WO2003018784A1

WO2003018784A1 - Process for producing antigen-specific human t cells and drugs

Info

Publication number: WO2003018784A1
Application number: PCT/JP2002/008499
Authority: WO
Inventors: Takashi Nishimura
Original assignee: Toray Industries, Inc.
Priority date: 2001-08-24
Filing date: 2002-08-23
Publication date: 2003-03-06

Abstract

A process for producing major histocompatibility complex class II-restricted antigen-specific human Th1 cells which enable a novel therapy for cancer or immune diseases. This process comprises starting with peripheral blood or bone marrow collected from a patient, culturing it in vitro using an exogenous cytokine without adding any disease-origin antigen outside, thus inducing dendrocytes, culturing CD4-positive T cells contained in the culture in the coexistence with the above dendrocytes and thus inducing into cytotoxic human Th1 cells. By administering the cytotoxic human Th1 cells thus produced to patients, cancer and immune diseases can be treated.

Description

Method for producing antigen-specific human T cells and pharmaceutical

Technical field

The present invention relates to a method for producing human Th1 cells, cells produced by the method, and pharmaceutical uses thereof. The human Thl cells of the present invention are particularly useful for immunotherapy of cancer, host versus graft reaction, and the like.

Background art

Treatment of cancer is one of the most important medical issues. To date, a variety of surgical, chemo, and radiation therapies have been tried, but the outcome has improved, but is still not at a satisfactory level. Since the 1980s, immunotherapy has also been attempted and progressed with improvements, and is expected as a new cancer treatment. Originally, it is thought that cancer cells can be eliminated by the immune function of the host.However, in patients who have a large amount of cancer cells in the body and are in an immunosuppressed state, the patient's own immune system alone is no longer sufficient for cancer. The cells cannot be eliminated. For this reason, immunotherapy is intended to treat cancer by administering various cytotoxic agents or immunostimulating substances from outside the body, or by enhancing the active immunity in the body by injecting activated lymphocytes. It is. To date, various methods have been tried, but the effect is still limited and further improvement is required (Fujimimoto T. et al: J. I. uno l 158: 5619, 199) Fu, Patent No. 2530966 and "Current State of Cellular Immunotherapy", History of Medicine, vol. 195, No. 1, 2000).

Recently, it has become clear that dendritic cells play a very important role in antigen presentation, the fundamental response of the immune response (Inaba. K. et al: Proc. Nat I. Acad Sc. USA 80: 6041, 1983, Banchereau J. et al: Nature 392: 245, 1998). In cancer immunotherapy, vaccine therapy for cancer using dendritic cells is currently receiving attention, and some of its promises have been reported (Sato Μ et al: Int. Immunol. 12: 335, 2000, "The Present Situation of Cellular Immunotherapy", History of Medicine, vo 1.195, No. 1, 2000). Specifically, after a cancer peptide antigen and dendritic cells are cultured for several hours to enhance antigen presentation ability, the dendritic cells are transfused into a patient, and the dendritic cells are further transferred to the patient. This method involves inducing cytotoxic T cells (GTL) from peripheral blood lymphocytes of patients as stimulator cells and injecting them into cancer patients. Clinical trials have already been conducted for melanoma, prostate cancer, breast cancer, etc. I have.

Such immunotherapy can specifically sensitize antigen-presenting cells when sensitizing dendritic cells and other antigen-presenting cells with cancer antigen-cancer antigen-derived peptides in vitro. Then, how efficiently the required amount of GTL can be induced, and how efficiently the GTL can reach the lesion in the cancer patient, are important matters for improving the therapeutic effect. The most rational antigen used to sensitize dendritic cells is the cancer cell antigen of the patient, but the cancer peptide antigen is the major histocompatibility complex of the patient.

(MHG) It is essential that it be able to be displayed on the molecule (MHG restriction), and the specific antigen used in this case is an MHG class I binding peptide (expressed on all nucleated cells and platelets, (A peptide derived from a cytoplasmic protein is presented to cytotoxic GD8-positive killer cells.) It is difficult to induce antigen-specific GTL with class I-binding peptide alone, and attempts to overcome this problem by using various adjuvants to enhance immunity have continued. However, a practical method has not yet been established. For example, trials using interleukin 2 (IL-2), IL-12, etc. (Ribas A et al: Cancer Res 57: 2865,

In 1997), only a small cancer reduction and survival benefit were obtained. In immunotherapy, of the two types of MHG molecules, not only MHG class I but also MHG class II (peptides mainly expressed on antigen presenting cells and derived from plasma membrane and extracellular proteins are converted to GD4-positive helper T cells. It is important to induce an immune response through (presentation), but this induction technique has not yet been established.

