JP2011051937A - Cancer therapeutic agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cancer therapeutic method, a cancer therapeutic agent effective for a living body and a system useful for cancer treatment. <P>SOLUTION: The cancer therapeutic method includes (A) a step to warm the cancer focus and/or the region including the cancer focus of a subject, and (B) a step to administer lymphocytes to the subject during the step (A) or after the step (A). The cancer therapeutic agent includes lymphocytes as an effective ingredient and is administered to a subject who has been treated with the warming treatment to the cancer focus and/or the region including the cancer focus during the warming treatment and/or after the warming treatment. Since the cancer therapeutic method and the cancer therapeutic agent have a high curing effect and yet put a small burden and stress on a subject and constitute a system effective for cancer treatment, this invention is also used after surgery, radiotherapy, and chemotherapy, and is highly useful in the medical field. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cancer treatment method, a therapeutic agent, and a treatment system useful in the medical field.

  Cancer treatment includes surgical treatment (surgery), radiation therapy, chemotherapy, immunotherapy (cell therapy and vaccine therapy), and the like. Among them, chemotherapy using an anticancer drug is generally used. In addition, thermotherapy (hyperthermia), which is a treatment of cancer from a different viewpoint, is also performed. Hyperthermia uses the action of cancer cells dying when the temperature is 42 to 43 ° C. or higher, and by heating the site including the local or peripheral area of the cancer lesion from the outside, This is a method of selectively damaging cancer cells to reduce the cancer lesion or to cure the cancer. In recent years, it has been known that there is a sensitizing action with radiation therapy and anticancer agents.

  In adoptive immunotherapy, which is a type of cell therapy often performed on cancer patients, the patient's own lymphocytes are cultured outside the body and the resulting lymphocytes are administered to the patient. There are various culture methods, including the addition of interleukin-2 (IL-2) and interleukin-15 (IL-15), which are lymphocyte growth factors, stimulation with anti-CD3 antibody or anti-CD3 antibody and anti-CD28. A combination of antibody costimulation and lymphocyte growth factor is mainly used. In addition, in order to educate lymphocytes so that tumors can be recognized or damaged, culturing with addition of antigen-presenting tumor cells, tumor antigen protein or tumor antigen peptide, or antigen-presenting cells treated with antigen is also performed. Yes.

  Among adoptive immunotherapy, in which lymphokine-activated cells obtained by expansion culture by the action of IL-2 and anti-CD3 antibody from cytotoxic T cells (CTL) or peripheral blood lymphocytes induced in vitro are transferred. , How to maintain the cytotoxic activity when expanding the above-mentioned cells, how efficiently lymphocytes can be expanded outside the body, how efficiently to expand lymphocytes with the ability suitable for treatment, etc. With respect to the above problem, the effects of using fibronectin and its fragments have already been studied by the present inventors (for example, Patent Documents 1 to 5). In addition, a method in which these adoptive immunotherapy and chemotherapy are used in combination has been studied (for example, Patent Document 6).

  In vaccine therapy, tumor antigen proteins and antigen peptides derived therefrom are formulated and used in admixture with adjuvants (incomplete Freund's adjuvant, CpG, etc.) for enhancing immunogenicity. In addition, derivatives (such as chimeric proteins with GM-CSF) for enhancing the immunogenicity of those antigens, those in which proteins or peptides are incorporated into antigen-presenting cells, DNA vaccines for expressing antigen genes, etc. They have been tested and are used alone or in admixture with adjuvants. All of the antigens used as the above vaccines are prepared and administered in vitro.

International Publication No. 03/080817 Pamphlet International Publication No. 2005/019450 Pamphlet JP 2007-061020 A International Publication No. 2007/020880 Pamphlet International Publication No. 2007/040105 Pamphlet International Publication No. 2008/143014 Pamphlet

  Anticancer agents exert their effects by killing cancer cells, but many kill normal cells as well as cancer cells. At this time, blood cells such as lymphocytes are killed, and the white blood cell count decreases. Therefore, anticancer drugs are administered in several cycles, assuming the metabolism of anticancer drugs in the patient's body, waiting for the recovery of these functions after a certain period of time, In some cases, sufficient killing effect cannot be obtained against cancer.

