HTLV-I specific CTL activator

Abstract

The present invention addresses the problem of providing an HTLV-I-specific CTL activator, and a method for producing the same.　In the present invention, peripheral blood mononuclear cells harvested from a subject infected with human T cell leukemia virus type I (HTLV-1) are cultured in a medium for animal cell culture, and the peripheral blood mononuclear cells obtained thereby are treated with an anti-cancer agent. The peripheral blood mononuclear cells thus obtained are used as an HTLV-I-specific CTL activator.

Description

[Technical field]

[0001] The present invention relates to an HTLV-I-specific CTL activator and a method for producing the same. [Background technology]

[0002] Adult T cell leukemia (ATL) is a malignant lymphoid tumor caused by human T cell leukemia virus type-1 (HTLV-1), and occurs in approximately 5% of HTLV-1-infected individuals. Among ATL, the acute and lymphomatous types (both types are collectively called "aggressive ATL") have a poor prognosis due to their rapid onset and high recurrence rate. The 3-year survival rate of aggressive ATL is 24% with chemotherapy and 33% with hematopoietic stem cell transplantation, and the median overall survival with new drugs such as mogamulizumab and lenalidomide, which have recently become available for clinical use in Japan, is also less than 2 years. Therefore, the establishment of a safe and effective treatment method for aggressive ATL is required.

[0003] On the other hand, in the case of ATL, the smoldering and chronic types (both types are collectively called indolent ATL) progress more slowly than aggressive ATL, but many cases worsen within a few years, and the long-term prognosis is still poor. However, it has been pointed out that chemotherapy for indolent ATL accelerates the progression of the disease, so the clinical principle is to observe the patient without treatment until signs of transformation to aggressive ATL (such as an increase in the concentration of lactate dehydrogenase (LDH) in the blood) appear and the patient actually transforms into an aggressive ATL. Although early diagnosis of ATL is possible, there is nothing that can be done for indolent ATL until the disease progresses, and there is a need to establish a safe, effective treatment method for indolent ATL that can be applied early.

[0004] In addition to ATL, HTLV-1 is also known as the causative virus of HTLV-I-associated myelopathy (HAM), tropical spastic paraparesis (TSP), and HTLV-1 uveitis (HU).

[0005] Furthermore, since 2010, the recommended level of HTLV-1 antibody testing in prenatal checkups has been raised, and those who test positive for antibodies are now notified of their infection. This is to reduce the frequency of vertical transmission of HTLV-1 to infants through breast milk by restricting breastfeeding for those who test positive for antibodies. However, even if infants are fed formula, a few percent of infants will still be infected with HTLV-1. In addition, since there are no established methods to reduce the risk of developing HTLV-1 infection or prevent it, the reality is that only counseling is provided to infected pregnant women themselves, and the psychological and social problems of being notified of the infection are immeasurable. This problem is also true for those who are found to be infected with HTLV-1 when donating blood. Therefore, it is urgent to establish a method to prevent the onset of the disease in those infected with HTLV-1.

[0006] The present inventors have confirmed that HTLV-1 Tax-specific CTL activity, which is responsible for the antitumor effect, is reduced in ATL patients, and have been conducting research into methods for activating HTLV-1 Tax-specific CTL. For example, Patent Document 1 discloses a peptide of the epitope site of Tax-specific CTL that can activate HTLV-1 Tax-specific CTL, and a vaccine for inducing immune response that contains such a peptide. The present inventors have actually developed a Tax peptide-pulsed dendritic cell vaccine that uses such a peptide as an antigen. In a preliminary clinical study of such dendritic cell vaccine therapy, two out of three cases showed good results of survival for more than four years, suggesting the effectiveness of immunotherapy (Non-Patent Document 1). Such immunotherapy has fewer side effects than chemotherapy, and is expected to be applied to ATL patients at an earlier stage in the future. However, at present, the types of epitopes that can be used are limited, and therefore the patients eligible for this treatment are limited to those with the human leukocyte antigen (HLA) types HLA-A0201, HLA-A2402, or HLA-A1101. In Japan, ATL patients with these HLA types account for approximately 65% ​​of all ATL patients, and such peptide vaccines cannot be used for the remaining approximately 35% of ATL patients. Therefore, there is a need to establish a more universal treatment method that can be applied to more ATL patients.

[0007] T cells mainly include CD4 positive T cells and CD8 positive T cells. CD4 positive T cells include CD4 positive helper T cells, and CD8 positive T cells include CD8 positive cytotoxic T cells (CD8+CTL). In general, in order to activate the response of CD8+CTL, antigen presenting cells (APCs) that have phagocytosed antigens are activated, and they must express costimulatory molecules (CD86, etc.) and produce cytokines such as IL-12, while presenting antigen peptides not only to MHC class II molecules but also to MHC class I molecules (i.e., "antigen cross-presentation"). However, when a protein antigen is immunized as an antigen, the protein antigen is mainly presented to MHC class II molecules and is rarely presented to MHC class I molecules. Therefore, when activating the CD8+CTL response, synthetic oligopeptides capable of binding to MHC class I are often used as antigens, but in this case, a separate adjuvant is required to activate the antigen presenting cells. Currently, only a limited number of adjuvants are available for use in humans, and their efficiency in inducing CD8+ CTL responses is uncertain.

[0008] Incidentally, the present inventors have found that a rat model orally infected with HTLV-1 exhibits immune tolerance to HTLV-1-specific T cells, that the amount of HTLV-1 provirus in the aforementioned rat model is inversely correlated with the HTLV-1-specific T cell response, and that the aforementioned rat model was inoculated with syngeneic HTLV-1-infected cells to restore the HTLV-1-specific T cell response (Non-Patent Documents 2 and 3). However, Non-Patent Documents 2 and 3 do not distinguish whether the T cell response is a response of CD4-positive T cells or a response of CD8-positive T cells, and do not disclose that inoculation of HTLV-1-infected cells activates the response of CD8-positive T cells. In addition, the present inventors cultured peripheral blood mononuclear cells (PBMCs) derived from ATL patients and confirmed Tax expression in the peripheral blood lymphocytes, and found that Tax expression was induced in about half of the ATL patients, and that costimulatory molecules such as CD80 were also induced (Non-Patent Document 4). In addition, Non-Patent Document 4 describes that when peripheral blood lymphocytes from an ATL patient were cultured and then fixed in formalin and administered to healthy rats, an HTLV-1-specific T cell response was induced in a CD4-predominant manner.

[0009] However, it was not previously known that PBMCs obtained by culturing PBMCs collected from HTLV-1-infected individuals, such as ATL patients, and treating the PBMCs with anticancer drugs can be administered to a subject to efficiently activate HTLV-1-specific CD8+ CTLs in the subject. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2006 / 035681 Brochure [Non-patent literature]

[0011] [Non-Patent Document 1] Br J Haematol, 169: 356-367, 2015 [Non-Patent Document 2] J Virol 77: 2956-2963. 2003 [Non-Patent Document 3] J Virol 80:7375-7381. 2006 [Non-Patent Document 4] Int J Cancer 114:257-267. 2005 Summary of the Invention [Problem to be solved by the invention]

[0012] An object of the present invention is to provide an HTLV-I-specific CTL activator and a method for producing the same. [Means for solving the problem]

[0013] In order to solve the above problems, the present inventors have intensively conducted the following research. First, CD8-positive Tax-specific CTLs and antigen-presenting cells with MHC-I matching those of the CTLs were prepared. Next, HTLV-1-infected T cells with MHC-I not matching those of both cells (CD8-positive Tax-specific CTLs and antigen-presenting cells) were cultured and then treated with an anticancer drug. The HTLV-1-infected T cells treated with the anticancer drug after the above-mentioned culture (hereinafter also referred to as "cultured infected T cells treated with an anticancer drug") were co-cultured with the above-mentioned antigen-presenting cells, and further fixed with formalin, and then co-cultured with the above-mentioned CTLs, whereby IFN-γ was produced. This showed that during the co-culture, the antigen-presenting cells took in the "cultured infected T cells treated with an anticancer drug" and presented the Tax antigen derived from HTLV-1 on MHC-I, and the CTLs recognized the Tax antigen and produced IFN-γ. In addition, when "cultured infected T cells treated with anticancer drugs" were co-cultured with immature dendritic cells derived from monocytes of healthy subjects, the expression of CD86 (a type of costimulatory molecule known to be necessary for activating the response of CD8 positive CTLs) from the immature dendritic cells was significantly increased. These results indicate that "cultured infected T cells treated with anticancer drugs" can be used as an HTLV-I-specific CTL activator.

