JP2010265327A - Method for producing cancer antigen-nonspecific targeted t cell and medicine - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cancer therapeutic agent useful for treating patients suffering from cancer in which information on a cancer antigen for the cancer antigen-specific vaccine therapy or cancer in which the type of human leuchocyte antigen (HLA) is incompatible with the vaccine therapy. <P>SOLUTION: An antigen-specific Th1 cell is produced by adding a type 1 cytokine to a small amount of peripheral blood obtained from the patient suffering from cancer and carrying out culture in the presence of an antigen activating the T cell other than the cancer antigen. A tumor-specific CTL is induced in a living body as a cancer-carrying host by administering the antigen used for inducing the antigen-specific Th1 cell and the antigen-specific Th1 cell in combination. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for producing a targeted T cell for use in the treatment of cancer, which consists of a T cell specific for an antigen other than cancer, and its application.

  In the present specification, cancer refers to a malignant neoplasm, and cancer and tumor are treated as synonymous.

  The main treatments for cancer include surgery, chemotherapy, and radiation therapy, but they cannot eradicate cancer cells. Although these therapeutic effects and the length of the subsequent non-recurrence period are thought to depend on the host's immunity, the host's immunity is affected by side effects of chemotherapy and radiation therapy, and the negative effects of these treatments Cannot be ignored.

  Therefore, from the idea of overcoming cancer by enhancing the host's defense mechanism, cancer treatments using in vitro substances such as bacterial components and plant-derived components that non-specifically enhance immune ability have been attempted. Treatment using cytokines, which are in vivo substances that regulate immunity, is also underway. These substances are called biological response modifiers (BRMs) and may be used alone to treat cancer, but may also be used in combination with surgery, chemotherapy, or radiation therapy.

  The bacterial cell components clinically applied as BRM include BCG, OK432 (Picibanil), Ancer, Bestin, and the like, and the plant-derived components include Lentinan, Sonifilan, and Kurestin.

  BCG is known as a live attenuated vaccine for the prevention of tuberculosis, but in order to remove the side effects of the vaccine, the active ingredient can be separated from the cell wall skeletal component (BCG-CWS) of the BCG strain or the culture supernatant of Mycobacterium tuberculosis. Tried. Tuberculin used for the inspection of tuberculosis infection, past and vaccination is a tuberculosis culture supernatant that has been filtered after overheating, but recently purified PPD (tuberculin purified protein derivative) has been used. .

  BCG is known to cause delayed type hypersensitivity (DTH) and induce helper T cell type 1 (Th1 cells). BCG-CWS and PPD also cause DTH in the same manner as BCG, and it has been shown that immunotherapy with BCG-CWS is effective in many cancers (see, for example, Non-Patent Document 1).

  In addition to BRM, there is also a method of enhancing the immunity of the host by removing blood cells from the living body, inducing immune cells with enhanced antitumor effects by adding cytokines, and then transferring them into the living body again. ing. Mononuclear cells isolated from peripheral blood are cultured in the presence of interleukin-2 (IL-2), so that lymphokine-activated killer having broad antitumor activity against natural killer (NK) cell-resistant tumors (Lymphokine Activated Killer, LAK) cells can be induced (see, for example, Non-Patent Document 2). Thereafter, a large amount of IL-2 became available by genetic recombination technology (see, for example, Non-Patent Document 3), and clinical application of adoptive immunotherapy using LAK cells against tumors was made, and its usefulness was demonstrated (for example, Non-Patent Document 4).

Furthermore, by adding an anti-CD3 monoclonal antibody (MoAb) to IL-2 and culturing, it became possible to culture a large amount of cells having LAK activity from mononuclear cells obtained from a small amount of peripheral blood (for example, Non-patent document 5).

  Peripheral T lymphocytes express CD3 molecules along with the T cell receptor (TCR) on the cell surface, and depending on the expression of CD4 or CD8 molecules, helper T (Th) cells or cytotoxic T cells (CTL). being classified.

  By using MoAb and magnetic beads for molecules expressed on the cell surface (for example, CD4 molecule, CD8 molecule), cells having the target cell surface antigen can be concentrated or removed (see, for example, Patent Document 1).

  Th cells are divided into Th1 cells that produce type 1 cytokines such as interferon-γ (IFN-γ) and IL-2, and Th2 cells that produce type 2 cytokines such as IL-4 and IL-10 (for example, Non-patent document 6), Th1 cells act as effectors of cellular immunity, and Th2 cells are responsible for regulation of humoral immunity. In addition, IFN-γ produced by Th1 cells suppresses Th2 cells, and IL-4 produced by Th2 cells suppresses Th1 cells (see, for example, Non-Patent Document 7).