  On the other hand, treatment with anticancer agents leads to a decrease in immune function due to a decrease in white blood cells including lymphocytes, and increases the risk of infectious diseases. In addition, recovery of the immune response to cancer cells may be delayed.

  In addition, hyperthermia (hereinafter, also referred to as hyperthermia) is less burdensome on the patient and exerts its effect by killing cancer cells due to temperature changes caused by the warming treatment. On the other hand, there are cases where a sufficient killing effect cannot be obtained.

  An object of the present invention is to provide a cancer treatment method, a cancer therapeutic agent, and a system useful for cancer treatment that are effective for a living body.

  As a result of diligent efforts to solve the above-mentioned problems, the present inventors combined thermoimmune therapy with adoptive immunotherapy using a lymphocyte, particularly preferably a cell population containing naive T-like cells, The inventors have found that tumor growth in a living body can be extremely efficiently suppressed, and have completed the present invention.

That is, if the present invention is outlined,
[1] A method for treating cancer comprising the following steps (A) and (B):
(A) a step of heating a cancer lesion part and / or a site including the cancer lesion part of the test individual, and (B) lymph to the test individual during the step (A) and / or after the step (A). Administering a sphere,
[2] The method according to [1], wherein step (A) is a heating treatment at 38 to 43 ° C.
[3] The method according to [1], wherein the step (B) is performed 1 hour to 10 days after the execution of the step (A).
[4] The method according to [1], wherein the lymphocyte is a lymphocyte culture obtained by culturing a cell population containing lymphocytes or lymphocyte progenitor cells collected from a test individual,
[5] The method according to [4], wherein the lymphocyte is a lymphocyte culture obtained by culturing in the presence of an anti-CD3 antibody,
[6] The method according to [5], wherein the lymphocyte is a lymphocyte culture obtained by culturing in the presence of fibronectin, fibronectin fragment, or a mixture thereof.
[7] The method according to any one of [1] to [6], wherein the lymphocyte is a naive T-like cell population isolated from a lymphocyte culture.
[8] To be administered during and / or after the above-mentioned heating treatment to a test individual who is carrying out a heating treatment to a cancer lesion part and / or a site including the cancer lesion part, A cancer therapeutic agent containing lymphocytes as an active ingredient,
[9] The therapeutic agent according to [8], which is administered to a test individual who is performing warming treatment 1 hour to 10 days after the start of the treatment,
[10] The therapeutic agent according to [8], wherein the lymphocyte is a lymphocyte culture obtained by culturing a cell population containing lymphocytes or lymphocyte progenitor cells collected from a subject individual,
[11] The therapeutic agent according to [10], wherein the lymphocyte is a lymphocyte culture obtained by culturing in the presence of an anti-CD3 antibody,
[12] The therapeutic agent of [11], wherein the lymphocyte is a lymphocyte culture obtained by culturing in the presence of fibronectin, a fibronectin fragment, or a mixture thereof,
[13] The therapeutic agent according to any one of [8] to [12], wherein the lymphocyte is a naive T-like cell population isolated from a lymphocyte culture.
[14] Includes a lymphocyte culture obtained by culturing a cell population containing lymphocytes or lymphocyte progenitor cells, and a device capable of warming a cancer lesion and / or a site containing a cancer lesion Cancer treatment system,
About.

  The system includes a transfer container in which a naive T-like cell population is enclosed, a means for acquiring image data indicating the position of the tumor, a means for heating the periphery of the tumor based on the image data, and a naive T-like cell population. A cancer treatment system that includes a device with a means for transferring. In the present invention, the test individual includes a human patient holding a cancer lesion and a non-human animal.

  INDUSTRIAL APPLICABILITY According to the present invention, there are provided a cancer treatment method, a cancer treatment agent, and a cancer treatment system that activate cellular immunity against cancer and has a high therapeutic effect. Moreover, since this invention has little physical burden and stress to a test individual | organism | solid, it can be used also after surgery, radiation therapy, or chemotherapy. Furthermore, it can also be used when cancer has recurred after the therapy.

