This is because when administered to a patient, the function can be exhibited without being excluded by the patient's own immunity.

Fourth Embodiment: Method for Inducing Immunocompetent Cells Containing Disease Antigen-Specific CD8 + CTL Next, the method for inducing immunocompetent cells of the present invention will be described. The method for inducing immunocompetent cells of the present invention is a method for inducing immunocompetent cells containing disease antigen-specific CD8 + CTL, and more specifically, sympathize antigen-presenting cells with bisphosphonates, bacterial cell wall skeletal components and disease antigens. And the step (ii) of culturing the antigen-presenting cells and lymphocytes in a mixed or simultaneous manner with or after the sensitization. Here, as described above, immunocompetent cells include T cells, iNKT cells, NK cells, B cells, monocytes, dendritic cells, macrophages, etc., and recognize the specificity of antigens and / or are specific. A cell having the ability to participate in an immune reaction or a cell group containing one or more of these cells.

  First, similarly to the first embodiment, an antigen-presenting cell (for example, a dendritic cell) is activated (step (i)). Then, at the same time as or after the sensitization, the antigen-presenting cells and the reaction cells are seeded in a culture vessel and mixed and cultured (step (ii)). As a result, the disease antigen-specific stimulus from the activated antigen-presenting cell can be received, and the disease antigen-specific CD8 + CTL in the reaction cell can be activated. Furthermore, the γδ T cells in the reaction cells are activated by stimulation from IPP (Isopentenyl diphosphate) -like molecules presented on the antigen-presenting cell surface by inhibition of the antigen-presenting cell metabolic system by sensitization with bisphosphonate. Can be IFNγ produced from γδT cells can help further activation and proliferation of the disease antigen-specific CD8 + CTL.

  In addition, antigen-presenting cells sensitized by bacterial cell wall skeleton components (peptidoglycan or the like), for example, immature dendritic cells, are stimulated through TLR2 / 4 that acts as a peptidoglycan receptor, and maturation is promoted. Unlike dendritic cells matured with TNFα, PGE2, etc., dendritic cells matured with BCG-CWS have phagocytic ability, so they can take in and process disease antigens such as cancer antigen proteins. Antigen molecules can be presented on HLA. Moreover, since the expression of costimulatory molecules such as CD40, CD83, CD80 and CD86 on dendritic cells is improved, CTL can be activated.

  In addition, the reactive cells here are, for example, human lymphocytes, and mononuclear cells derived from peripheral blood are preferable. It is preferable that this reaction cell is derived from autologous origin or the same origin as HLA.

  The culture vessel is not particularly limited, and culture plates, petri dishes, flasks, bags, etc. that are usually used in the field can be used. The density | concentration which seed | inoculates each cell group can be freely set according to the condition to implement.

When the antigen-presenting cell is a dendritic cell, the mixed culture of the dendritic cell and the reactive cell can be performed using, for example, an AIM-V medium. In addition to AIM-V medium, commercially available media used for cell culture, such as RPMI-1640 medium, DMEM, TIL, KBM, and IMEM, can be used. Further, 5% to 20% of bovine serum, FBS, serum such as human plasma, cytokines, etc. may be added as necessary. Culturing is performed at 34 ° C. to 38 ° C., preferably 37 ° C., for example, under 2% to 10%, preferably 5% CO ₂ conditions. The culture period is not particularly limited, but is preferably 5 to 21 days, and more preferably 7 to 14 days. The number of dendritic cells and reaction cells to be seeded can be set according to the container to be seeded and the application. Further, the ratio of mixing dendritic cells and reactive cells can be appropriately set according to the situation, and is not particularly limited. For the purpose of increasing the ratio of disease antigen-specific CD8 + CTL in the reaction cells, the ratio of reaction cells to dendritic cells is preferably 20: 1 to 2: 1.

  By such an induction method of the present invention, it is possible to prepare (produce) the immunocompetent cells of the present invention containing disease antigen-specific CD8 + CTL. In addition, the immunocompetent cells of the present invention thus obtained can be immunized as immunocompetent cells containing disease antigen-specific CD8 + CTLs at a higher rate by being stimulated directly or repeatedly with the activated antigen-presenting cells of the present invention. It can be used for cell therapy and can be expected to have a higher therapeutic / preventive effect on cancer and infectious diseases.