[0014] In addition, it was shown that the addition of a histone deacetylase inhibitor to the culture medium when culturing HTLV-1-infected T cells increases IL-12 production from antigen-presenting cells (preferably dendritic cells) and also improves cross-presentation efficiency.

[0015] In addition, it was shown that peripheral blood mononuclear cells obtained by removing CD8 positive cells from peripheral blood mononuclear cells taken from subjects infected with HTLV-1 and culturing the cells, and then treating the cells with anticancer drugs, showed a higher proportion of cells expressing Tax protein, an antigen derived from HTLV-1.

[0016] The present inventors have discovered these things and have completed the present invention.

[0017] That is, the present invention provides (1) An HTLV-I-specific CTL activator for administration to a subject infected with human T-cell leukemia virus type I (HTLV-1), comprising peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from the subject in an animal cell culture medium and treating the peripheral blood mononuclear cells with an anticancer drug; or (2) The HTLV-I-specific CTL activator according to (1) above, wherein the peripheral blood mononuclear cells are obtained by culturing peripheral blood mononuclear cells collected from a subject infected with HTLV-1 for one day or more; (3) The HTLV-I-specific CTL activator according to (1) or (2) above, wherein the animal cell culture medium contains IL-2, IL-15, or both; (4) The HTLV-I-specific CTL activator according to any one of (1) to (3) above, wherein the animal cell culture medium contains a histone deacetylase inhibitor; (5) The HTLV-I-specific CTL activator according to any one of (1) to (4) above, wherein the peripheral blood mononuclear cells collected from a subject infected with HTLV-1 are peripheral blood mononuclear cells obtained by removing CD8 positive cells from peripheral blood mononuclear cells collected from a subject infected with HTLV-1; (6) The HTLV-I-specific CTL activator according to any one of (1) to (5) above, wherein the peripheral blood mononuclear cells collected from a subject infected with HTLV-1 are peripheral blood mononuclear cells activated with a CD3 antibody, or with both a CD3 antibody and a CD28 antibody, after removing CD8-positive cells from the peripheral blood mononuclear cells collected from the subject infected with HTLV-1; (7) The HTLV-I-specific CTL activator according to any one of (1) to (6) above, wherein the peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from a subject infected with HTLV-1 in an animal cell culture medium and treating the peripheral blood mononuclear cells with an anticancer drug are peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from a subject infected with HTLV-1 in an animal cell culture medium and treating the peripheral blood mononuclear cells with an anticancer drug, and then contacting the peripheral blood mononuclear cells with an antibody capable of binding to HTLV-1-infected cells; or (8)(a) culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type 1 (HTLV-1) in an animal cell culture medium; (b) treating the peripheral blood mononuclear cells obtained by the culture in the step (a) with an anticancer drug and then collecting the peripheral blood mononuclear cells; and (c) mixing the peripheral blood mononuclear cells collected in step (b) with a pharma- ceutically acceptable carrier to prepare a formulation; A method for producing an HTLV-I-specific CTL activator for administration to a subject, comprising the steps of: (9) The method for producing an HTLV-I-specific CTL activator according to (8) above, wherein the animal cell culture medium contains IL-2, IL-15 or both; or (10) The method for producing an HTLV-I-specific CTL activator according to (8) or (9) above, wherein the animal cell culture medium contains a histone deacetylase inhibitor; or (11) The method for producing an HTLV-I-specific CTL activator according to any one of (8) to (10) above, wherein the step (a) is a step of culturing peripheral blood mononuclear cells obtained by removing CD8 positive cells from peripheral blood mononuclear cells collected from a subject infected with HTLV-1 in an animal cell culture medium; or (12) The method for producing an HTLV-I-specific CTL activator according to any one of (8) to (11) above, wherein the step (a) is a step of removing CD8-positive cells from peripheral blood mononuclear cells collected from a subject infected with HTLV-1, and then culturing the peripheral blood mononuclear cells activated with a CD3 antibody or with both CD3 and CD28 antibodies in an animal cell culture medium; Regarding. Effect of the Invention

[0018] According to the present invention, it is possible to provide an HTLV-I-specific CTL activator and a method for producing the same. It is also expected that according to the present invention, it is possible to provide a relatively inexpensive and effective immunotherapy to many patients, regardless of the HLA type of the patient suffering from a disease caused by HTLV-1 infection. It is also expected that according to the present invention, it is possible to provide a therapy that can be applied in an early stage to smoldering or chronic ATL (indolent ATL), which is currently observed without treatment as a rule due to the lack of a safe and effective therapy. [Brief description of the drawings]