The present invention includes the following steps (A) and (B):
(A) a step of heating a cancer lesion part and / or a site including the cancer lesion part of the test individual, and (B) lymph to the test individual during the step (A) and / or after the step (A). Administering a sphere;
I will provide a. That is, the present invention includes thermotherapy in step (A) and adoptive immunotherapy in step (B).

  In the present specification, “warming treatment” refers to locally heating a cancer lesion or heating a site including a cancer lesion. The temperature for heating (the temperature reached by the cancer lesion by heating: the target temperature for heating) is preferably 38 to 44 ° C, preferably 41 to 43 ° C, more preferably 42.5 ° C. The time for heating is preferably 10 minutes to 2 hours, and preferably 30 to 50 minutes in one heating treatment.

  The method for heating is not particularly limited as long as the cancer lesion can be locally heated or the site including the cancer lesion can be heated. For example, hot water bath, heater, high frequency, microwave, or radio wave is used. The method used is mentioned. Moreover, it can be simply performed by using a commercially available local heating device (for example, Thermotron RF8 (manufactured by Yamamoto Vinita)). The cancer lesion may be heated from any direction, up, down, left, or right, or may be heated from all directions with respect to a local region or a site including the cancer lesion.

  In the present specification, “lymphocyte” means a cell of the immune system represented by T cell and B cell, and a cell population containing the cell. Although the present invention is not particularly limited, a culture obtained by culturing a cell population containing lymphocytes or lymphocyte progenitor cells (hereinafter also referred to as lymphocyte culture) is exemplified. In a preferred embodiment of the present invention, a lymphocyte culture containing naive T-like cells described below is used.

  The lymphocyte may be any of those derived from cells collected from the subject individual (autologous lymphocytes) and those derived from cells collected from a donor other than the subject individual (donor lymphocytes). For the former, those derived from the former test subject themselves are used. When collecting lymphocytes from the test individual, the timing may be either before or after step (A).

  In the present specification, the “naive T-like cell population” means a naive T cell and / or a naive T cell surface antigen marker CD45RA, CD62L, CCR7, CD27, CD28 and the like (hereinafter referred to as naive T-like cell population). A cell population containing a high ratio of cells).

  The cell population containing a high amount of the naive T-like cell population may be a culture prepared by an artificial cell culture operation. Lymphocytes can be artificially expanded by culturing in the presence of anti-CD3 antibodies. It is also known that lymphocyte cultures obtained by culturing fibronectin, fibronectin fragments, or mixtures thereof in combination with anti-CD3 antibodies are rich in naive T-like cells. Therefore, the use of the lymphocyte culture obtained by the method is exemplified as one preferred embodiment of the present invention. Although the present invention is not particularly limited, the lymphocyte used in the present invention has a fibronectin fragment, preferably a site selected from a VLA-4 binding region, a VLA-5 binding region, and a heparin binding region. Recombinant fibronectin fragments are used. In a particularly preferred embodiment of the present invention, lymphocytes or lymphocytes in a medium containing IL-2 in the presence of a recombinant fibronectin fragment (eg, CH-296 described below) and an anti-CD3 antibody (eg, OKT-3). By culturing a cell population containing progenitor cells, a lymphocyte culture suitable for use in the present invention is prepared. The culture method is also described in Patent Documents 1 to 3. The cell population containing the lymphocytes or lymphocyte progenitor cells is not particularly limited to the present invention, as long as it contains CD4 positive or CD8 positive T cells and their progenitor cells, For example, peripheral blood mononuclear cells (PBMC), umbilical cord blood mononuclear cells, bone marrow cells, spleen cells, hematopoietic stem cells and the like can be used. For culture, any of those collected from a living body can be used as they are or in a cryopreserved state.

  The anti-CD3 antibody and fibronectin fragment are dissolved in the medium so that they coexist, and an appropriate solid phase, for example, a container (petri dish, flask, bag, etc.) used for culture, or a cell such as a bead, membrane, slide glass, etc. You may fix | immobilize and use for the culture | cultivation support | carrier. The carrier is used by immersing it in a culture solution in cell culture equipment during cell culture. The immobilization of the component can be performed by the same method as the immobilization of the fibronectin fragment described in WO 97/18318 pamphlet and WO 00/09168 pamphlet.