  The advantage of preparing the immunocompetent cells of the present invention in vitro is that a large amount of cancer cells and infectious cells can be directly killed by repeatedly stimulating reactive cells with the activated antigen-presenting cells of the present invention. The immunocompetent cells can be easily prepared.

Fifth Embodiment: Drug containing the immunocompetent cell of the present invention Next, a drug containing the immunocompetent cell of the present invention will be described.

  First, the immunocompetent cells of the present invention containing the disease antigen-specific CD8 + CTL obtained in the fourth embodiment are collected by centrifugation or the like. Next, the collected cells are washed. The liquid to be washed is isotonic, and any liquid can be used as long as it can be used as a medicine. Thereafter, it is preferable to use physiological saline, PBS or the like in consideration of administration to a patient. Then, the immunocompetent cells containing the recovered disease antigen-specific CD8 + CTL can be used as a medicine by being suspended in physiological saline. Moreover, a cytokine etc. can be added as needed. The medicament is particularly preferably used as a therapeutic / preventive agent for cancer and / or infectious diseases. Accordingly, as a related embodiment, the present invention includes the use of the activated antigen presenting cells of the present invention for the manufacture of a medicament for cancer and / or infection.

Sixth Embodiment: Treatment / Prevention Method Using Immunocompetent Cells of the Present Invention Next, a treatment / prevention method using the immunocompetent cells of the present invention will be described.

  Treatment and prevention of cancer and / or infectious diseases by administering the immunocompetent cells of the present invention obtained in the fourth embodiment or the medicament of the present invention obtained in the fifth embodiment. It can be performed.

The number of cells to be administered can be appropriately selected according to the administration method and the patient's condition, and is not particularly limited. Usually, the number of doses per administration is preferably 10 ⁸ to 10 ¹² / person, more preferably 10 ⁹ / person or more. The number of doses varies depending on the patient's condition, but usually 4-6 doses are taken as one course. The administration interval is not particularly limited, but for example, it is preferable to administer once every two weeks or once a month. The method of administration is not particularly limited, but it can be injected by intravenous, subcutaneous, intracutaneous injection, direct injection into regional lymph nodes, direct injection into the lesion, or systemic administration as an infusion It is also possible. Alternatively, it can be injected from an artery near the lesion. Moreover, it is preferable that the immunocompetent cell of the present invention is derived from a patient's self or another family who shares the same HLA. Because it is derived from the patient's own origin or from another family that shares the same HLA, when administered to the patient, it is possible to exert its function without being excluded by the patient's own immune system. .

Seventh embodiment: Activator for antigen-presenting cells Next, the activator for antigen-presenting cells of the present invention will be described.

  The antigen-presenting cell activator of the present invention contains a bisphosphonate and a bacterial cell wall skeleton component. The antigen-presenting cell activator of the present invention can be added to a medium to promote activation of antigen-presenting cells when culturing antigen-presenting cells ex vivo. The amounts of the bisphosphonate and the bacterial cell wall skeleton component can be appropriately set so that the final concentration is the amount described in the first embodiment.

  Alternatively, the antigen-presenting cells present in the body can be activated by suspending in an appropriate solvent such as physiological saline and administering the suspension in the body. The dose for administration into the body can be appropriately set based on the amounts described in the package inserts of bisphosphonate and bacterial cell wall skeleton components.

  Hereinafter, the present invention will be described in detail with reference to examples. However, it goes without saying that the present invention is not limited to this.

<Example 1-1: Collection and preparation of dendritic cells>
75 mL of peripheral blood was collected from a healthy donor. Mononuclear cells were obtained from the peripheral blood of this healthy person. At this time, the mononuclear cell layer was recovered using a specific gravity solution for blood cell separation. In order to remove platelets and the like from the collected cells, the cells were washed several times with AIM-V supplemented with 10% FBS, and then monocytes (CD14 positive cells) were isolated by MACS.