To establish a cell immunity control method that is useful for the treatment of cancer, allergy, and autoimmune diseases for which a sufficient immunoregulation method has not yet been established, it is necessary to activate GTL with MHG class I-binding antigen It is necessary to activate Th1 cells with class II binding antigen peptides. This is because this cell population is very useful for immunotherapy, has high cytotoxicity and cytokine-producing ability, and is MHG class II-restricted, so it is a strong cell against target cancer cells and cells associated with pathogenesis. Injury G This is because a D4 positive GT can be obtained. In other words, in order to obtain a high therapeutic effect in immunotherapy, activation of antigen-specific Th1 cells is followed by MHG class II-restricted antigen-specific killer T cells (GD4 positive GT1J or GD4 positive helper). It is extremely important to create an environment in which Th1-driven systemic cellular immunity, which is the induction of Nokiller cells, is activated.

On the other hand, recent advances in immunity research on T cells have revealed that helper T cells are divided into two subtypes, TM and Th2, and that many immune responses are controlled in the mouse system ( Mosmann, TR: Ann. NY Acd. Sci. 664: 89, 1992). It was also revealed that a similar helper T cell subset exists in humans, and it has been speculated that the balance between Th1 and Th2 plays an important role in the development of a number of diseases including cancer (Sal game, P. et. Science, 254: 279, 1991, Abbas. AK et al. Nature 383: 787, 1996). According to them, Th1 and Th2 are in a functional balance with each other, and when this balance is maintained, they are healthy, but when Th1 becomes excessive, autoimmune diseases, rheumatoid arthritis, etc. In multiple sclerosis and the like, it is said that excess Th2 causes cancer, immunodeficiency, allergic disease and the like. This method of immunoregulation based on TM ZTh2 balance is also expected to lead to effective treatment of cancer, but no efficient method for inducing Thl cells and Th2 cells has been established, and specific clinical applications have not been established. Not yet done. In clinical application to humans, the concept of Th1 / Th2 balance was originally proposed in mouse experiments. It is necessary to verify whether they have the ability to induce T cells and produce cytokines that control cancer cytotoxicity and immune balance that have functions applicable to immunotherapy. This verification is not yet sufficient.

In addition to the cancer immunotherapy described above, bone marrow transplantation and hematopoietic stem cell transplantation can also be considered as one of the immunotherapy using Th1 cells. Bone marrow transplants are used to treat leukemia and aplastic anemia, and hematopoietic stem cell transplants are used in solid tumors after high-dose chemotherapy or intense radiation therapy (myeloablative hematopoietic stem cell transplants) and in patients with solid cancers. After a small amount of chemotherapy or radiation therapy (non-myeloablative hematopoietic stem cell transplantation), 02 08499

Four

Attempts have also been made for inborn errors of metabolism that depend on blood cells. In the case of bone marrow transplantation or hematopoietic stem cell transplantation, host-graft reaction (HVGD) occurs, and weak graft-versus-host reaction (GVHD) is known to lead to poor graft survival or recurrence of the underlying disease. Whether GVHD occurs depends on chance and controlling GVHD, which is key to the success of bone marrow transplantation and hematopoietic stem cell transplantation, is a major medical challenge (Yabe. Et al.). a: Bone Marrow Transp l antat i on. 24: 29, 1999) ₀

In practice, immunotherapy requires the isolation of antigen-presenting cells and antigen sensitization, followed by cultivation, and separate isolation and culturing of T cells, which requires a great deal of labor and high know-how. Desired. In addition, there remain issues such as difficulties in obtaining antigens such as cancer cells and cancer peptides, complicated procedures for handling them, and adequate facilities. In addition, the use of patient cells poses medical problems, such as the invasion and burden on patients, and the fact that it is virtually impossible to draw blood from patients who have become extremely anemic due to chemotherapy or radiation therapy. is there. Therefore, in the case of using blood cells derived from a patient, it is extremely important for practical use that the target cells are easily and efficiently induced from a small amount of sample.