The culture may be performed under known lymphocyte culture conditions except for the use of anti-CD3 antibodies and fibronectin fragments. For example, it is possible to 37 ° C., and cultured under conditions, such as 5% CO _2. In addition, a fresh medium can be added to the cell culture medium for dilution at an appropriate time interval, the medium can be exchanged for a fresh one, or the cell culture equipment can be exchanged. The culture period is usually about 2 to 15 days. The culture medium used and other components used at the same time can be appropriately set depending on the type of lymphocyte to be produced, as will be described later. Serum and plasma can also be added to the medium. The amount added to these media is not particularly limited, but is 0 to 20% by volume, preferably 0 to 5% by volume. The origin of serum or plasma may be either self (meaning that the origin is the same as the cell being cultured) or non-self (meaning that the origin is different from the cell being cultured). Things are used.

  Naïve T-like cell populations isolated or isolated from lymphocyte cultures can also be used in the present invention. The means for obtaining a naive T-like cell population from the culture obtained by the above-described culture method is not particularly limited. For example, the naive T-cell surface antigen marker is used as an index for separation or simple separation by a known method. Can be separated. For example, a cell population rich in naive T-like cells can be prepared by using a substance that specifically binds to the surface antigen marker, such as an anti-CD62L antibody.

  Furthermore, the “lymphocyte” used in the present invention may be one into which a foreign gene has been introduced. The term “foreign gene” means a gene that is artificially introduced into a naive T-like cell population to be transfected, and includes genes derived from the same species as the naive T-like cell population to be introduced. . The foreign gene is not particularly limited. For example, a gene encoding a protein (for example, an enzyme, a cytokine, or a receptor), an antisense nucleic acid, siRNA (small interfering RNA), or a ribozyme can be used. . Moreover, you may introduce | transduce simultaneously the suitable marker gene which enables selection of the cell by which gene introduction was carried out. For example, after introducing a gene that encodes an enzyme related to resistance to an anticancer drug to impart drug resistance to lymphocytes, or to introduce a gene that confers sensitivity to a specific drug and transplanted to a living body Lymphocytes can be removed by administration of the drug. Gene transfer can be performed by a known method, for example, a method using a viral vector.

  In the present invention, the body of the test individual after step (A) contains a large amount of free cancer antigens from killed cancer cells in the blood, and these are contained in antigen-presenting cells such as macrophages and dendritic cells. It is eaten and a state in which a large amount of cancer antigen is presented, and a CTL having cancer antigen-specific cytotoxic activity is easily induced. Moreover, it is expected that the immunosuppressed state of the cancer patient is canceled by the step (A). Therefore, by administering the naive T-like cell population in the step (B), the cells come into contact with the cancer antigen in the test individual released from the immunosuppressed state, and specifically kill the cancer cells of the test individual. There is the advantage of being guided by a CTL with the ability to do. Furthermore, the naïve T-like cell population can survive for a long time in the subject individual.

  (B) A process is rapidly implemented after (A) process is implemented with respect to a test individual | organism | solid. “Implemented promptly” as used herein refers to a range in which a desired effect can be obtained by administering lymphocytes in step (B), with an appropriate interval during and / or after step (A). (B) Implementing a process is included. For example, the interval between step (A) and step (B) is, for example, 1 hour to 10 days, preferably 3 hours to 8 days, more preferably 3 hours to 24 hours after the execution of step (A). Moreover, what is necessary is just to implement (B) process after (A) process start, when implementing (B) process in (A) process. Moreover, as a preferred embodiment of the present invention, a method for treating cancer in which the combination of both steps (A) and (B) is repeated a plurality of times is exemplified. For example, peripheral blood mononuclear cells are collected from a test individual before the implementation of step (A), and the peripheral blood mononuclear cells are used as a material to produce a lymphocyte culture by a known culture method as described above. A naive T-like cell population is separated or isolated from the culture, and step (B) is performed at an appropriate time using the cell population. Such a course including both steps (A) and (B) can be performed a plurality of times.