  During the initial Th cell activation, Th1 cells are differentiated by the presence of IL-12 (see, for example, Non-Patent Document 8), and Th2 cells are differentiated by the presence of IL-4 (for example, Non-Patent Document 9). reference).

  Th2 cell-derived cytokines are dominant in tumor sites (see, for example, Non-Patent Document 10), and in cancer immunotherapy, it is also important to correct Th1 / Th2 balance and activate Th1-driven cellular immunity, In addition to BCG-derived components, BRMs that induce the production of Th1-type type 1 cytokines are known (see, for example, Non-Patent Document 11).

Patent No. 2530966

Hayashi et al., 1998, Porc. Japan Acad. , 74 (B): 50-55 Grimm et al., 1982, J. Am. Exp. Med. 155: 1823-1842 Taniguchi et al., 1983, Nature, 302: 305-310. Rosenberg et al., 1985, New Engl. J. et al. Med. 313: 1485-1492 Ochoa et al., 1987, J. MoI. Immunol. 138: 2728-2733. Mosmsnn et al., 1986, J. MoI. Immunol. 136: 2348-2357. Maggi et al., 1992, J. MoI. Immunol. 148: 2142-1247. Seder et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 10188-10192. Hsieh et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6065-6069 Huang, 1995, Cancer Res. 55: 3847-3853. Fujimoto et al., 1997, J. MoI. Immunol. 158: 5619-5626.

  In the cancer-bearing state, the Th1 / Th2 balance is lost, and Th2 cell-derived cytokines are dominant. Therefore, activation of cellular immunity driven by Th1 is necessary.

  In order to induce tumor-specific T cells in vitro, it is necessary to obtain the patient's own tumor tissue by surgery. Recently, it has been possible to induce tumor-specific T cells using tumor antigen peptides, but limited known tumor antigen peptides and limited types of major histocompatibility complexes (also called human leukocyte antigens). Only patients with) can be indicated.

  Further, in order to induce tumor-specific T cells, a large amount of blood is required to obtain APC (antigen presenting cells) to be used for the induction and much time, and the amount necessary for cell therapy in vitro. It is difficult to obtain tumor specific T cells.

  Therefore, an object of the present invention is to provide a cancer therapeutic agent and a cancer treatment method capable of activating cellular immunity and inducing tumor-specific T cells in cancer patients.

  The present inventors have found that effective treatment of cancer is possible by administering an antigen other than a cancer antigen in combination with T cells activated by this antigen. That is, this invention provides the cancer therapeutic agent containing the T cell activated with the antigen for administering to a patient in combination with antigens other than a cancer antigen. The present invention also provides a cancer therapeutic agent comprising the antigen for administration to a patient in combination with T cells activated by an antigen other than the cancer antigen. Preferably, the T cells are derived from a patient to whom the cancer therapeutic agent of the present invention is to be administered. In a preferred embodiment, antigens other than cancer antigens are administered in cancer tissues, in the vicinity of cancer tissues, in the vicinity of regional lymphoid tissues, and the like. T cells activated by the antigen are administered into cancer tissue, in the vicinity of cancer tissue, in the vicinity of regional lymph tissue, intravenously, and the like.

  Preferably, in the therapeutic agent for cancer of the present invention comprising a T cell activated by an antigen, the T cell is a Th1 cell specific for this antigen.

  In the cancer therapeutic agent of the present invention, preferably, the antigen is a bacterial cell component, for example, a group consisting of PPD, BCG-CWS, BCG, OK432, Ancer, Bestin, Corynebacterium parvum, LC9018, WPG, and Nocardia rubra cell wall skeleton More selected.

  Preferably, the antigen is a plant-derived component, and is selected from the group consisting of, for example, lentinan, soniferan and krestin.

  Also preferably, the antigen is a chemically synthesized substance and is selected from the group consisting of, for example, MDP, MDP-Lys, levamisole and cimetidine.

  In another aspect, the present invention provides a method for producing a cancer therapeutic agent of the present invention comprising T cells activated by an antigen. The method includes activating T cells with the antigen. Preferably, the T cell is derived from a patient to which the cancer therapeutic agent of the present invention is administered. In the method of the present invention, preferably, the T cell is activated by culturing the T cell in the presence of the antigen and IL-2.