These problems are common issues not only in cancer treatment and bone marrow transplantation, but also in intractable diseases targeted for immunotherapy in the future.

As described above, immunotherapy has many challenges, including not only the concept verification but also the development of corresponding medical technologies, and simple and useful treatments including these have not yet been established. There is no present.

As mentioned above, in cancer therapy and immunotherapy such as bone marrow transplantation, the development of safe and simple methods to induce MHG class II-restricted GTL and Th1 cells, and their application to treatment, is a major medical challenge. It has become a challenge.

Disclosure of the invention

An object of the present invention is to provide a method for producing human Th1 cells, which solves these problems, and enables a new therapeutic method for cancer and immune diseases, a medicament comprising cells produced by the production method, An object of the present invention is to provide a use for a disease that can be treated with cells produced by the production method. As a result of intensive studies, the present inventors have made it possible to easily obtain MHG class II-restricted antigen-specific human Th1 cells having cytotoxic activity using exogenous cytokines from peripheral blood and bone marrow cells of patients. The present inventors have found that the human Th1 cells can be used for cancer treatment, bone marrow transplantation, and the like, and have completed the present invention. That is, the present invention provides a method for inducing dendritic cells by using peripheral blood or bone marrow collected from a patient as a material, and culturing the cells in vitro without adding a disease-derived antigen from the outside and using an exogenous cytokine. A major histocompatibility complex class 11-restricted antigen, which comprises culturing CD4 positive T cells contained in a substance in the presence of the dendritic cells and inducing human Th1 cells having cytotoxic activity. A method for producing a specific human Th1 cell is provided. The present invention also provides a human Th1 cell produced by the method of the present invention, and provides a medicine or a pharmaceutical composition containing the human Th1 cell. For example, it provides drugs for treating cancer, drugs for treating malignant diseases of the blood or hematopoietic system, and drugs for controlling host versus graft reactions. Specific examples of therapeutic agents for malignant diseases of the blood or hematopoietic system include various therapeutic agents for leukemia, malignant lymphoma, multiple myeloma, myelodysplastic syndrome, or aplastic anemia. Further, the present invention provides a use of the above-mentioned cell of the present invention for producing a cancer therapeutic agent, a therapeutic agent for blood or hematopoietic malignancy, or a host versus graft reaction control agent. Further, the present invention provides a method for treating cancer, blood or hematopoietic malignant disease, which comprises administering an effective amount of the above-described cell of the present invention to each patient of cancer, immune disease, blood or hematopoietic malignant disease. I will provide a. The present invention also provides a method for controlling a host-graft reaction, which comprises administering an effective amount of the above-described cell of the present invention to a patient who wants to control the host-graft reaction.

The antigen-specific Th1 cells obtained by the method of the present invention also produce cytokines, and can exert specific cytotoxic activity against target cells and precise cytotoxic activity against target cells, so that therapeutic effects on patients are obtained. Can induce a Th1-driven systemic cellular immune state with Moreover, since the obtained Th1 cells are derived from the T lymphocytes of the patient, even if they are returned to the body of the patient, no problem such as harmful rejection occurs. Therefore, effective immunotherapy for various cancers including leukemia can be performed. It is also useful as a method for controlling host-graft reactions. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a method for inducing activated T cells using leukemia cells as an example. The commonly used conventional method (A) and the method (B) of the present invention are shown.

FIG. 2 is a diagram showing the results of identification of lymphocyte surface antigens before and after culture in Example 1. After culturing, pure CD4-positive cells are obtained. GD4-positive and ZGD8-negative helper T cells, which had only 0.92% before culture (before culture), were induced to 94.66% after culture (after culture). It turns out that it is a pure CD4 positive helper killer cell population.

FIG. 3 shows the cytotoxicity of T lymphocytes against autologous cancer obtained by the method of the present invention (Th1 condition) or the method of the comparative example (Th0 condition and Th2 condition) in Examples 1 to 3. FIG. ThO; ThO-type helper T cell induction condition; Th0 / 1; ThO and Thl-type helper T cell induction condition; Th1; Th1-type helper T cell induction condition; Th2; Th2-type helper T cell induction condition In this case, the expression is as follows, and it can be seen that the cytotoxicity is highest when induced by Th1 type.