  Differentiation from the resulting monocytes (dendritic cell precursor cells) to dendritic cells was induced. Monocytes were cultured in AIM-V medium containing 500 U / mL GM-CSF (IMMUNEX), 500 U / mL IL-4 (Osteogenetics GmbH), 5% AB serum in AIM-V. Culturing was carried out for 5 days to prepare immature dendritic cells.

<Example 1-3: Preparation of dendritic cells sensitized with zoledronic acid, BCG-CWS and disease antigen peptide and preparation of reaction cells>
To a suspension of immature dendritic cells derived from peripheral blood prepared as described above, ZOMETA (Novartis Pharma) was added as 0.1 μM as zoledronic acid. At the same time, BCG-CWS (20 μg / mL) was added and cultured for 24 hours to prepare antigen-presenting cells (APC).

  The remaining cells (CD14-negative cell population, mainly T cell population) after isolation of CD14-positive cells for dendritic cell preparation, as 10% FBS, 10% dimethyl sulfoxide ( Cells frozen and stored in AIM-V medium supplemented with DMSO) were thawed, washed and used.

Melanoma antigen MART-1 modified antigen A27L (amino acid sequence: ELAGIGILTV) is added as a disease antigen peptide to a final concentration of 2 μg / mL, and mixed lymphocyte culture reaction using APC and lymphocytes (derived from the same donor as the antigen-presenting cells) (Mixed Lymphocyte Reaction; MLR) was performed. Culturing was performed at 37 ° C. under 5% CO ₂ for 7 days and 14 days. In order to detect antigen-specific CD8-positive T cells after 7 days and 14 days, a tetramer assay was performed.

  14 days after the start of MLR, the ratio of A27L-specific CD8 + CTL (disease antigen-specific CD8 + CTL) to be contained in all cells was measured with a flow cytometer using a labeled antibody and an antigen (A27L) -specific tetramer.

  As a procedure for calculating the proportion of HLA-A * 0201 A27L tetramer-positive and CD8-positive cells, PE-labeled A27L tetramer (MBL) was first added to the cells washed with PBS after culturing. After staining for 20 minutes at room temperature in the dark, FITC-labeled anti-CD8 antibody (BD Pharmingen) was added and stained at 4 ° C. for 20 minutes in the dark. The isotype of each antibody was used for control. Epis XL / MCL was used for cell measurement, and Epis 32 was used for analysis of measurement results.

  The results are shown in Table 1 below. As shown in the table, it was confirmed that DC sensitized with BCG-CWS increased the number of A27L positive cells in 14-day culture with lymphocytes compared to imDC. In addition, the effect was enhanced by adding zoledronic acid.

In addition, the cytotoxic activity of lymphocytes induced by MLR was measured by terascan assay using the JCOCB cell line (HLA-A * 0201 + Mart1 +) as a target cell.

<Measurement of cytotoxic activity>
MLR was performed in the same manner as in Example 1.

The lymphocytes on the 14th day after the start of MLR were collected. HLA-A * 0201-positive and MART1-positive human melanoma cell line JCOCB was labeled with calcein (Dojindo Laboratories) and used as target cells. The collected lymphocytes were used as effector cells, mixed so that the effector cells: target cells were 20: 1 or 40: 1, and seeded on a 96-well half plate. Cultivation was performed for 4 hours at 37 ° C. and 5% CO ₂ .

  The cells after the mixed culture were collected, and the tendency intensity in the well was measured using Terascan (Minervatech) to measure the cytotoxic activity.

  Table 2 below shows the results of measuring the percentage of apoptotic cells by A27L antigen-specific CD8 + CTL induced by activated dendritic cells sensitized with ZOMETA, BCG-CWS and A27L. ImDC-induced lymphocytes also have cytotoxic activity against cell lines, and the effect is enhanced by adding zoledronic acid. Furthermore, it was confirmed that DC sensitized with BCG-CWS has stronger cytotoxic activity.

  As described above, the activated antigen-presenting cell of the present invention can induce disease antigen-specific CD8 + CTL very efficiently compared to dendritic cells sensitized with only conventional peptides. Therefore, by administering to a patient as a composition for administration, disease antigen-specific CD8 + CTL is induced in vivo, and a therapeutic / preventive effect on cancer and infectious diseases can be expected. Furthermore, in vitro, it can be used as a composition for inducing disease antigen-specific CD8 + CTL.