In another aspect, the method of the present invention further comprises the step of separating Th1 and Tc1 cells activated by the antigen. Preferably, the step of separating Th1 cells and Tc1 cells imparted with antigen specificity is performed by using antibody magnetic beads or a cell sorter. In another embodiment, the method of the present invention further comprises the step of mixing the separated Th1 cells and Tc1 cells in any ratio.

  In yet another aspect, the present invention provides a composition for cancer treatment and a kit for cancer treatment comprising an antigen other than a cancer antigen and the T cell of the present invention activated by the antigen.

  According to the present invention, administration of an antigen other than a cancer antigen and a T cell activated by the antigen to a cancer-bearing host can induce tumor-specific CTL in the living body and reject the tumor. it can. In particular, the present invention is useful for use in cancer patients for whom cancer antigen information for cancer antigen-specific vaccine therapy is not available and / or cancer patients whose human leukocyte antigen (HLA) type is not compatible with vaccine therapy. is there.

Cell proliferation by this method Proportion of CD4 + and CD8 + cells in culture Intracellular cytokine staining of CD4 positive cells Comparison of IFN-γ production ability by co-culture with stimulating cells reacted with PPD or stimulating cells not reacted with PPD Changes in tumor size of tumor-bearing mice in each treatment group Survival rate of tumor-bearing mice in each treatment group

  The present invention provides a novel method for treating cancer comprising administering to a patient a combination of an antigen other than a cancer antigen and a T cell activated by the antigen. By administering these in combination, tumor-specific CTL is induced in vivo and the tumor can be rejected.

  As an antigen other than a cancer antigen, a bacterial cell component, a plant-derived component, a chemically synthesized substance, or the like can be used. Examples of the cell component include, but are not limited to, PPD, BCG-CWS, BCG, OK432, Ancer, Bestin, Corynebacterium parvum, LC9018, WPG, and Nocardia rubra cell wall skeleton. Examples of plant-derived components include, but are not limited to, lentinan, sonifiran and krestin. Examples of chemically synthesized materials include, but are not limited to MDP, MDP-Lys, levamisole and cimetidine.

  The T cell activated by the antigen is preferably a Th1 cell specific for this antigen. T cells activated by an antigen can be produced by the following method.

  Mononuclear cells collected from human peripheral blood by a method such as specific gravity centrifugation are added to an antigen-added medium (for example, AIM-V: Invitrogen, no serum added, or the same as human AB type serum or cultured cells) Blood group serum, preferably autoserum, is added in an amount of 0.1 to 30%, preferably 5 to 10% (the serum herein includes plasma or plasma-treated serum, or non-human serum or plasma) Incubate at The concentration of the antigen is 0.1-100 μg / ml, preferably 1.0-10 μg / ml. Furthermore, at an appropriate time during culture (for example, after 1 to 7 days, preferably after 3 to 5 days), IL-2 at a final concentration of 0.5 to 500 IU / ml, preferably 5 to 100 IU / ml is added to the medium. Activate T cells. Furthermore, it is preferable to perform re-stimulation with cells inactivated by mitomycin C (MMC) treatment of the same mononuclear cells reacted with the same antigen at an appropriate time in the culture. In this way, T cells specific for the antigen can be induced.

  Particularly preferably, Th1 cells specific for an antigen are induced by culturing mononuclear cells under conditions where Th1 cells proliferate preferentially. The step of inducing Th1 cells comprises mononuclear cells in the presence of anti-CD3 antibody, IL-2, and IL-12, preferably the presence of anti-CD3 antibody, IL-2, IL-12 and anti-IL-4 antibody. More preferably, it is carried out by culturing in the presence of anti-CD3 antibody, IL-2, IL-12, anti-IL-4 antibody and IFN-γ.

Induction of Th cells is confirmed by performing dual-color staining of the obtained cells with fluorescently labeled anti-CD4 antibody and anti-CD8 antibody and measuring the ratio of CD4 positive cells and CD8 positive cells with a flow cytometer. can do. Furthermore, the induction of Th1 cells can be confirmed by measuring the production of IFN-γ after stimulating the obtained cells with anti-CD3 antibody in the presence of brufeldin A.

  The specificity of Th1 cells for antigens was determined by co-culturing the Th1 cells with inactivated mononuclear cells or lymphoblast (LCL) cells reacted with the same antigen and treated with mitomycin C. It can be evaluated by measuring the amount of IFN-γ in the culture supernatant. As a comparative control, Th1 cells cocultured with cells inactivated by mitomycin C treatment of mononuclear cells or LCL cells of the same person not reacted with antigen are used.