FIG. 4 is a diagram showing the amounts of IFN-r production obtained in Examples 1 to 3 by the method of the present invention (Th1 condition) or the method of the comparative example (Th0 condition and Th2 condition). ThO: In the case of ThO type helper T cell induction condition, Thl; In the case of Th1 type helper T cell induction condition, Th2; In the case of Th2 type helper T cell induction condition,

It turns out that FN-r production is high.

FIG. 5 is a diagram showing MHG class II antigen restriction of Example 3. Inhibition of IFN-r production by autologous cancer cell stimulation and cytotoxicity against autologous cancer cells by antibodies against MHG class 11 antigens shown by Th1 cells induced from IM patients. The MHG class 11 antibody inhibits IFN- production and cytotoxic activity and clearly shows MHG class 11 restriction.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention preferably induces dendritic cells from granulocyte macrophage colony stimulating factor (GM-GSF) and IL-3 from a patient's bone marrow or peripheral blood without externally adding an antigenic substance. Then, by adding IL-2 to the culture system, a small amount of GD4-positive T cells were allowed to interact with dendritic cells and proliferated. Cytotoxic activity against autologous cancer cells and production of IFN-χ can be obtained by culturing CD4-positive T cells with Th1-inducible cytokines such as IL-12 and interferon τ (IFN-r). The following are the Th1 cell type GD4-positive helper killer cells and the method of inducing them. These Th1-type GD4-positive helper killer cells have strong cytotoxicity and high IFN-T production ability, so they elicit an immune response in the patient and induce a Th1-driven systemic cellular immune state You can expect the ability to do it. Since the immunotherapeutic activity is extremely high, it can be used to treat cancer and control host-graft reactions. Also, since the patient's original autologous cells are used, it is highly safe and can be expected to avoid serious side effects.

Fig. 1 shows an example of a conventional method for sensitizing cancer antigens followed by a method for inducing activated T cells (Fig. 1A), using leukemia as an example. 1 shows the present invention (FIG. 1B) using killer cells. In the present invention, cytokines are added over time in a single culture system that does not require a foreign cancer antigen, which is conventionally required, and is performed without mixing with another cell. This makes it possible to obtain MHG class II-restricted Th1-type CD4-positive helper killer cells very simply and efficiently.

The cells used here are not particularly limited as long as they are peripheral blood or bone marrow of various cancer patients, but can be preferably obtained from blood cancer patients such as leukemia and malignant lymphoma. The peripheral blood or bone marrow may be any of a fresh sample, a cryopreserved sample, and a cryopreserved sample. Since the amount of the sample required for the present invention may be a very small amount of about a few mL of bone marrow or peripheral blood, it may be a small sample for examination or a residual sample, or may be a frozen sample. Therefore, it can be applied to cancer patients with extreme anemia and leukemia patients with few normal blood cells, and has the advantage that it can be applied to many target diseases with little burden on patients. For example, it is possible to induce 10 ⁷ to 10 ⁸ cells required for normal adoptive immunotherapy from 1 to 2 mL of leukemia blood.

As for peripheral blood, whole blood may be cultured, or only white blood cell components may be separated and cultured, but the latter is more efficient and preferable. Further, mononuclear cells may be separated from leukocyte components. In the case of bone marrow, the whole cells that make up the bone marrow are cultured. Alternatively, mononuclear cells may be separated and cultured therefrom. Peripheral blood and its leukocyte components and bone marrow cells include monocytes, dendritic cells, hematopoietic stem cells or immature dendritic cells, GD4-positive cells, and target cells such as blood cancer cells. Cancer cells are also included. However, Th1-driven systemic cell-mediated immunity is not formed in which peripheral blood and bone marrow remain in a state where a sufficient therapeutic effect can be obtained. Is obtained.

In general, in leukemia and lymphoma, as shown in Figure 2, normal bone marrow cells and peripheral blood obtained from a patient have very few normal CD4-positive T cells at several percent (see Example 1). Conventional methods do not allow sufficient isolation of GD4-positive T cells, and subsequent induction of helper T cells is virtually impossible. According to the present invention, Th1 cells can be prepared from blood from a very small amount, which was conventionally impossible or extremely difficult to prepare. In addition, the ability to induce cells from frozen lymphocytes greatly reduces the burden on patients. For example, compared with the conventional method, where blood is collected at a time of about 20 mL and blood is collected once every two weeks, this method freezes lymphocytes with 5 mL of blood at a time. Store and make 5 servings per 1 mL. Patients with leukemia or solid cancer are often anemic, and frequent blood collections place a heavy burden on patients, but this law greatly reduces the burden.