  Further, the step (A) may be a single treatment or may be repeated a plurality of times. The number of treatments and conditions are determined in consideration of, for example, damage to the test individual. For example, the step (A) can be performed for 40 minutes to 50 minutes a day, this can be performed for 6 days, and the process can proceed to the next step (B). Here, all the temperatures to be heated may be the same temperature, or may be appropriately changed within an effective range.

In the step (B), the dose of lymphocytes administered to the test individual and various conditions thereof can be set according to the immune state. Although this invention is not specifically limited, For example, per adult day, it is preferably 1 × 10 ⁵ to 1 × 10 ¹² cells / day, more preferably 5 × 10 ^{5 to} 5 × 10 ¹¹ cells / day. More preferably, 1 × 10 ⁶ to 1 × 10 ¹¹ cells / day are exemplified. Usually, lymphocytes are administered to veins, arteries, subcutaneous, intraperitoneal, etc. by injection or infusion.

  The cancer to which the treatment method of the present invention is applied is not particularly limited. For example, solid cancers such as esophageal cancer, lung cancer, myeloma, ovarian cancer, head and neck cancer, malignant melanoma, stomach cancer, colon cancer, rectal cancer, pancreatic cancer, biliary tract cancer, hepatocellular carcinoma Is exemplified. The treatment method of the present invention can also be applied after excision of tumor tissue.

  According to the present invention, cancer thermotherapy using lymphocytes is provided, particularly for cancers such as stomach cancer, colon cancer, rectal cancer, pancreatic cancer, biliary tract cancer, and hepatocellular carcinoma. The method of the present invention combines thermotherapy and lymphocyte administration as adoptive immunotherapy. Cancer is killed by shrinking cancer cells by heating, which is a physical cancer treatment, and cancer immunity is strengthened by administering lymphocytes. Furthermore, one effect of hyperthermia enhances general immunity.

  Administered lymphocytes come into contact with tumor antigens released by the destruction of cancer cells by heating, and tumor antigens presented to macrophages and dendritic cells that have high viability against anticancer drugs This induces high specific cytotoxicity against the cancer to be treated.

  Next, the cancer therapeutic agent used for the cancer treatment method of the present invention will be described. The therapeutic agent of the present invention is characterized by containing lymphocytes as its active ingredient. In addition, a cancer therapeutic agent to be administered to a test individual who is performing heating during or after heating treatment to a cancer lesion and / or a site containing the cancer lesion is there. Although the present invention is not particularly limited, for example, a preparation containing lymphocytes to which instructions for use in the method for treating cancer of the present invention are attached is included in the present invention.

  The lymphocytes that are active ingredients of the therapeutic agent of the present invention include lymphocytes administered to the test individual in the step (B) in the cancer treatment method of the present invention, particularly preferably a naive T-like cell population. Is mentioned. The material of the lymphocyte may be any of autologous lymphocytes derived from the subject individual, donor lymphocytes collected from a donor other than the subject individual, and from a culture obtained by subjecting to artificial cell culture operations. As described above, it may be separated or isolated.

  The therapeutic agent of the present invention can be prepared as an infusion or injection by mixing lymphocytes as an active ingredient with known organic or inorganic carriers, excipients, stabilizers and the like suitable for parenteral administration. There is no particular limitation on the administration method, and the same means as for known pharmaceuticals containing cells, for example, intravenous administration by injection or infusion can be used.

  Furthermore, the present invention provides a cancer therapeutic system comprising the therapeutic agent of the present invention and a device capable of warming a cancer lesion part and / or a site containing the cancer lesion part. As another embodiment, the therapeutic agent of the present invention, that is, the reagents used for the preparation of the naïve T-like cell population, and a cancer lesion and / or a device capable of warming a site containing the cancer lesion A cancer treatment system consisting of Examples of reagents used for the preparation of naive T-like cell populations include anti-CD3 antibodies, fibronectin fragments, IL-2, and lymphocyte culture media. A therapeutic system including a cell culture container (such as a culture container coated with an anti-CD3 antibody and a fibronectin fragment) in which a necessary amount of these reagents is housed can be provided. Further, the system includes a transfer container in which a naive T-like cell population is enclosed, a means for acquiring image data indicating the position of the tumor, a means for heating the periphery of the tumor based on the image data, and a naive T-like cell population Cancer treatment system including a device equipped with means for transferring

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not limited to these description at all.