Claims (38)

  A method for activating antigen-presenting cells, comprising the step of co-sensitizing the antigen-presenting cells with a bisphosphonate, a bacterial cell wall skeleton component and a disease antigen.   The method for activating antigen-presenting cells according to claim 1, wherein the antigen-presenting cells are selected from the group consisting of dendritic cells, immature dendritic cells, and artificial antigen-presenting cells. The bisphosphonate is a compound represented by the following general formula (I), a salt thereof, and a hydrate thereof,

In the above formula (I), R is a hydrogen atom or a lower alkyl group, and R ₁ and R ₂ are each independently a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, a thiol group, an aryl group, or a substituent. Selected from the group consisting of a substituted aryl group, an alkyl group or a substituted alkyl group, a lower alkylamino group, an aralkyl group, a cycloalkyl group and a heterocyclic group, or R ₁ and R ₂ are one of the same cyclic structure Forming part,
Examples of the substituent in R ₁ and R ₂ include halogen atoms, lower alkyl groups, hydroxyl groups, thiol groups, amino groups, alkoxy groups, aryl groups, arylthio groups, aryloxy groups, alkylthio groups, cycloalkyl groups, and heterocyclic rings. The method for activating an antigen-presenting cell according to claim 1 or 2, which is selected from the group consisting of formula groups.   The bisphosphonate is selected from the group consisting of zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, salts thereof, and hydrates thereof. The method for activating an antigen-presenting cell according to one item.   The method for activating an antigen-presenting cell according to any one of claims 1 to 4, wherein a concentration of the bisphosphonate in the sensitization is 0.001 to 20 µM.   The method for activating an antigen-presenting cell according to claim 5, wherein the concentration of the bisphosphonate in the sensitization is 0.001 to 5 µM.   The method for activating an antigen-presenting cell according to any one of claims 1 to 6, wherein the bacterial cell wall skeleton component is BCG-CWS.   The disease antigen is a group consisting of cancer antigen or infectious disease antigen protein, cancer antigen or infectious disease antigen peptide, cell lysate of cancer cell or infectious disease cell, apoptotic cell, necrotic cell, and heat-treated product thereof. The method for activating an antigen-presenting cell according to any one of claims 1 to 7, which is selected from:   The method for activating an antigen-presenting cell according to any one of claims 1 to 8, wherein the concentration of the disease antigen in the sensitization is 0.01 to 20 µg / mL.   The method for activating antigen-presenting cells according to claim 9, wherein the concentration of the disease antigen in the sensitization is 0.1 to 2 μg / mL.   A method for producing activated antigen-presenting cells, which comprises treating antigen-presenting cells with the activation treatment method according to any one of claims 1 to 10.   An activated antigen-presenting cell produced by the production method according to claim 11.   A pharmaceutical composition for cancer and / or infection, comprising the activated antigen-presenting cell according to claim 12.   The pharmaceutical composition according to claim 13, wherein the antigen-presenting cell is derived from an autologous or another family having the same HLA.   A method for preventing and / or treating cancer and / or infectious diseases, comprising administering the activated antigen-presenting cell according to claim 12.   The method for prevention and treatment according to claim 15, wherein the antigen-presenting cell is derived from autologous origin or from another family similar to HLA. An immunocompetent cell induction method comprising the following steps (i) and (ii):
(I) sensitizing antigen-presenting cells with bisphosphonates, bacterial cell wall skeletal components and disease antigens.
(Ii) A step of mixing and culturing the antigen-presenting cells and lymphocytes simultaneously with or after the sensitization.   19. The method for inducing immunocompetent cells according to claim 18, wherein the induced immunocompetent cells comprise disease antigen-specific CD8 + CTL.   The method for inducing immunocompetent cells according to claim 17 or 18, wherein the antigen-presenting cells are selected from the group consisting of dendritic cells, immature dendritic cells, and artificial antigen-presenting cells. The bisphosphonate is a compound represented by the following general formula (I), a salt thereof, and a hydrate thereof,