[0019] [Figure 1] Figure 1 shows the results of fixing / permeabilizing PBMCs from an acute ATL patient (aATL) before culture (Day 0) or after 3 days (Day 3) of culture, staining the intracellular Tax protein with a primary antibody (anti-Tax antibody or a control mouse anti-IgG antibody), and then detecting the HTLV-1 Tax protein in the cells stained with a secondary antibody (A488-labeled anti-mouse IgG). The left panel of Figure 1 shows the results on Day 0, and the right panel of Figure 1 shows the results on Day 3. [Diagram 2]Figure 2 shows the results of detecting intracellular Tax protein in PBMCs from an acute ATL patient (aATL), which were cultured for one day (day 1) either as is (Whole) or after removing CD8-positive cells (CD8(-)). The left panel of Figure 2 (Whole day 1) shows the results when aATL PBMCs were cultured for one day as is. The right panel of Figure 2 (CD8(-) day 1) shows the results when CD8-positive cells were removed from aATL PBMCs and the PBMCs were cultured for one day. [Diagram 3] Figure 3 shows the results of removing CD8-positive cells from PBMCs of a chronic ATL patient (cATL), stimulating them with CD3 and CD28 antibodies, and culturing the PBMCs for 24 days, after which intracellular Tax protein in the PBMCs was detected. [Figure 4] FIG. 4 shows the results of detecting intracellular Tax protein in aATL PBMC-derived cell line (ILT-A line) or cATL PBMC-derived cell line (ILT-B line) after culturing them in a medium containing SAHA (suberoylanilide hydroxamic acid) or DMSO (control) for 2 days. The lower left panel of FIG. 4 shows the results when the ILT-A line was cultured in a medium containing IL-15 and SAHA. The upper left panel of FIG. 4 shows the results when the ILT-A line was cultured in a medium containing IL-15 and DMSO. The lower right panel of FIG. 4 shows the results when the ILT-B line was cultured in a medium containing IL-2 and SAHA. The upper right panel of FIG. 4 shows the results when the ILT-B line was cultured in a medium containing IL-2 and DMSO. [Diagram 5] FIG. 5 shows an evaluation system constructed by the present inventors, which evaluates activation and cross-presentation of antigen-presenting cells by HTLV-1-infected cells. [Figure 6]FIG. 6 shows the results of measuring the IFN-γ concentration (pg / mL) in the culture supernatant using the evaluation system constructed by the present inventors. "ILT-MMC alone" in FIG. 6 represents the IFN-γ concentration in the co-culture supernatant with CTL when only MMC-treated ILT-A cells (HTLV-1-infected T cell line derived from an ATL patient) are cultured as the initial culture. "ILT-Formalin alone" in FIG. 6 represents the IFN-γ concentration in the co-culture supernatant with CTL when only formalin-treated ILT-A cells (HTLV-1-infected T cell line derived from an ATL patient) are cultured as the initial culture. "THP1 alone" in FIG. 6 represents the IFN-γ concentration in the co-culture supernatant with CTL when only THP1 cells, which are antigen-presenting cells, are cultured as the initial culture. "ILT-MMC+THP1" in Fig. 6: IFN-γ concentration in the supernatant of co-culture with CTL when MMC-treated ILT-A cells and THP1 cells (antigen-presenting cells) were cultured as the initial culture. "ILT-Formalin+THP1" in Fig. 6: IFN-γ concentration in the supernatant of co-culture with CTL when formalin-treated ILT-A cells and THP1 cells (antigen-presenting cells) were cultured as the initial culture. "None (CTL alone)" in Fig. 6: IFN-γ concentration when neither ILT-A cells nor THP1 cells (antigen-presenting cells) were cultured during the initial culture (i.e., when only Tc-M1, a CTL, was cultured). [Figure 7]FIG. 7 shows the results of contacting MMC-treated ILT-A cells with plasma from an ATL patient, a HAM / TSP patient, or a non-infected individual, co-culturing with THP1 cells (antigen-presenting cells), fixing with formalin, and then measuring the IFN-γ concentration (pg / mL) in the co-culture supernatant with CTL. "ILT alone" in FIG. 7 shows the results when MMC-treated ILT-A cells were used but THP1 cells were not used. "ILT+APC" in FIG. 7 shows the results when MMC-treated ILT-A cells and THP1 cells were used without plasma or antibody. "SN-1" and "SN-2" in FIG. 7 show the results when plasma from a non-infected individual was used in addition to MMC-treated ILT-A cells and THP1 cells. "ATL-1", "ATL-2", and "ATL-3" in FIG. 7 show the results when plasma from an ATL patient was used in addition to MMC-treated ILT-A cells and THP1 cells. "HAM-1," "HAM-2," and "HAM-3" in Fig. 7 show the results when plasma from a HAM / TSP patient was used in addition to MMC-treated ILT-A cells and THP1 cells. "Poteligeo" in Fig. 7 shows the results when mogamulizumab, an anti-CCR4 antibody, was used in addition to MMC-treated ILT-A cells and THP1 cells. "APC alone" in Fig. 7 shows the results when THP1 cells were used without using MMC-treated ILT-A cells. [Figure 8]FIG. 8 shows the results of co-culturing ILT-A cells treated with plasma from an HTLV-1-infected individual after MMC treatment with THP1 cells, fixing the cells with formalin, and measuring the IFN-γ concentration (pg / mL) in the co-culture supernatant with CTLs. "10(5)" in FIG. 8 shows the results when the above ILT-A cells and THP1 cells were co-cultured at a ratio of 1:1 (ratio of the number of cells). "5×10(4)" in FIG. 8 shows the results when the above ILT-A cells and THP1 cells were co-cultured at a ratio of 0.5:1 (ratio of the number of cells). "2.5×10(4)" in FIG. 8 shows the results when the above ILT-A cells and THP1 cells were co-cultured at a ratio of 0.25:1 (ratio of the number of cells). In addition, in Figure 8, the black bars ("MMC-ILT / (+)plasma") represent the results when "MMC-treated ILT-A cells" were contacted with plasma from an HTLV-1-infected individual, the gray bars ("MMC-ILT / (-)plasma") represent the results when "MMC-treated ILT-A cells" were contacted with plasma from a non-infected individual, and the open bars ("MMC-ILT") represent the results when "MMC-treated ILT-A cells" were not contacted with plasma from an HTLV-1-infected individual. [Figure 9]FIG. 9 shows the results of co-culture of antigen-presenting cells (THP1 cells) treated with a reverse transcriptase inhibitor (zidovudine: AZT) with ILT-A cells treated with MMC for 16 hours, fixing all the cells obtained with a formalin solution, and co-culture with HLA-A2-restricted CD8-positive Tax-specific CTL (Tc-M1) for 16 hours, and measuring the IFN-γ concentration (pg / mL) in the culture supernatant. "ILT 1×10(5)" in FIG. 9 shows the results when the above ILT-A cells and THP1 cells were co-cultured at a ratio of 1:1 (ratio of the number of cells). "ILT 3×10(4)" in FIG. 9 shows the results when the above ILT-A cells and THP1 cells were co-cultured at a ratio of 0.3:1 (ratio of the number of cells). "Medium" on the horizontal axis of FIG. 9 shows the results when ILT-A cells were not used. In addition, in Figure 9, the black bars ("THP1-AZT") represent the results when AZT was added to the medium and THP1 cells, etc. were cultured, the gray bars ("THP1") represent the results when AZT was not added to the medium, and the open bars ("Medium") represent the results when THP1 cells were not used. [Figure 10] FIG. 10 shows the results of measuring the expression intensities of CD83 and CD86 in the HLA-A2-negative dendritic cell fraction by a flow cytometer after co-culture of HLA-A2-negative healthy immature dendritic cells with MMC-treated HLA-A2-positive ILT-C strain for 24 hours and then staining with anti-HLA-A2 antibody and anti-human CD83 antibody or anti-human CD86 antibody. The gray results show the results of staining with a control antibody (mouse IgG1). FIG. 10, lower left panel: shows the results of measuring the expression of CD83 in cells obtained by co-culture of healthy immature dendritic cells with MMC-treated ILT-C strain. FIG. 10, upper left panel: shows the results of measuring the expression of CD83 in cells obtained by culturing healthy immature dendritic cells without using MMC-treated ILT-C strain. FIG. 10, lower right panel: shows the results of measuring the expression of CD86 in cells obtained by co-culture of healthy immature dendritic cells with MMC-treated ILT-C strain. Figure 10, upper right panel: shows the results of measuring CD86 expression in cells obtained by culturing immature dendritic cells from healthy donors without using the MMC-treated ILT-C line. [Figure 11]FIG. 11 shows the results of measuring the IL-12 concentration (pg / mL) in the culture supernatant of ILT-A or ILT-B cells cultured for 24 hours in a medium containing SAHA (a type of HDAC inhibitor) or a medium containing DMSO (control medium), treating the cells obtained with MMC, and then co-culturing with dendritic cells. "ILT-A SAHA" in FIG. 11 shows the results when cells obtained by culturing ILT-A cells in a medium containing SAHA and treating them with MMC were used. "ILT-A DMSO" in FIG. 11 shows the results when cells obtained by culturing ILT-A cells in a medium containing DMSO and treating them with MMC were used. "ILT-B SAHA" in FIG. 11 shows the results when cells obtained by culturing ILT-B cells in a medium containing SAHA and treating them with MMC were used. "ILT-B DMSO" in FIG. 11 shows the results when cells obtained by culturing ILT-B cells in a medium containing DMSO and treating them with MMC were used. The right side of each item shows the results when co-cultured with dendritic cells (black bars ("DC")), and the left side of each item shows the results when dendritic cells were not used ("Medium"). However, the actual results for "Medium" were all below the detection limit (nd). [Figure 12] FIG. 12 shows the results of measuring the IL-12 concentration (pg / mL) in the culture supernatant of ILT-B cells cultured for 24 hours in a medium containing VPA (a type of HDAC inhibitor) or a medium containing DMSO (control medium), treated with MMC, and then co-cultured with dendritic cells. "VPA" in FIG. 12 shows the results when cells obtained by culturing ILT-B cells in a VPA-containing medium and treating them with MMC were used. "DMSO" in FIG. 12 shows the results when cells obtained by culturing ILT-B cells in a DMSO-containing medium and treating them with MMC were used. Note that the right side of each item shows the results when co-cultured with dendritic cells (black bars ("DC")), and the left side of each item shows the results when dendritic cells were not used ("Medium"). However, the actual results for "Medium" were all below the detection limit (nd). [Figure 13]FIG. 13 shows the results of measuring the IFN-γ concentration (pg / mL) in the coculture supernatant with CTLs after the cells obtained by culturing ILT-A cells or ILT-B cells in a medium containing SAHA (a type of HDAC inhibitor) or a medium containing DMSO (control medium) for 24 hours were treated with MMC, cocultured with dendritic cells, and fixed with formalin. "ILT-A SAHA" in FIG. 13 shows the results when the cells obtained by culturing ILT-A cells in a medium containing SAHA and treating them with MMC were used. "ILT-A DMSO" in FIG. 13 shows the results when the cells obtained by culturing ILT-A cells in a medium containing DMSO and treating them with MMC were used. "ILT-B SAHA" in FIG. 13 shows the results when the cells obtained by culturing ILT-B cells in a medium containing SAHA and treating them with MMC were used. "ILT-B DMSO" in FIG. 13 shows the results when the cells obtained by culturing ILT-B cells in a medium containing DMSO and treating them with MMC were used. "Medium" on the horizontal axis of Figure 13 represents the results when only medium or dendritic cells (DC) were co-cultured with CTLs without using ILT cells. Note that the right side of each item represents the results when co-cultured with dendritic cells (solid black bars ("DC")), and the left side of each item represents the results when dendritic cells were not used (open bars ("Medium"). Note that "nd" represents below the detection limit. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention relates to [1] An HTLV-I-specific CTL activator (or a composition for activating HTLV-I-specific CTL) for administration to a subject infected with human T-cell leukemia virus type I (HTLV-1), comprising peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from the subject in an animal cell culture medium and treating the peripheral blood mononuclear cells with an anticancer drug; or [2] (a) culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type 1 (HTLV-1) in an animal cell culture medium; (b) treating the peripheral blood mononuclear cells obtained by the culture in the step (a) with an anticancer drug and then collecting the peripheral blood mononuclear cells; and (c) mixing the peripheral blood mononuclear cells collected in step (b) with a pharma- ceutically acceptable carrier to prepare a formulation; a method for producing an HTLV-I-specific CTL activator (or an HTLV-I-specific CTL activator or a composition for activating an HTLV-I-specific CTL), for administration to a subject, comprising the steps of: The present invention includes the following embodiments. In the present specification, the HTLV-I-specific CTL activator can be used as a composition for activating HTLV-I-specific CTL.