In addition, in bone marrow transplantation and blood stem cell transplantation, cells provided from donors can be similarly cultured by this method, and the host-graft reaction can be appropriately controlled, thereby improving graft survival and treating underlying diseases. Can be connected to

The production method of the present invention can be carried out in a single step by adding the above-described various cytotoxic agents to a medium, or a first step of inducing dendritic cells, and GD4-positive T cells contained in a culture. Is cultured in the presence of the above-mentioned dendritic cells, and a second step of inducing human Th1 cells having cytotoxic activity can also be performed.

When performed in a single step, the cytodynamic force used for stimulating cells in the present invention is preferably at least GM-GSF, IL-3, IL-2 and IFN-. Other cytokines, such as IL-12, may be used as needed. In addition, culture period The period is not particularly limited as long as the required number of dendritic cells or Th1-type CD4-positive helper / killer T cells are induced, but is usually performed for 3 to 8 weeks.

When performed in two steps, the first step is performed in the presence of granulocyte macrophage colony stimulating factor and / or interleukin 3, and the second step is to add interleukin 2 to the culture obtained in the first step. And growing the CD4-positive T cells while interacting with the dendritic cells, and further adding interferon r and / or interleukin 12 to obtain human Th1 having cytotoxic activity. Inducing into cells. In the step of expanding CD4-positive T cells in the second step, the culture obtained in the first step is preferably interleukin 2 and granulocyte-macrophage coagulation stimulating factor or interleukin 3, more preferably Granulocyte macrophage colony stimulating factor and interleukin 3 may be further added to interleukin 2 to the culture obtained in the step, and the culture may be continued. The second step can be carried out in one step or in two steps, but when it is carried out in two steps, the step of expanding GD4-positive T cells and human Th1 cells having cytotoxic activity Add the cytokines used in any of the steps of induction. The culture period is not particularly limited as long as the required number of dendritic cells and Th1-type GD4-positive helper killer T cells are induced, but usually the first step is 1 to 7 days, preferably 2, The first stage of the second step is 2 to 6 weeks, preferably 2 to 3 weeks, and the second stage of the second step is 2 to 7 days, preferably 2 days.

In any of the above methods and any steps, any cytokine may be used, such as a natural type or a genetically modified type, as long as it has characteristics that have been confirmed to be safe and bioactive. However, it is preferable to use a standard that has been used for medical purposes and that has been assured of the required quality in the minimum amount required. The concentration of the cytokine to be added is not particularly limited in any of the methods and steps described above, as long as dendritic cells and TM-type GD4-positive helper killer T cells are induced. The concentration is preferably about 10-1000 ng / mL, more preferably about 20-500 ng / mL. When a plurality of cytokins are used, the concentration of each cytokine is usually preferably 1 ng / nl or more. Culture is white It can be performed using a well-known medium usually used for culturing blood cells. The culture temperature is not particularly limited as long as leukocyte proliferation is possible, but the human body temperature of about 37 ° C is most preferable. Also, the gaseous environment in the culture is not limited especially as long as the proliferation of white blood cells, it is preferable to vent the 5% G0 _2. The equipment used for cell separation and culture can be appropriately used. It is preferable that safety is confirmed for medical use, and that the operation is stable and simple. In particular, for cell culture devices, irrespective of general containers such as petri dishes, flasks, bottles, etc., stacked containers, multi-stage containers, roller bottles, spinner bottles, bag type incubators, hollow fiber columns, etc. can also be used. .

The following procedure is considered as a specific treatment form. That is, blood is collected from a patient with the target disease, lymphocyte and leukocyte components are separated, culture is started, and the Th1-type GD4-positive helper killer T cells induced 2 to 4 weeks later are infused into the patient themselves . The burden on the patient is basically only blood collection and transfusion, and the cancer cells are suppressed by the induced Th1-type GD4-positive helper killer T cells, and as a result are treated. In bone marrow transplantation and blood stem cell transplantation, cells provided by donors are similarly cultured, and host-graft reactions are controlled, leading to improved graft survival and treatment of primary diseases be able to.