Example 1 Examination of effects of combined use of transfected naive T-like cells and hyperthermia using mouse syngeneic tumor model (1) Preparation of spleen lymphocytes from tumor-bearing mice The right groin of 6-week-old female C57BL / 6 mice Was shaved about 9 square centimeters under anesthesia, and 0.1 mL of hB16F10, which is a highly metastatic melanoma suspended in RPMI 1640 medium (manufactured by Sigma) at 4 × 10 ⁶ cells / mL, was inoculated subcutaneously. . Six days later, the spleen was removed and ground in a RPMI 1640 medium using a glass slide, and the ground spleen was collected in a tube. Thereafter, spleen cells filled up to 45 mL with the same medium were allowed to stand on ice for 5 minutes and then transferred to a new tube through a 40 μm cell strainer (Becton Dickinson). The cell suspension thus obtained was centrifuged to remove the supernatant, and the cells precipitated as a hemolysis operation were treated with ACK buffer (0.15 M NH ₄ Cl, 0.01 M KHCO ₃ , 0.01 mM Na ₂ EDTA, pH 7). .4) Suspended in 2 mL. The cell suspension was further suspended by adding 2 mL of ACK buffer, and then RPMI1640 medium was added so that the cell suspension became 50 mL. The resulting cell suspension was centrifuged and the supernatant was removed. The cells were suspended in 10 mL of RPMI 1640 medium and transferred to a new tube through a cell strainer. RPMI1640 medium was added to the cell suspension to a volume of 40 mL, and the cells were collected by centrifugation. Cells were suspended in a mixture of equal amounts of RPMI 1640 medium and CP-1 (Kyokuto Pharmaceutical Co., Ltd.) containing 8% human serum albumin (HSA, formulation name; Buminate: manufactured by Baxter), and liquid nitrogen until used. Saved in.

(2) Immobilization of anti-mouse CD3 antibody and human CH-296 fragment Anti-mouse CD3 antibody and human CH-296 fragment [RetroNectin (registered trademark), manufactured by Takara Bio Inc .: And described as CH-296]. That is, an ACD-A solution (Terumo) containing anti-mouse CD3 antibody (final concentration 5 μg / mL, R & D Systems) was added to a 12-well cell culture plate (Corning) at 800 μL / well at 4 ° C. Incubated overnight. Next, CH-296 was added to each well to a final concentration of 5 μg / mL, and further incubated at room temperature for 5 hours. Immediately before use, the ACD-A solution containing antibody / CH-296 was removed by suction from the culture equipment, and each well was washed twice with Dulbecco's PBS (manufactured by Nissui Pharmaceutical Co., Ltd., hereinafter referred to as DPBS) and once with RPMI 1640 medium. The test was conducted.

(3) Purification of spleen lymphocytes with nylon fiber In order to increase the purity of the spleen lymphocytes prepared in Example 1- (1), purification was performed using nylon fibers. A column was prepared by filling a 20 mL syringe (manufactured by Terumo) with 1.5 g of nylon fiber (manufactured by Wako Pure Chemical Industries), equilibrated with DPBS, and sterilized at 121 ° C. for 20 minutes. This column was equilibrated with RPMI1640 medium containing 10% fetal bovine serum (Invitrogen, hereinafter referred to as FBS), and incubated at 37 ° C. for 1 hour in a 5% CO ₂ incubator. The spleen lymphocytes prepared in Example 1- (2) were suspended in 2-3 mL of RPMI 1640 medium containing 10% FBS so as not to exceed 5 × 10 ⁸ cells, applied to the column, and then 37% in a 5% CO ₂ incubator. Incubated for an additional hour at 0 ° C. The eluted cells were collected by adding 15-20 mL of 10% FBS-containing RPMI 1640 medium, which had been kept at 37 ° C., to the column.