  Further, in the population of activated T cells obtained as described above, Th1 cells and Tc1 cells imparted with antigen specificity may be separated. This step can be performed by isolating CD4 positive cells or CD8 positive cells having antigen specificity using a magnetic bead or cell sorter to which a CD4 or CD8 antibody is bound. The Th1 cells and Tc1 cells thus separated can be mixed at an arbitrary ratio so as to obtain an optimum effect in the treatment of cancer.

  The antigen-specific T cells obtained according to the present invention as described above can be used for the treatment of cancer in combination with an antigen. Examples of cancers that can be treated using the antigen-specific T cells of the present invention include squamous cell carcinoma, lung cancer, bladder cancer, head and neck cancer, melanoma, ovarian cancer, cervix, prostate cancer, breast cancer, and genitourinary cancer. And digestive organ cancer. In particular, the antigen-specific Th1 cell of the present invention is a vaccine in which cancer antigen information for cancer antigen-specific vaccine therapy cannot be obtained and / or human leukocyte antigen (HLA) type is a cancer antigen peptide. Useful for the treatment of cancer patients who are not compatible with therapy.

  By administering an antigen in a cancer patient's cancer tissue, in the vicinity of a cancer tissue, in the vicinity of a regional lymphoid tissue, etc. Antigen-specific T cells that have infiltrated the tumor are activated by the antigen. This activates Th1-led cellular immunity that is non-specific to the cancer antigen in the tumor site, and causes non-specific destruction of the tumor. As non-specific tumors are destroyed, tumor-specific CTLs (cytotoxic T cells) are induced in vivo during the treatment process.

  Examples of the present invention are shown below, but the present invention is not limited to these examples.

  Mononuclear cells collected from human peripheral blood by specific gravity centrifugation are added with PPD (Nippon BCG Manufacturing Co., Ltd.) to a concentration of 2.5 μg / ml, and cultured to reach 20 U / ml on the fourth day. Culture was continued with the addition of IL-2.

On days 7 and 14, the same mononuclear cells reacted with PPD were restimulated with inactivated cells treated with mitomycin C (MMC), and further cultured in the presence of IL-2. To induce PPD-specific T cells. More than 1 × 10 ¹⁰ cells were obtained from 30 ml of peripheral blood (FIG. 1).

  Cells in culture were appropriately collected, dual-color staining was performed using fluorescently labeled anti-CD4 antibody and anti-CD8 antibody, and the ratio of CD4 positive cells and CD8 positive cells was measured with a flow cytometer.

  As a result, the ratio of CD4 positive cells increased and the ratio of CD8 positive cells decreased, indicating that CD4 positive helper T cells proliferate predominantly by this method (FIG. 2).

  Furthermore, some of the collected cells were stimulated with anti-CD3 antibody in the presence of brufeldin A for 4 hours, and CD4 on the cell surface and IFN-γ and IL-4 accumulated in the cells were used for the respective fluorescently labeled antibodies. 3 color dyeing was performed.

  As a result, most of CD4 positive cells and CD8 positive cells produced IFN-γ, most CD4 positive cells were Th1 cells, and most CD8 positive cells were Tc1 cells (FIG. 3).

  The same mononuclear cells or lymphoblast (LCL) cells reacted with PPD were inactivated by treatment with mitomycin C, and after co-culture with the PPD-specific T cells obtained above for 24 hours, The amount of IFN-γ in the liquid was measured. As a comparative control, cells inactivated by mitomycin C treatment of mononuclear cells or LCL cells of the same person not reacted with PPD were co-cultured with the PPD-specific T cells obtained above.

  As a result, co-culture with the same mononuclear cells or LCL cells reacted with PPD showed strong production of IFN-γ, and with co-culture with the same mononuclear cells or LCL cells not reacted with PPD, IFN -Production of γ was hardly observed (Fig. 4).

  From the above results, it was shown that PPD-specific T cells are easily induced in humans by this method.

C57BL / 6 mice were treated with E. coli. G7 cancer cell line (cell in which ovalbumin OVA is expressed as a virtual cancer antigen by gene transfer into EL-4 cancer cell line) is inoculated intradermally to create a tumor-bearing mouse model, and the tumor diameter reaches 8-10 mm treatment started after (2 × 10 ⁶ to about 10 days if the E.G7 cancer cell lines were inoculated intradermally flank).

  E. PPD was used as an antigen unrelated to the antigen of the G7 cancer cell line. In addition, PPD-specific Th1 cells administered at the same time were immunized with 100 μg of PPD emulsified in advance using Freund's complete adjuvant in C57BL / 6 mice at a dose of 100 μg / mouse, and Freund's non-reactivity several times at 2-week intervals PPD emulsified with complete adjuvant was hyperimmunized to 100 μg per mouse, and induced from lymph node cells collected 7 to 10 days after the final immunization.