In the above formula (I), R is a hydrogen atom or a lower alkyl group, R ₁ and R ₂ are each, independently, a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, a thiol group, an aryl group or a substituted Selected from the group consisting of a substituted aryl group, an alkyl group or a substituted alkyl group, a lower alkylamino group, an aralkyl group, a cycloalkyl group and a heterocyclic group, or R ₁ and R ₂ are one of the same cyclic structure Forming part,
Examples of the substituent in R ₁ and R ₂ include halogen atoms, lower alkyl groups, hydroxyl groups, thiol groups, amino groups, alkoxy groups, aryl groups, arylthio groups, aryloxy groups, alkylthio groups, cycloalkyl groups, and heterocyclic rings. The method for inducing immunocompetent cells according to claim 17, which is selected from the group consisting of formula groups.   21. Any of the claims 17-20, wherein the bisphosphonate is selected from the group consisting of zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, their salts, and their hydrates. The method for inducing immunocompetent cells according to claim 1.   The method for inducing immunocompetent cells according to any one of claims 17 to 21, wherein the concentration of the bisphosphonate in the sensitization is 0.001 to 20 µM.   The method for inducing immunocompetent cells according to claim 22, wherein the concentration of the bisphosphonate in the sensitization is 0.001 to 5 µM.   The method for inducing immunocompetent cells according to any one of claims 17 to 23, wherein the bacterial cell wall skeleton component is BCG-CWS.   18. The disease antigen is selected from the group consisting of cancer antigen or infectious disease antigen protein, peptide thereof, cell lysate of cancer cell or infectious disease cell, apoptotic cell, necrotic cell, and heat-treated product thereof. 25. The method for inducing immunocompetent cells according to any one of 24 to 24.   The method for inducing immunocompetent cells according to any one of claims 17 to 25, wherein the concentration of the disease antigen in the sensitization is 0.01 to 20 µg / mL.   27. The method of inducing immunocompetent cells according to claim 26, wherein the concentration of the disease antigen in the sensitization is 0.1 to 2 [mu] g / mL.   A method for producing immunocompetent cells, the method comprising producing immunocompetent cells by the induction method according to any one of claims 17 to 27.   An immunocompetent cell produced by the production method according to claim 28.   30. A pharmaceutical composition for cancer and / or infection, comprising the immunocompetent cell of claim 29.   The pharmaceutical composition for cancer and / or infectious disease according to claim 30, wherein the immunocompetent cells are autologous or derived from another family having the same HLA.   A method for preventing and / or treating cancer and / or infectious diseases, comprising administering the immunocompetent cell according to claim 29.   33. The method for preventing and / or treating cancer and / or infectious diseases according to claim 32, wherein the immunocompetent cells are autologous or derived from another family having the same HLA.   An activator for an antigen-presenting cell comprising a bisphosphonate and a bacterial cell wall skeleton component.   35. The antigen-presenting cell activator according to claim 34, wherein the antigen-presenting cell is selected from the group consisting of dendritic cells, immature dendritic cells, and artificial antigen-presenting cells. The bisphosphonate is a compound represented by the following general formula (I), a salt thereof, and a hydrate thereof,

In the above formula (I), R is a hydrogen atom or a lower alkyl group, and R ₁ and R ₂ are each independently a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, a thiol group, an aryl group, or a substituent. Selected from the group consisting of a substituted aryl group, an alkyl group or a substituted alkyl group, a lower alkylamino group, an aralkyl group, a cycloalkyl group and a heterocyclic group, or R ₁ and R ₂ are one of the same cyclic structure Forming part,
Examples of the substituent in R ₁ and R ₂ include halogen atoms, lower alkyl groups, hydroxyl groups, thiol groups, amino groups, alkoxy groups, aryl groups, arylthio groups, aryloxy groups, alkylthio groups, cycloalkyl groups, and heterocyclic rings. 36. The activator for an antigen-presenting cell according to claim 34 or 35, selected from the group consisting of a formula group.   The bisphosphonate is selected from the group consisting of zoledronic acid, pamidronic acid, alendronic acid, risedronic acid, ibandronic acid, incadronic acid, etidronic acid, salts thereof, and hydrates thereof. The activator of an antigen-presenting cell according to one item.   The activator for an antigen-presenting cell according to any one of claims 34 to 37, wherein the bacterial cell wall skeleton component is BCG-CWS.