(4) Expansion culture of mouse T cell population 10% FBS, 0.1 mM NEAA mixture (manufactured by Cambrex), 1 mM sodium pyruvate (manufactured by Cambrex), 50 μM 2-mercaptoethanol (manufactured by Invitrogen), containing 0.2% HSA The lymphocytes prepared in Example 1- (3) were suspended in GT-T503 medium (manufactured by Takara Bio Inc.) (hereinafter referred to as culture medium) so as to be 3 × 10 ⁶ cells / mL. The culture medium was added at 1.9 mL / well to the anti-mouse CD3 antibody and human CH-296 immobilized plate prepared in Example 1- (2), and the above cell suspension was added at 0.5 mL / well. After addition, the cells were cultured at 37 ° C. in a 5% CO ₂ incubator (culture day 0). On the third day of culture, the cell suspension is diluted with a culture medium to 3.6 × 10 ⁵ cells / mL, and added to a new 225 cm ² cell culture flask (Corning) with nothing immobilized. The whole amount was transferred. At this time, mouse IL-2 (manufactured by R & D Systems) was added to a final concentration of 100 U / mL, and mouse IL-7 (manufactured by R & D Systems) was added to a final concentration of 10 ng / mL. Cells expanded and cultured on the 6th day of culture were collected and subjected to the test in the following syngeneic tumor model.

(5) Separation of naive T-like cells and effector T-like cells of the expanded mouse T cell population The cells obtained in Example 1- (4) were collected, and the required amount was collected, and then 500 × g, room temperature And centrifuged for 5 minutes to remove the supernatant. The obtained cells were suspended in DPBS (hereinafter referred to as 0.5% BSA / DPBS) containing 0.5% BSA and 2 mM disodium ethylenediaminetetraacetate so as to be 1.11 × 10 ⁸ cells / mL. did. 10 μL of CD62L (L-selectin) microbead (mouse) per 1 × 10 ⁷ cells (mouse) (Milteny Biotech) was added to the cell solution, and 15 minutes with occasional stirring at 4 ° C. in the dark. Incubated. Next, 1 mL of 0.5% BSA / DPBS per 1 × 10 ⁷ cells was added to the cell solution, centrifuged at 500 × g for 5 minutes at room temperature to remove the supernatant, and then 0.5 mL per 1 × 10 ⁸ cells was used. 0.5% BSA / DPBS was added, sufficiently suspended, and allowed to stand on ice to obtain a CD62L microbead-labeled cell solution. Subsequently, an LS column (manufactured by Miltenyi Biotech, hereinafter referred to as a separation column) was installed in a VarioMACS ^™ separator (made by Miltenyi Biotech, hereinafter referred to as a separation device), and 3 mL of 0.5% BSA / DPBS. Rinse with. After the CD62L microbead-labeled cell solution was added to the column and eluted, further rinsed with 9 mL of 0.5% BSA / DPBS, and the CD62L ^- cells obtained by collecting the eluted fraction were designated as effector T-like cells. did. The column was removed from the separation apparatus, 5 mL of buffer was added, and the CD62L ⁺ cells obtained by extruding and collecting with a plunger attached to the separation column were used as naive T-like cells.

(6) Hyperthermia treatment and transfer of T cell population in C57BL / 6-hB16F10 syngeneic tumor model The right buttock of a 6-week-old female C57BL / 6 mouse was shaved under anesthesia to about 9 square centimeters Was inoculated with 0.1 mL of hB16F10 suspended in RPMI1640 medium to 4 × 10 ⁶ cells / mL. Then, the group setting is performed as follows, the non-treatment group is A group, the thermotherapy group is B group, the thermotherapy and naive T-like cell combination therapy group is C group, and the thermotherapy and effector T-like cell combination therapy group is D group. It was. Hyperthermia was performed 3 days and 4 days after tumor inoculation except for group A. For the thermal treatment, a thermal apparatus that radiates far infrared rays from heating elements installed at the top and bottom was used. A wire mesh cage containing mice was placed in a thermal apparatus, and after preirradiation at 60 ° C. for 10 minutes, thermal treatment was subsequently performed at 43 ° C. for 50 minutes. On day 6 and day 10 after tumor inoculation, each mouse T cell prepared in Example 1- (5) was prepared in RPMI 1640 medium to 3.5 × 10 ⁸ cells / mL and 2 × 10 ⁸ cells / mL, respectively. Naive T-like cells and effector T-like cells were administered to each individual of group C and group D from the tail vein. Further, 0.2 mL of physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.) was administered to each individual of Group A and Group B from the tail vein.