[0021] In addition, the present invention provides, as another aspect, [3] (a) culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type 1 (HTLV-1) in an animal cell culture medium; (b) treating the peripheral blood mononuclear cells obtained by the culture in the step (a) with an anticancer drug and then collecting the peripheral blood mononuclear cells; and (c) administering the peripheral blood mononuclear cells collected in the step (b) (or an HTLV-I-specific CTL activator or composition for activating the HTLV-I-specific CTLs containing the peripheral blood mononuclear cells) to the subject; A method for preventing or treating a disease caused by HTLV-1 infection in a subject, comprising the steps of: [4] (a) culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type 1 (HTLV-1) in an animal cell culture medium; (b) treating the peripheral blood mononuclear cells obtained by the culture in the step (a) with an anticancer drug and then collecting the peripheral blood mononuclear cells; and (c) administering the peripheral blood mononuclear cells collected in the step (b) (or an HTLV-I-specific CTL activator or composition for activating the HTLV-I-specific CTLs containing the peripheral blood mononuclear cells) to the subject; A method for activating anti-HTLV-1 specific CTL in a subject, comprising the steps of: [5] "Peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type I (HTLV-1) in an animal cell culture medium and treating the resulting peripheral blood mononuclear cells with an anticancer drug" for use in the prevention or treatment of diseases caused by HTLV-1 infection; or [6] Use of "peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type I (HTLV-1) in an animal cell culture medium, and treating the resulting peripheral blood mononuclear cells with an anticancer drug" for the manufacture of a preventive or therapeutic agent (or a pharmaceutical composition for the prevention or treatment) for a disease caused by HTLV-1 infection; The present invention also includes the following aspects:

[0022] (peripheral blood mononuclear cells) The "peripheral blood mononuclear cells" in the present invention are not particularly limited, so long as they are peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells (hereinafter also referred to as "cultured infected PBMCs") obtained by collecting peripheral blood mononuclear cells from a subject infected with human T-cell leukemia virus type I (HTLV-1) (hereinafter also referred to as "PBMCs derived from an infected subject") in an animal cell culture medium, and treating the resulting peripheral blood mononuclear cells with an anticancer agent (hereinafter also referred to as "cultured infected PBMCs treated with an anticancer agent").

[0023] In the present invention, types of peripheral blood mononuclear cells (PBMCs) include T cells, B cells, NK cells, and monocytes derived from peripheral blood, and among these, T cells derived from peripheral blood are particularly preferred.

[0024] Specific examples of the above "subject infected with human T-cell leukemia virus type I (HTLV-1)" include subjects suffering from a disease caused by HTLV-1 (hereinafter also referred to as "HTLV-1 disease") and subjects infected with HTLV-1 but not suffering from HTLV-1 disease (i.e., not yet developed HTLV-1 disease). Of these, from the viewpoint of enjoying the effects of the present invention to the fullest extent, subjects with low Tax-specific CTL activity are preferred. Examples of the above HTLV-1 disease include adult T-cell leukemia (ATL), HAM, TSP, and HU, with ATL being preferred.

[0025] Known methods can be used to determine whether a subject is infected with HTLV-1, including the Western blot method or line blot method for examining whether anti-HTLV-1 antibodies are present in the subject's blood, and a nucleic acid detection method (PCR method) for specifically detecting HTLV-1 viral DNA (proviral DNA) in the genome of the subject's PBMC.

[0026] The method for collecting PBMCs from a subject infected with HTLV-1 is not particularly limited, and a preferred example is a method for collecting PBMCs by applying a known density gradient centrifugation method to peripheral blood collected from the subject. A commercially available human lymphocyte separation solution can be suitably used for the density gradient centrifugation method.

[0027] A preferred example of peripheral blood mononuclear cells collected from a subject infected with HTLV-1 ("PBMCs from an infected subject") is PBMCs infected with HTLV-1. A cell population of PBMCs collected from a subject infected with HTLV-1 may contain PBMCs infected with HTLV-1, and all individual cells in the cell population do not have to be infected with HTLV-1, but the higher the percentage of infected cells, the better, and examples of the number of cells infected with HTLV-1 among the PBMCs in such a cell population include 5% or more, 15% or more, and 40% or more of the PBMCs.

[0028] In addition, preferred examples of PBMC derived from an infected subject include PBMC (cell population of PBMC) obtained by removing CD8 positive cells from PBMC (cell population of PBMC) collected from a subject infected with HTLV-1. When PBMC (cell population of PBMC) from which CD8 positive cells have been removed is used as PBMC derived from an infected subject, it is preferable because the proportion of cells expressing Tax protein, an antigen derived from HTLV-1, increases when the PBMC is cultured. Such a proportion is preferably 20% or more, 35% or more, 50% or more, etc.

[0029] Furthermore, preferred examples of PBMCs derived from an infected subject include PBMCs (PBMC cell populations) obtained by removing CD8-positive cells from PBMCs (PBMC cell populations) collected from a subject infected with HTLV-1 and stimulating the PBMCs (PBMC cell populations) with a CD3 antibody or with both CD3 and CD28 antibodies. When such stimulated PBMCs (PBMC cell populations) are used as PBMCs derived from an infected subject, the proportion of cells expressing Tax protein, an antigen derived from HTLV-1, increases when the PBMCs are cultured, which is preferable.

[0030] (Animal cell culture medium) The "animal cell culture medium" used in the present invention is not particularly limited as long as it is a liquid medium suitable for the growth of peripheral blood mononuclear cells (preferably peripheral blood T cells), and basal animal cell culture media such as RPMI1640 medium, RPMI medium, DMEM medium, IMEM medium, and MEM medium can be used. A mixed medium of these basal animal cell culture media can also be used. Commercially available basal animal cell culture media can be used.

[0031] The animal cell culture medium of the present invention may be a serum-free medium or a serum-containing medium. A preferred example of such a serum-containing medium is a medium in which 5 to 20% by weight of serum (e.g., FCS) has been further added to the above-mentioned basal animal cell culture medium.

[0032] The animal cell culture medium of the present invention may not contain either IL-2 (interleukin-2) or IL-15 (interleukin-15), but preferably further contains one or two substances selected from the group consisting of IL-2 (interleukin-2) and IL-15 (interleukin-15). IL-2 and IL-15 are suitable for continuing the culture of peripheral blood mononuclear cells for a long period of time, and therefore can be particularly preferably used when PBMC derived from an infected subject is cultured for, for example, 2 days or more. The concentration of IL-2 in the animal cell culture medium of the present invention can be, for example, 3 to 300 u / mL, preferably 6 to 150 u / mL, and more preferably 15 to 60 u / mL, and the concentration of IL-15 can be, for example, 1 to 100 ng / mL, preferably 2 to 50 ng / mL, and more preferably 5 to 20 ng / mL. Commercially available IL-2 and IL-15 can be used.

[0033] In addition, the animal cell culture medium of the present invention may not contain a histone deacetylase (HDAC) inhibitor, but preferably further contains an HDAC inhibitor. If an HDAC inhibitor is contained in a medium for culturing PBMC derived from an infected subject, the IL-12 production from antigen-presenting cells (APCs) increases when the PBMCs are co-cultured with APCs, and the cross-presentation efficiency of APCs is also improved, which is preferable.

[0034] The HDAC inhibitor is not particularly limited as long as it is a substance that has an inhibitory effect on HDAC, and examples thereof include suberoylanilide hydroxamic acid (SAHA), valproic acid (VPA), trichostatin A (TSA), scriptaid, oxamflatin, trapoxin, phenylbutyrate, MS-275 (Entinostat), pyroxamide, etc., among which SAHA and VPA are preferred. In addition, two or more types of HDAC inhibitors may be used in combination. In addition, these HDAC inhibitors can be commercially available products.