When using the Th1 cells obtained by the present invention for therapy, it is preferable to administer Thl cells to peripheral blood by intravenous injection, infusion, or the like. In the case of solid cancer, it may be administered to peripheral blood or directly into cancer tissue. The number of cells to be administered can be appropriately set depending on the patient's condition, etc., but when administered to peripheral blood, it is usually about 10 ⁷ to 10 ¹¹ cells per adult patient, preferably 10 ⁸ to 10 ⁹ About one. Example

Next, the present invention will be described more specifically with reference to examples.

Example 1 Production of Th1 cells (Part 1)

Human acute lymphocytic leukemia (ALL) separating mononuclear from bone marrow cells or peripheral blood of patients, 2 χ 10 ⁶ cells Roh including mL concentration in 10% human serum AIM-V medium (GI BG0 - BRL), seeded in a 12-well plate, seeded with GM-GSF (30 ng / mL) and IL-3 (30 ng / mL). ) Was added and cultured for 2 days to induce dendritic cells. Thereafter, IL-2 (100 U / mL (10 ng / mD) was added to the culture solution, and cultured for 14 to 21 days to obtain a large amount of GD4-positive T cells. The cells were stained with GD4 antibody and Fluorescein isot hiocyanate-labeled anti-human GD8 antibody, and confirmed by flow cytometry (see Fig. 2) (Immunological Experiment Procedures, 1995, Namedo Co., Ltd.). The GD4-positive T cells were further cultured for two days in a culture solution containing -12 (40 U / mL (4 ng / mL)) and! FN-r (30 ng / mL). If the volume exceeds 5 × 10 ⁶ mL, collect the cells with a glass pipette while stirring, re-appear with approximately 1 × 10 ⁶ mL of the above-mentioned cytokine-supplemented culture medium, and renew the plate. The obtained CD4-positive T cells were collected in a centrifuge tube using a glass pipette with stirring, and centrifuged. Recovered.

For comparison, Th2 conditions were added at the concentration of IL-4 (30 ng / mL) from the beginning of the culture, and no IL-12 and IFN-r were added at the final stage, or ThO conditions (ThO conditions). Cells or Th1 / 2 progenitor cells) at the final stage without -12 and IFN-r were also cultured and recovered in the same manner as above.

Example 2 Production of Th1 cells (Part 2)

The human chronic myelogenous leukemia (CIVIL) patients bone marrow cells or peripheral blood from mononuclear isolated minute, the 2Kai10 ⁶ pieces Roh including mL concentration in 10% human serum AIM-V medium (manufactured by GIBC0-BRL Co.) The cells were opaque, seeded on a 12-well plate, added with GM-GSF (30 ng / mL) and IL-3 (30 ng / mL), and cultured for 2 days to induce dendritic cells. Thereafter, IL-2 (100 U / mL (10 ng / mD) was added to the culture solution, and cultured for 14 to 21 days to obtain a large amount of CD4-positive T cells. (4 ng / mD) and culture medium containing IFN-r (30 ng / mL) and continued culturing for another 2 days, and CD4-positive T cells were collected in the same manner as in Example 1. The cells were cultured under Th2 conditions and ThO conditions in the same manner as in 1, and collected in the same manner as described above.

Example 3 Production of Th1 cells (Part 3)

Human acute monocytic leukemia (AMoL (M4)) mononuclear than bone marrow cells or peripheral blood of a patient is separated, 2x10 containing ^six mL concentration in 10% human serum AIM-V medium (GIBG 0-BRL), seeded on a 12-well plate, added GM-GSF (30 ng / mL) and IL-3 (30 ng Ml), and cultured for 2 days to induce dendritic cells. Thereafter, -2 (100 U / mL (10 ng / mD) was added to the culture solution, and the mixture was cultured for 14 to 21 days to obtain a large amount of GD4-positive T cells. mL (4 ng / mL)) and IFN-r (30 ng / mL) were further cultured for 2 days in a culture solution containing the same, and GD4-positive T cells were collected in the same manner as in Example 1. Therefore, the cells were cultured under Th2 and ThO conditions in the same manner as in Example 1. The cells were collected in the same manner as described above, and IL-12 (40 U / mL (4 ng / mD) and IFN-r (30 ng / mL) were continuously added, and CD4-positive T cells were collected in the same manner as described above. It was bad condition.