(7) Evaluation of anti-tumor activity after hyperthermia treatment and T cell administration in C57BL / 6-hB16F10 syngeneic tumor model The tumor size on day 17 after tumor inoculation in each individual was measured using electronic calipers. The results are shown in Table 1. The numbers in the table indicate the average value of each group N = 4-5. The tumor size was calculated using the following formula.
Tumor size = R1 × R1 × R2 × 0.5 (R1 is short diameter R2 is long diameter).

Moreover, the tumor weight on the 17th day after tumor inoculation in each individual was measured. The results are shown in Table 2. In addition, the number in a table | surface shows the average value of each group N = 4-5.

  As is clear from Tables 1 and 2, tumor size and weight were compared in the syngeneic tumor model by combining hyperthermia and naive T-like cells in combination with hyperthermia and effector T-like cells. Both were small and resulted in high antitumor activity. From this, the tumor growth suppression effect by the combined effect with a hyperthermia was shown by combining administration of a hyperthermia treatment, expansion culture, and administration of the isolated naive T-like cell population. In addition, it was confirmed that adoptive immunotherapy using a naive T-like cell population is extremely effective for the treatment of cancer in combination with hyperthermia.

  INDUSTRIAL APPLICABILITY According to the present invention, a cancer treatment method, a cancer therapeutic agent, and a system effective for cancer treatment having high therapeutic effects are provided. This method is extremely useful in the medical field because it can be used even after surgical treatment, radiation therapy, or chemotherapy because there is little physical burden and stress on the test individual.

Claims (14)

A method for treating cancer comprising the following steps (A) and (B):
(A) a step of heating a cancer lesion part and / or a site including the cancer lesion part of the test individual, and (B) lymph to the test individual during the step (A) and / or after the step (A). Administering a sphere.   The method according to claim 1, wherein the step (A) is a heating treatment at 38 to 43 ° C.   The method according to claim 1, wherein the step (B) is carried out 1 hour to 10 days after the step (A).   The method according to claim 1, wherein the lymphocyte is a lymphocyte culture obtained by culturing a cell population containing lymphocytes or lymphocyte progenitor cells collected from a subject individual.   The method according to claim 4, wherein the lymphocyte is a lymphocyte culture obtained by culturing in the presence of an anti-CD3 antibody.   The method according to claim 5, wherein the lymphocyte is a lymphocyte culture obtained by culturing in the presence of fibronectin, a fragment of fibronectin, or a mixture thereof.   The method according to any one of claims 1 to 6, wherein the lymphocyte is a naive T-like cell population isolated from a lymphocyte culture.   Lymphocytes to be administered during and / or after the above-mentioned warming treatment to a test subject who is performing warming treatment on the cancer lesion and / or the site containing the cancerous lesion A cancer therapeutic agent as an active ingredient.   The therapeutic agent of Claim 8 for administering to the test individual | organism | solid which is implementing the warming treatment 1 hour-10 days after the said treatment start.   The therapeutic agent according to claim 8, wherein the lymphocyte is a lymphocyte culture obtained by culturing a cell population containing lymphocytes or lymphocyte progenitor cells collected from a test individual.   The therapeutic agent according to claim 10, wherein the lymphocyte is a lymphocyte culture obtained by culturing in the presence of an anti-CD3 antibody.   The therapeutic agent according to claim 11, wherein the lymphocyte is a lymphocyte culture obtained by culturing in the presence of fibronectin, a fragment of fibronectin, or a mixture thereof.   The therapeutic agent according to any one of claims 8 to 12, wherein the lymphocyte is a naive T-like cell population separated from a lymphocyte culture.   Cancer including a lymphocyte culture obtained by culturing a cell population containing lymphocytes or lymphocyte progenitor cells, and a device capable of heating a cancer lesion and / or a site containing the cancer lesion Treatment system.