[0035] The concentration of the HDAC inhibitor in the medium for animal cell culture in the present invention can be appropriately set depending on the type of HDAC inhibitor used, and in the case of SAHA, for example, the concentration can be 0.1 to 20 μM, preferably 0.25 to 10 μM, and more preferably 0.5 to 2 μM, and in the case of VPA, the concentration can be 0.1 to 20 mM, preferably 0.25 to 10 mM, and more preferably 0.5 to 2 mM.

[0036] When PBMCs of the present invention are stimulated with a CD3 antibody, or a CD3 antibody and a CD28 antibody, the amount of CD3 antibody or CD28 antibody added to the animal cell culture medium of the present invention is not particularly limited, but immunobeads having CD3 antibody attached thereto can be added in a number that is 0.3 to 2 times (preferably 0.5 to 1.5 times, more preferably 0.8 to 1.2 times) the number of PBMC cells, or immunobeads having CD3 antibody and CD28 antibody attached thereto can be added in a number that is 0.3 to 2 times (preferably 0.5 to 1.5 times, more preferably 0.8 to 1.2 times) the number of PBMC cells.

[0037] (Method of culturing PBMC from infected subjects) The method for culturing PBMC derived from an infected subject is not particularly limited as long as the cells are cultured in an animal cell culture medium. Except for using the above-mentioned animal cell culture medium, normal culture conditions for PBMC (e.g., temperature conditions, carbon dioxide concentration conditions, culture period, etc.) can be used.

[0038] The temperature conditions mentioned above include, for example, 30 to 39°C, preferably 35 to 39°C.

[0039] The carbon dioxide concentration condition may be, for example, 3 to 7%, 4 to 6%, or the like.

[0040] The lower limit of the aforementioned culture period can be, for example, 1 day or more, and from the viewpoint of increasing the ratio of cells expressing the Tax protein derived from HTLV-1, preferably 3 days or more, more preferably 5 days or more. The upper limit of the culture period is not particularly limited, but can be 6 months or less, 3 months or less, or 1 month or less. More specifically, the aforementioned culture period can be 1 day to 6 months, 1 day to 3 months, 1 day to 1 month, 2 days to 6 months, 2 days to 3 months, 2 days to 1 month, 3 days to 6 months, 3 days to 3 months, 3 days to 1 month, 5 days to 6 months, 5 days to 3 months, 5 days to 1 month, etc.

[0041] When PBMCs derived from an infected subject are cultured in an animal cell culture medium, the number of PBMCs derived from an infected subject per 1 mL of medium used at the start of culture is, for example, 10 5 ~10 7 pieces / mL, 5×10 5 ~5×10 6 Examples include units / mL.

[0042] (Anti-cancer drugs) In cultured infected PBMCs, expression of HTLV-1 antigens is increased compared to PBMCs derived from infected subjects. When such cultured infected PBMCs are administered to an HTLV-1 infected person as an HTLV-I specific CTL activator, it is considered that the HTLV-1 neutralizing antibodies already present in the body fluids of the infected person will act, and new HTLV-I infection based on cultured infected PBMCs will not occur basically. Furthermore, in the present invention, cultured infected PBMCs are treated with an anticancer drug, so that the cultured infected PBMCs themselves are almost killed, and therefore there is almost no possibility of new HTLV-I infection occurring, and even if there is any, it is considered to be extremely limited and transient.

[0043] The term "anticancer agent" as used herein is not particularly limited as long as it is a substance that has the activity of inhibiting the survival and / or proliferation of cultured infected PBMCs, and for convenience, it also includes substances that are not usually used as anticancer agents, but preferably includes anticancer agents.

[0044] Such anticancer agents include antitumor antibiotics, alkylating agents, platinum preparations, DNA replication inhibitors such as topoisomerase inhibitors; metabolic antagonists such as folate metabolic antagonists, pyrimidine metabolic inhibitors, and purine metabolic inhibitors; microtubule inhibitors such as microtubule polymerization inhibitors and microtubule depolymerization inhibitors; molecular targeted therapeutic agents; and the like.

[0045] Examples of the antitumor antibiotic include mitomycin C (MMC), doxorubicin, epirubicin, daunorubicin, bleomycin, and sarkomycin, and among these, mitomycin C is preferable.

[0046] Examples of the alkylating agent include nitrogen mustards such as cyclophosphamide, ifosfamide, melphalan, busulfan, and thiotepa; and nitrosoureas such as nimustine, ranimustine, dacarbazine, procarbazine, temozolomide, carmustine, streptozotocin, and bendamustine.

[0047] Examples of the platinum agents include cisplatin, carboplatin, oxaliplatin, and nedaplatin.

[0048] Examples of the topoisomerase inhibitor include irinotecan, etoposide, doxorubicin, epirubicin, ofloxacin, ciprofloxacin, and levofloxacin.

[0049] Examples of the folate metabolic antagonist include dihydropteroate synthase inhibitors such as sulfadiazine, sulfamethoxazole, and diaphenylsulfone; dihydrofolate reductase inhibitors such as methotrexate, trimethoprim, and pyrimethamine; and the like.

[0050] Examples of the pyrimidine metabolism inhibitors include thymidylate synthase inhibitors such as fluorouracil (5-FU) and flucytosine (FC).

[0051] Examples of the above-mentioned purine metabolism inhibitors include IMPDH inhibitors such as 6-mercaptopurine and azathioprine; adenosine deaminase inhibitors such as pentostatin; ribonucleotide reductase inhibitors such as hydroxyurea; purine analogs such as thioguanine, fludarabine phosphate, and cladribine, and nucleotide analogs such as pyrimidine analogs such as cytarabine and gemcitabine; and the like.

[0052] Examples of the microtubule polymerization inhibitor include vinblastine, vincristine, vindesine, and fordesine.

[0053] Examples of the microtubule depolymerization inhibitor include paclitaxel, docetaxel, and the like.

[0054] An example of the molecular targeted therapeutic agent is mogamulizumab.

[0055] Two or more of these anticancer drugs may be used in combination. Furthermore, commercially available anticancer drugs can be used.

[0056] (Treatment of cultured infected PBMC with anticancer drugs) In the present invention, the method for treating cultured, infected PBMCs with an anticancer agent is not particularly limited as long as it is a method for contacting cultured, infected PBMCs with an anticancer agent, and a preferred example is a method in which an anticancer agent is added to a solution (e.g., a medium) containing cultured, infected PBMCs, and then allowed to stand for a certain period of time.

[0057] The concentration of the anticancer drug in a solution such as a medium is not particularly limited as long as it can inhibit the survival and / or proliferation of cultured infected PBMCs, and can be appropriately set by a person skilled in the art depending on the type of anticancer drug used. For example, when mitomycin C is used, the concentration of mitomycin C in the medium can be, for example, 5 to 500 μg / mL, preferably 10 to 250 μg / mL, and more preferably 25 to 100 μg / mL.

[0058] The time for which the cultured, infected PBMCs are contacted with the anticancer drug is not particularly limited, as long as it is possible to inhibit the survival and / or proliferation of the cultured, infected PBMCs, and examples of the time include 30 minutes to 24 hours, 30 minutes to 8 hours, and 30 minutes to 2 hours.

[0059] After treating the culture-infected PBMCs with an anticancer drug, it is preferable to wash the culture-infected PBMCs with a medium, etc., in order to remove the anticancer drug from the culture-infected PBMCs. After such washing, the culture-infected PBMCs can be collected by centrifugation, etc.

[0060] (Contacting HTLV-1-infected cells with an antibody capable of binding to the cells) The cultured infected PBMCs treated with an anticancer drug do not need to be further contacted with an antibody capable of binding to HTLV-1-infected cells, but since this may increase phagocytosis by antigen-presenting cells, it is preferable to further contact them with an antibody capable of binding to HTLV-1-infected cells.

[0061] In the present invention, examples of "antibodies capable of binding to HTLV-1-infected cells" include mogamulizumab and antibodies contained in the blood of HTLV-I-infected subjects.

[0062] A method for contacting the cultured infected PBMCs treated with an anticancer drug with an antibody capable of binding to HTLV-1-infected cells includes a method in which the cultured infected PBMCs treated with an anticancer drug are placed in a solution containing the antibody. When using an antibody contained in the blood of a subject infected with HTLV-I, the plasma of the subject can be used.