Example 4 Properties of Cells (Part 1)

The cytotoxic activity of the CD4-positive T cells obtained in Examples 1 to 3 on leukemia cells of the patient was examined. A specific method for examining the cytotoxic activity was as follows: the leukemia cells stored in a frozen state of the patient were frozen and thawed, then cultured, radiolabeled with ⁵¹ Cr according to a standard method during the experiment, and the GD4 positive obtained in the patient was obtained. T cells and radiolabeled leukemia cells were mixed and cultured at each effector cell-to-target cell ratio, and after 4 hours, the radioactivity released into the culture supernatant was measured to determine the cytotoxic activity. (Ishi 1995, Nankodo Co., Ltd.).

The results are shown in Figure 3. As shown in FIG. 3, GD4-positive T cells obtained from each patient by the method of the present invention (Th1 condition or ThO / 1 condition) are high on leukemia cells of each patient and show cytotoxic activity. Was.

Example 5 Properties of cells (Part 2)

In addition, the IFN-r producing ability of the GD4-positive T cells obtained in Examples 1 to 3 was examined. This was specifically performed as follows. Processing the leukemic cells obtained from patients with mitomycin C (50 g / mL) ( ( Ltd.) Kyowa Hakko), obtained from the My Bok mycin C treatment leukemia cells (5 χ 10 ⁴ cells) and patient The obtained CD4-positive Τ cells (2χ10 ⁵ ) were mixed and cultured, and 48 hours later, IFN-r produced in the culture supernatant was measured using an IFM-r ELISA kit (manufactured by R & D). Fig. 4 shows the results. As shown in FIG. 4, the GD4-positive T cells obtained by the method of the present invention (Th1 condition) showed IFN-r-producing ability, indicating that they were Th1-type cells.

These results enabled efficient induction of GD4-positive helper killer cells, which are considered promising for cancer immunotherapy and GVHD control.

Example 6 Properties of cells (Part 3)

The suppression of IFN-τ production and cytotoxic activity against autologous cancer cells by antibodies against MHG class II by stimulation of autologous cancer cells exhibited by the Th1 cells of the present invention obtained in Experimental Example 3 was examined. This was specifically performed as follows. The test was performed in the same manner as in the test system performed in Example 4 and Example 5, and the antiserum against MHG class I or antiserum against class II was added to the mixed culture system at a dilution of 100-fold, and the test was performed. .

Fig. 5 shows the results. Figure 5 shows that the production of IFM-τ by stimulation of autologous cancer cells was completely suppressed by the addition of antiserum to MHC Class II (a), and the cytotoxic activity against autologous cancer cells was also reduced by the addition of antiserum to MHG Class II. As a result, it was reduced by about 40% (b). This indicates that the induced GD4-positive T cells are restricted to MHC class II.

Reference example 1

Mouse ovalbumin (OVA) antigen-specific lymphocytes, together with antigen-presenting cells (mouse splenocytes), were combined with OVA peptide (10 μg / mL), IL-12 (20 U / mL), IFN-r (1 ng / ml). Cultured in RPMT 1640 medium supplemented with (mL), IL-2 (20 U / mL), anti-IL-4 antibody (50 g / mL), and fetal calf serum (10%) to induce OVA antigen-specific Th1 cells did. In 6 mice, 2 内 10 ⁶ cancer cells (A20-0VA) expressing the OVA antigen by gene transfer were transplanted intradermally into mice, and when the 0VA antigen-expressing carcinoma grew to 6-8 mm, 2-10 ⁷ induced 0V A antigen-specific Th1 cells were transferred to the tumor-bearing mice. As a result, disappearance of cancer was observed in all cases, and a marked antitumor effect was observed by injection into antigen-specific Thl cells.

Reference example 1 The effect of Th1 cell control on transplant cell engraftment was investigated in a mouse bone marrow transplantation model. That is, the 5 X 1 0 ⁷ or lymphocytes G57BL / 6 mice BDF1 mice transplanted with intravenous injection was measured the induced IFN-r. As a result, 5 to 7 days after transplantation, IFN-r was detected in the serum from 50 Opg / mL to 200 Opg / mL in serum, and 10 to 4 days later. All cells were replaced by cells from transplanted mice, whereas when IFN-r was 1 Opg / nt or less, transplanted lymphocytes did not survive. Was found to be important for colonization of the transplanted cells, indicating that graft-versus-host disease (GVHD) and transplantation during allogeneic hematopoietic cell transplantation in patients with hematopoietic tumors, such as leukemia patients. This is considered to be an important finding in controlling hematopoietic leukemia lymphoma (graft-versus-leukemia / lymphoma: GVL).