  CD4 positive T cells were collected from lymph node cells of PPD immunized mice using magnetic bead-labeled anti-mouse CD4 antibody and co-cultured with antigen presenting cells (APC) in the presence of 2.5 μg / ml of PPD. In this experiment, M57-treated C57BL / 6 mouse spleen cells were used as APC. Furthermore, 100 IU / ml IL-2, 20 IU / ml IL-12, 1 ng / ml IFN-γ, 50 μg / ml anti-IL-4 antibody were added and cultured from the 4th day of culture (type 1 culture conditions). .

  On the 7th day of the culture, APC cultured overnight in the presence of 2.5 μg / ml PPD was mixed with MMC treated so as to be 1: 1, and further cultured under type 1 culture conditions. On the 14th day of culture, the same operation was performed, and thereafter, the cells were confirmed to be IFN-γ producing cells by intracellular cytokine staining, and PPD-specific Th1 cells were obtained.

The treatment was divided into the following 3 groups (5 animals in each group). 1. 1. Administration of tail vein alone with PPD-specific Th1 cells (2 × 10 ⁷ / mouse) 2. PPD (10 μg / animal) was administered intratumorally alone; PPD-specific Th1 cells (2 × 10 ⁷ / animal) tail vein administration and PPD (10 μg / animal) intratumoral administration in combination. This treatment was performed 3 times every 2 days.

  As a result, in the combined group of PPD-specific Th1 cells and PPD, severe necrosis occurred in the tumor site, and complete regression of the tumor was observed. On the other hand, no significant antitumor effect was observed in the group administered with PPD-specific Th1 cells alone, and no necrosis due to inflammation was observed in the group administered with PPD alone, but no significant antitumor effect was observed (FIG. 5).

In addition, the survival rate of each treatment group Following up to day 60 after inoculation of the G7 cancer cell line, all patients died on days 34 to 36 in the PPD-specific Th1 cell-only administration group and PPD-only administration group, whereas PPD-specific Th1 cells and PPD In the combination group, 1 case died on the 40th day, but the remaining 4 cases survived until the 60th day (FIG. 6).

Claims (18)

A therapeutic agent for cancer comprising T cells activated by the antigen for administration to a patient in combination with an antigen other than the cancer antigen. A cancer therapeutic agent comprising the antigen for administration to a patient in combination with T cells activated by an antigen other than the cancer antigen. The cancer therapeutic agent according to claim 1 or 2, wherein the T cell is a Th1 cell specific to the antigen. The cancer therapeutic agent according to any one of claims 1 to 3, wherein the antigen is a bacterial cell component. The cancer therapeutic agent according to claim 4, wherein the bacterial cell component is selected from the group consisting of PPD, BCG-CWS, BCG, OK432, Ancer, Bestin, Corynebacterium parvum, LC9018, WPG, and Nocardia rubra cell wall skeleton. The cancer therapeutic agent according to any one of claims 1 to 3, wherein the antigen is a plant-derived component. The cancer therapeutic agent according to claim 6, wherein the plant-derived component is selected from the group consisting of lentinan, sonifiran and krestin. The cancer therapeutic agent according to any one of claims 1 to 3, wherein the antigen is a chemically synthesized substance. The cancer therapeutic agent according to claim 8, wherein the chemically synthesized substance is selected from the group consisting of MDP, MDP-Lys, levamisole and cimetidine. The method for producing a cancer therapeutic agent according to claim 1, comprising activating T cells with the antigen. 11. The method of claim 10, wherein the T cell is derived from the patient. The method according to claim 10 or 11, wherein the step of activating T cells comprises culturing T cells in the presence of the antigen and IL-2. The method according to any one of claims 10 to 12, further comprising a step of separating Th1 cells and Tc1 cells activated by the antigen. The method according to claim 13, wherein the step of separating Th1 cells and Tc1 cells activated by the antigen is performed by using antibody magnetic beads. The method according to claim 13, wherein the step of separating Th1 cells and Tc1 cells activated by the antigen is performed using a cell sorter. The method according to any one of claims 13 to 15, further comprising a step of mixing the separated Th1 cells and Tc1 cells in an arbitrary ratio. A composition for cancer treatment, comprising an antigen other than a cancer antigen and T cells activated by the antigen. A cancer treatment kit comprising an antigen other than a cancer antigen and T cells activated by the antigen.