[0063] (HTLV-I specific CTL activator) The HTLV-I-specific CTL activator of the present invention may contain only cultured infected PBMCs treated with an anticancer drug (i.e., peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type I (HTLV-1) in an animal cell culture medium and treating the resulting peripheral blood mononuclear cells with an anticancer drug), or may further contain other components (i.e., optional components).

[0064] The HTLV-I-specific CTL activator of the present invention may be prepared by a conventional method such as mixing cultured infected PBMCs treated with an anticancer drug with a pharma- ceutically acceptable carrier, etc. Such a preparation may be a solid preparation or a liquid preparation, with the liquid preparation being preferred.

[0065] Such liquid preparations include solutions, suspensions, syrups, slurries, emulsions, etc. Liquid carriers that can be used in such liquid preparations include any suitable organic or inorganic solvents, such as saline, buffered saline (e.g., phosphate buffered saline, Tris buffered saline), saline solution, etc. These solvents are preferably sterilized.

[0066] The content of "cultured infected PBMCs treated with anticancer drugs" in the HTLV-I-specific CTL activator of the present invention can be about 0.1 to 100% by weight, preferably about 1 to 99% by weight, and more preferably 10 to 90% by weight of the total activator. The content of "cultured infected PBMCs treated with anticancer drugs" in the HTLV-I-specific CTL activator of the present invention can be, for example, 10 5 ~10 7 pieces / mL, 5×10 5 ~5×10 6 Examples include units / mL.

[0067] (Dosage) The dose of the HTLV-I-specific CTL activator of the present invention is appropriately adjusted depending on the state of the disease, the age, weight, etc. of each patient. The number of "cultured infected PBMCs treated with an anticancer drug" in the drug per administration to a subject is, for example, 10 5 ~10 7 pcs, preferably 5 x 10 5 ~5×10 6 It is preferable to repeatedly administer such a dose once every few days, weeks, or months, a total of two or more times (for example, 2 to 10 times).

[0068] (Administration Method) The method of administration of the HTLV-I-specific CTL activator of the present invention is not particularly limited as long as it provides an HTLV-I-specific CTL activating effect, but examples include subcutaneous administration, intradermal administration, and intramuscular administration, with subcutaneous administration being preferred.

[0069] (Target of administration) The subject to which the HTLV-I-specific CTL activator of the present invention is administered is preferably the same subject from which the PBMCs derived from the infected subject were collected, in which case the HTLV-I-specific CTL activator can activate the HTLV-I-specific CTLs of the subject without causing a rejection reaction.

[0070] (Application of the present invention) The HTLV-I-specific CTL activator of the present invention is capable of activating HTLV-I-specific CTL in patients with HTLV-1 disease, and is therefore considered to have a therapeutic effect against HTLV-1 disease. Such therapeutic effects include an effect of improving symptoms of HTLV-1 disease, an effect of suppressing the transformation of HTLV-1 disease (particularly ATL) into blast crisis, and an effect of preventing recurrence after chemotherapy. In addition, the HTLV-I-specific CTL activator of the present invention is considered to have an effect of suppressing the onset of HTLV-1 disease in subjects infected with HTLV-I but not developing HTLV-1 disease, by activating HTLV-I-specific CTL.

[0071] Therefore, the HTLV-I-specific CTL activator (composition for activating HTLV-I-specific CTL) of the present invention or cultured infected PBMCs treated with an anticancer agent can also be used as an agent for improving (or a pharmaceutical composition for improving) a disease caused by HTLV-1 infection, an agent for suppressing the progression to blast crisis (or a pharmaceutical composition for suppressing the progression) of a disease caused by HTLV-1 infection, an agent for suppressing the onset of a disease caused by HTLV-1 infection (or a pharmaceutical composition for suppressing the onset), an agent for preventing or treating (or a pharmaceutical composition for preventing or treating) a disease caused by HTLV-1 infection, etc.

[0072] (HTLV-I specific CTL activation) In the present invention, "HTLV-I-specific CTL activation" (activating HTLV-I-specific CTL) means improving the cytotoxic activity of HTLV-I-specific CTL against HTLV-1, and includes, for example, inducing cross-presentation of antigen-presenting cells (preferably dendritic cells) in the body of a subject administered the HTLV-I-specific CTL activator of the present invention or "cultured infected PBMC treated with an anticancer drug," and then inducing HTLV-I-specific CTLs from naive CD8-positive T cells that have no cytotoxic activity, thereby improving the cytotoxic activity of HTLV-I-specific CTL against HTLV-1 in the body of the subject.

[0073] Improved cytotoxic activity of HTLV-I-specific CTL against HTLV-1 means that when peripheral blood mononuclear cells treated with an anticancer drug are administered to an infected subject in which the normal HTLV-I-specific CTL activity is weakened, the cytotoxic activity of HTLV-I-specific CTL against HTLV-1 in the subject is improved compared to before administration.

[0074] (Method of producing HTLV-I specific CTL activator) The method for producing the HTLV-I-specific CTL activator of the present invention includes the following steps: (a) culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type 1 (HTLV-1) in an animal cell culture medium; (b) treating the peripheral blood mononuclear cells obtained by the culture in the step (a) with an anticancer drug and then collecting the peripheral blood mononuclear cells; and (c) mixing the peripheral blood mononuclear cells collected in step (b) with a pharma- ceutically acceptable carrier to prepare a formulation; There is no particular limitation as long as it has the above structure.

[0075] The above step (a) is not particularly limited as long as it is a step of culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type I (HTLV-1) in an animal cell culture medium. The method of such culturing is as described above. Among such step (a), a preferred example is a step of stimulating peripheral blood mononuclear cells obtained by removing CD8 positive cells from peripheral blood mononuclear cells collected from a subject infected with HTLV-1 with a CD3 antibody or both CD3 and CD28 antibodies, and then culturing the cells in an animal cell culture medium. Furthermore, examples of a method for stimulating peripheral blood mononuclear cells obtained by removing CD8-positive cells from peripheral blood mononuclear cells collected from a subject infected with HTLV-1 with a CD3 antibody, or with both CD3 and CD28 antibodies, include a method of culturing peripheral blood mononuclear cells from which CD8-positive cells have been removed in the animal cell culture medium of the present invention to which immunobeads having CD3 antibody attached, or immunobeads having CD3 and CD28 antibodies attached, have been added in an amount of 0.3 to 2 times (preferably 0.5 to 1.5 times, and more preferably 0.8 to 1.2 times) the number of PBMCs (peripheral blood mononuclear cells from which CD8-positive cells have been removed).

[0076] The above step (b) is not particularly limited as long as it is a step of treating the peripheral blood mononuclear cells obtained by the culture in the above step (a) with an anticancer drug and then collecting the peripheral blood mononuclear cells. The method of treating with the anticancer drug and the method of collecting the peripheral blood mononuclear cells are as described above.

[0077] A preferred example of the above step (b) is a step of treating the peripheral blood mononuclear cells obtained by the culture in step (a) with an anticancer drug, contacting them with an antibody capable of binding to HTLV-1-infected cells, and then collecting the peripheral blood mononuclear cells.

[0078] The above step (c) is not particularly limited as long as it is a step of mixing the peripheral blood mononuclear cells collected in the above step (b) with a pharma- ceutically acceptable carrier to prepare a pharmaceutical preparation. Such a method is as described above.

[0079] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0080] Test 1. [Test for induction of Tax antigen expression by culturing HTLV-1-infected T cells] HTLV-1-infected mononuclear cells are present in the peripheral blood of HTLV-1-infected individuals (including ATL patients). However, no HTLV-1 antigen is detected in infected mononuclear cells immediately after isolation from peripheral blood. In order to use HTLV-1-infected peripheral blood mononuclear cells as a vaccine antigen, the expression of the HTLV-1 Tax antigen is required. Therefore, the following test was performed to investigate how the Tax antigen is expressed by culturing HTLV-1-infected peripheral blood mononuclear cells.

[0081] (1) PBMC culture method Peripheral blood was collected from two patients with acute ATL (aATL). Peripheral blood mononuclear cells (PBMCs) were separated from the peripheral blood by density gradient method using Ficoll-Paque (registered trademark), a human lymphocyte density separation solution. 5 × 10 PBMCs were directly added to RPMI 1640 medium (hereinafter referred to as "IL-2-supplemented medium") supplemented with 10% FCS (fetal calf serum) and rhIL-2 (Immunace (registered trademark), manufactured by Shionogi Pharmaceutical Co., Ltd.) to a concentration of 10 u / mL. 5 The cells were cultured at 37°C in a CO2 incubator for several days to several weeks.