Claims

Scope

1. Dendritic cells are induced and cultured by using peripheral blood or bone marrow collected from a patient as a material, and culturing the cells in vitro without adding a disease-derived antigen from the outside and using an exogenous site force-in. Major histocompatibility complex class 11 restriction, including culturing CD4-positive T cells contained in cells in the presence of the dendritic cells and inducing human Th1 cells having cytotoxic activity For producing human Th1 cells specific for a specific antigen.

2. The method according to claim 1, wherein the patient's disease is cancer.

3. The method _{c according to} claim 1, wherein the patient's disease is a blood or hematopoietic malignancy.

4. The method according to claim 2, wherein the disease of the patient is leukemia, malignant lymphoma, multiple myeloma, myelodysplastic syndrome, or aplastic anemia.

5. The exogenous cytokine is a group obtained by adding at least interferon and / or interleukin 12 to granulocyte macrophage colony-stimulating factor, interleukin 3 and interleukin 2 at least. The method described in paragraph 1.

6. The dendritic cell is induced by culturing the bone marrow cell or leukocyte component in the presence of at least one cytokine selected from the group consisting of granulocyte macrophage colony stimulating factor and intermouthin 3. Any of 1 to 5 above.

7. To culture GD4-positive T cells contained in the culture in the presence of the dendritic cells and to induce human Th1 cells having cytotoxic activity, the GD4-positive T cells are interleukin The method according to any one of claims 1 to 6, further comprising the step of: proliferating while interacting with the dendritic cells in the presence of the cells; and inducing human Th1 cells under culture conditions in which interferon r and no or interleukin 12 are added. The method of paragraph 1.

9. The first step of inducing dendritic cells, and the second step of culturing CD4-positive T cells contained in the culture in the presence of the above-mentioned dendritic cells to induce human Th1 cells having cytotoxic activity 6. The method according to claim 1, comprising the steps of:

10. The first step comprises: granulocyte macrophage colony stimulating factor and / or Carried out in the presence of interleukin 3, wherein the second step comprises adding interleukin 2 to the culture obtained in the first step and continuing the culture to grow GD4-positive T cells; and 10. The method according to claim 9, further comprising the step of: adding cron r and / or interleukin 12 to induce the cells to have human Th1 cells having cytotoxic activity. 11. A cell produced by the method according to any one of claims 1 to 10.

12. A medicament comprising the cell according to claim 11.

13. A pharmaceutical composition comprising the cell of claim 11.

14. A therapeutic agent for cancer comprising the cell according to claim 11.

15. A therapeutic drug for malignant diseases of the blood or hematopoietic system containing the cells of claim 11.

16. The blood or hematopoietic malignancy according to claim 15, wherein the blood or hematopoietic malignancy is a therapeutic drug for leukemia, malignant lymphoma, multiple myeloma, myelodysplastic syndrome or aplastic anemia. Therapeutic drugs.

17. A host-graft reaction control agent comprising the cell according to claim 11.

18. Use of the cell according to claim 11 for producing a therapeutic agent for cancer.

19. Use of the cell according to claim 11 for producing a therapeutic drug for cancer of malignant diseases of blood or hematopoietic system.

20. The use according to claim 19, wherein the blood or hematopoietic malignancy is leukemia, malignant lymphoma, multiple myeloma myelodysplastic syndrome or aplastic anemia. 21. Use of the cell according to claim 1 for producing a host-graft reaction controlling drug.

22. A method for treating cancer, comprising administering an effective amount of the cell according to claim 11 to a patient with cancer or an immune disease.

23. A method for treating a patient with a blood or hematopoietic malignancy using an effective amount of the cell according to claim 11.

24. The therapeutic method according to claim 23, wherein the blood or hematopoietic malignancy is a leukemia, malignant lymphoma, multiple myeloma myelodysplastic syndrome or aplastic anemia patient.

25. A method for controlling a host-graft reaction, comprising administering an effective amount of the cell according to claim 11 to a patient in whom it is desired to control the host-graft reaction.