[0082] (2) Cultivation method of PBMCs from which CD8 positive cells have been removed Moreover, instead of adding the above-mentioned PBMCs directly to the IL-2-supplemented medium, the above-mentioned PBMCs from which CD8-positive cells had been removed were added to the IL-2-supplemented medium and cultured for several days to several weeks in a CO2 incubator at 37°C. Note that immunobeads (Dynabeads (registered trademark), ThermoFisher) to which CD8 antibodies had been attached were used to remove CD8-positive cells from the PBMCs.

[0083] (3) Confirmation of Tax antigen expression in PBMCs after culture After culturing PBMCs derived from aATL patients for 3 days using the method described above in (1), the PBMCs were subjected to flow cytometry to detect intracellular Tax protein in the PBMCs. The method for detecting Tax protein by flow cytometry is as follows.

[0084] After 3 days of culture, PBMCs were fixed with Permeabilization / Fixation Buffer (eBioscience) for 30 minutes and then washed with Permeabilizing buffer (eBioscience). Mouse anti-HTLV-1 Tax antibody LT-4 (Lee B, Tanaka Y, Tozawa H. 1989. Monoclonal antibody defining tax protein of human T-cell leukemia virus type-I. Tohoku J Exp Med 157:1-11.) was added to these PBMCs as a primary antibody and incubated on ice for 30 minutes. After washing these PBMCs, Alexa488-anti-mouse IgG antibody was added as a secondary antibody and PBMCs were stained for 30 minutes. After washing these PBMCs, HTLV-1 Tax protein in PBMCs was detected using a cell analyzer MACSQuant (Miltenyi Biotec) (right panel of Figure 1 (Day 3)).

[0085] On the other hand, as a control, similar flow cytometry was performed on PBMCs before culture derived from an aATL patient using the method (1) above to detect HTLV-1 Tax protein in the PBMCs (left panel of Figure 1 (Day 0)).

[0086] As shown in Figure 1, Tax protein was not detectable at all in PBMCs before culture, but after 3 days of culture, Tax protein was detectable in more than half of the PBMCs. These results indicate that intracellular expression of the Tax antigen is induced when PBMCs from aATL patients are cultured.

[0087] (4) Effect of depleting CD8 positive cells from PBMCs PBMCs from aATL patients were cultured for 1 day using the method described in (1) above, and then the PBMCs were subjected to flow cytometry using the method described in (3) above to detect intracellular Tax protein in the PBMCs. The results are shown in the left panel of Figure 2.

[0088] In addition, CD8 positive cells were removed from PBMCs derived from aATL patients by the method described in (2) above, and the PBMCs were then cultured for one day by the method described in (1) above. The PBMCs were subjected to flow cytometry by the method described in (3) above to detect intracellular Tax protein in the PBMCs. The results are shown in the right panel of Figure 2.

[0089] As can be seen from the results in Figure 2, the percentage of cells in which Tax protein was detected (7.15%) was higher when PBMCs were cultured for one day after removing CD8-positive cells than the percentage (5.33%) when PBMCs were cultured for one day as is. This result shows that removing CD8-positive cells from infected PBMCs before culturing is preferable in terms of increasing the percentage of cells expressing Tax protein, an antigen derived from HTLV-1.

[0090] (5) Longer-term culture of PBMCs depleted of CD8 positive cells CD8 positive cells were removed from PBMCs derived from chronic ATL patients using the same method as in (2) above, and then immunobeads (Dynabeads, ThermoFisher) bearing CD3 and CD28 antibodies were added to the medium in the same number as the PBMCs to stimulate the PBMCs, and the PBMCs were cultured for 24 days using the method in (1) above. The PBMCs were subjected to flow cytometry using the method in (3) above to detect intracellular Tax protein in the PBMCs. The results are shown in Figure 3.

[0091] As can be seen from the results in Figure 3, when CD8 positive cells were removed from PBMCs and then the cells were cultured for 24 days, Tax protein was detectable in approximately 60% or more of the PBMCs.

[0092] (6) Effects of histone deacetylase (HDAC) inhibitors The present inventors have previously established T cell lines expressing Tax protein by long-term culture of PBMCs from ATL patients in the presence of 30 u / mL IL-2 (rhIL-2; manufactured by Peprotec) or 10 ng / mL IL-15 (rhIL-15; manufactured by Peprotec) (J Virol 79:10088-10092 and PLoS Pathog. 2017 Sep 14;13(9):e1006597.). Among such cell lines established by the present inventors, the ILT-A line and the ILT-B line were prepared. The ILT-A line is an IL-15-dependent cell line derived from the PBMCs of an acute ATL patient, and the ILT-B line is an IL-2-dependent cell line derived from the PBMCs of a chronic ATL patient. We attempted to confirm how histone deacetylase (HDAC) inhibitors, which are known to have anticancer effects, affect the expression of Tax protein when ILT-A and ILT-B cells were cultured.

[0093] Suberoylanilide hydroxamic acid (SAHA) was added to the IL-2 or IL-15-added medium described in (1) or (6) above to prepare a SAHA-containing medium containing 0.5 μM SAHA. Dimethyl sulfoxide (DMSO) was added to the IL-2 or IL-15-added medium described in (1) or (6) above to prepare a DMSO-containing medium (control medium) containing 0.001% by weight DMSO.

[0094] ILT-A cells were cultured in SAHA-containing medium and control medium for 2 days each at 37°C in a CO2 incubator. Flow cytometry was performed on the ILT-A cells using the method described in (3) above to detect Tax protein in the ILT-A cells. A similar experiment was performed using ILT-B cells instead of ILT-A cells to detect Tax protein in the ILT-B cells. These results are shown in Figure 4. The results in Figure 4 indicate that the efficiency of induction of Tax protein expression was improved when the PBMC cell line was cultured in the presence of an HDAC inhibitor compared to when it was cultured in the absence of an HDAC inhibitor.

[0095] Test 2. [Induction of antigen cross-presentation by HTLV-1-infected cells] In general, in order to activate the response of CD8+CTL, it is necessary that antigen-presenting cells that have phagocytosed antigens are activated, and that they express costimulatory molecules (CD86, etc.) and produce cytokines such as IL-12, while presenting antigen peptides not only to MHC class II molecules but also to MHC class I molecules (i.e., "antigen cross-presentation"). Therefore, the present inventors have investigated an in vitro evaluation system for whether cross-presentation occurs, and constructed such an evaluation system (Figure 5). In this evaluation system, CD8-positive Tax-specific CTLs (Tc-M1) and antigen-presenting cells (THP1) that match MHC-I are used. Then, HTLV-1-infected T cells (ILT-A or ILT-B), which do not match MHC with the CTLs or the antigen-presenting cells, are treated with mitomycin C (MMC) (a type of anticancer drug) and co-cultured with the above-mentioned CTLs and antigen-presenting cells. During such co-culture, when the antigen-presenting cells take up the HTLV-1-infected T cells and present the HTLV-1-derived Tax antigen on MHC-I (cross-presentation), the CD8-positive Tax-specific CTLs recognize the presented Tax antigen and produce IFN-γ in the culture medium. The level of IFN-γ concentration in the culture medium supernatant can be used to evaluate the degree of CTL activation by presentation. Specifically, the evaluation of cross-presentation using this evaluation system was performed as follows.

[0096] The CD8+ Tax-specific CTL used was the HLA-A2-restricted CD8+ Tax-specific CTL Tc-M1 strain. The Tc-M1 strain was derived by culturing PBMCs from a HAM / TSP patient in the presence of 100u / mL rhIL-2 and repeatedly stimulating them with autologous HTLV-1-infected cells at 2-week intervals (International immunology 3:761-767). The human monocyte-derived THP1 cell line was used as the HLA-A2-positive antigen-presenting cell (Virology 318:17-23). The antigen cells used were "ILT-A cells (HTLV-1-infected T cell line derived from an ATL patient) that do not have HLA-A2, which were cultured for 2 days in IL-15-supplemented medium, then treated with 50 μg / mL MMC (manufactured by Kyowa Hakko Kirin Co., Ltd.) (a type of anticancer drug) at 37°C for 30 minutes, and then washed" (hereinafter simply referred to as "MMC-treated ILT-A cells"), or "ILT-A cells (HTLV-1-infected T cell line derived from an ATL patient) that do not have HLA-A2, which were cultured for 2 days in IL-15-supplemented medium, then treated with 1% by weight formalin for 15 minutes, and then washed."

[0097] The above-mentioned antigen cells not having HLA-A2 were co-cultured with HLA-A2 positive THP1 cell line for 16 to 20 hours. All cells obtained from the co-culture were fixed with 1% by weight aqueous formalin solution at room temperature for 15 minutes, and then the washed cells were co-cultured with the above-mentioned HLA-A2 restricted CD8 positive Tax specific CTL (Tc-M1) for 16 hours in RPMI1640 medium containing 25u / mL IL-2 and 10% FCS. The supernatant of the culture solution obtained by the co-culture was collected, and the IFN-γ concentration in the supernatant was measured by ELISA (BD OptiEIA (registered trademark)). The IFN-γ concentration was used as an index of CTL activity.

[0098] As a control experiment, the antigen cells were not co-cultured with the antigen-presenting cells, but instead co-cultured with the aforementioned HLA-A2-restricted CD8-positive Tax-specific CTL (Tc-M1) for 16 hours, and the IFN-γ concentration in the supernatant of the resulting culture medium was measured.

[0099] The measurement results of these IFN-γ concentrations are shown in Figure 6. In Figure 6, "ILT-MMC alone" represents the IFN-γ concentration in the culture supernatant when only MMC-treated ILT-A cells (HTLV-1-infected T cell line derived from an ATL patient) were co-cultured with CTLs as the initial culture, "ILT-Formalin alone" represents the IFN-γ concentration in the culture supernatant when only formalin-treated ILT-A cells (HTLV-1-infected T cell line derived from an ATL patient) were co-cultured with CTLs as the initial culture, and "THP1 "Alone" represents the IFN-γ concentration when only THP1 cells, which are antigen-presenting cells, were co-cultured with CTLs in the initial culture, "ILT-MMC+THP1" represents the IFN-γ concentration produced by CTLs when MMC-treated ILT-A cells and THP1 cells (antigen-presenting cells) were cultured in the initial culture, "ILT-Formalin+THP1" represents the IFN-γ concentration produced by CTLs when formalin-treated ILT-A cells and THP1 cells (antigen-presenting cells) were cultured in the initial culture, and "None (CLT alone)" represents the IFN-γ concentration when neither ILT-A cells nor THP1 cells (antigen-presenting cells) were cultured in the initial culture (i.e., when only Tc-M1, which is a CTL, was cultured).

[0100] The results in Figure 6 show the following: CD8-positive Tax-specific CTL (Tc-M1) was hardly activated when co-cultured only with MMC-treated ILT-A cells. In addition, CD8-positive Tax-specific CTL (Tc-M1) was hardly activated when co-cultured only with THP1 cells (antigen-presenting cells). However, CD8-positive Tax-specific CTL (Tc-M1) was activated (produced IFN-γ) when co-cultured with "cells obtained by co-culture of ILT-A cells and THP1 cells." From these findings, it was considered that when MMC-treated HTLV-1-infected T cells (e.g., MMC-treated ILT-A cells) were co-cultured with antigen-presenting cells (e.g., THP1 cells), the antigen-presenting cells took up the HTLV-1-infected T cells and presented the Tax antigen derived from HTLV-1 on MHC-I. In other words, it was considered that the antigen-presenting cells cross-presented the Tax antigen. As can be seen from the results of "ILT-Formalin+THP1" in Figure 6, even when CTLs were co-cultured with "formalin-fixed HTLV-1-infected T cells co-cultured with antigen-presenting cells," the CTLs were hardly activated.

[0101] Study 3. [Effects of HTLV-1 neutralizing antibodies and reverse transcriptase inhibitors] In the aforementioned Test 2, it was shown that CD8+ Tax-specific CTL (Tc-M1) was activated (produced IFN-γ) when co-cultured with "cells obtained by co-culture of ILT-A cells and THP1 cells". To confirm that this phenomenon in Test 2 was not the result of HTLV-1 newly infecting antigen-presenting cells from ILT-A cells, which are HTLV-1-infected cells, and HTLV-1 antigens generated from the newly infected cells, the new infection of HTLV-1 was inhibited with HTLV-1 neutralizing antibodies or reverse transcriptase inhibitors, and then a cross-presentation evaluation test in Test 2 was performed. Specifically, the following experiments were performed to examine the effect of HTLV-1 neutralizing antibodies and reverse transcriptase inhibitors on the efficiency of cross-presentation of Tax antigen by HTLV-1-infected cells.

[0102] (1) Effect of HTLV-1 neutralizing antibodies The blood of HTLV-1-infected individuals contains high concentrations of HTLV-1 neutralizing antibodies, which are expected to bind to cells expressing HTLV-1 antigens. Mogamulizumab (trade name Potelidio (registered trademark); manufactured by Kyowa Hakko Kirin Co., Ltd.), an anti-CCR4 antibody, also binds to HTLV-1-infected cells. The following experiment was conducted to investigate the effects of these antibodies binding to HTLV-1-infected cells.

[0103] ILT-A cells were cultured in IL-15-added medium for 2 days, and then treated with 50 μg / mL MMC (Kyowa Hakko Kirin Co., Ltd.) (a type of anticancer drug) at 37°C for 30 minutes, followed by washing to obtain "MMC-treated ILT-A cells (MMC-treated ILT-A cells)." Meanwhile, aqueous solutions containing 0.5% by weight of plasma from three ATL patients, three HAM / TSP patients, and two non-infected individuals were prepared to obtain plasma aqueous solutions. Approximately 10% of the aforementioned "MMC-treated ILT-A cells" were added to the aqueous plasma solutions. 5 The MMC-treated ILT-A cells were washed and then incubated for approximately 10 min at 4 °C for 10 min. 5 The cells were co-cultured with THP1 cells at 100x the amount of each cell. All cells obtained from the co-culture were fixed with 1% by weight aqueous formalin solution at room temperature for 15 minutes, and the washed cells were then co-cultured with the above-mentioned HLA-A2-restricted CD8-positive Tax-specific CTL (Tc-M1) for 16 hours. The supernatant of the culture solution obtained by the co-culture was collected, and the IFN-γ concentration in the supernatant was measured by ELISA (BD OptiEIA (registered trademark)). The IFN-γ concentration was used as an index of CTL activity.

[0104] In addition, a similar experiment was performed using an aqueous solution containing 10 μg / mL of the anti-CCR4 antibody mogamulizumab (trade name Poteligeo (registered trademark); manufactured by Kyowa Hakko Kirin Co., Ltd.) instead of the aqueous solution containing 0.5% by weight of patient plasma, and the IFN-γ concentration in the supernatant was measured.

[0105] The results of measuring IFN-γ concentrations in these experiments are shown in Figure 7. As can be seen from the results in Figure 7, the IFN-γ concentration was not significantly decreased when treated with plasma from HTLV-1-infected individuals ("ATL-1", "ATL-2", "ATL-3", "HAM-1", "HAM-2", "HAM-3" in Figure 7) or when treated with anti-CCR4 antibody ("Poteligeo" in Figure 7). These results indicate that the induction of Tax antigen cross-presentation by HTLV-1-infected cells is not due to new infection with HTLV-1. In addition, no significant difference was observed between treatment with plasma from HTLV-1-infected individuals ("ATL-1", "ATL-2", "ATL-3", "HAM-1", "HAM-2", "HAM-3" in Figure 7), treatment with plasma from non-infected individuals ("SN-1", "SN-2" in Figure 7), and treatment with anti-CCR4 antibody ("Poteligeo" in Figure 7).

[0106] (2) Effect of the ratio of the number of MMC-treated ILT-A cells to THP1 cells The ILT-A cells were cultured in IL-15-supplemented medium for 2 days (as described above), then treated with 50 μg / mL MMC (Kyowa Hakko Kirin Co., Ltd.) (a type of anticancer drug) at 37°C for 30 minutes and then washed to obtain "MMC-treated ILT-A cells." The MMC-treated ILT-A cells were co-cultured with THP1 cells at a cell number ratio of 1:1, 0.5:1, or 0.25:1, and the IFN-γ concentration was measured in the same manner as described above in “(1) Effect of HTLV-1 neutralizing antibodies.” In this study, ATL-1 plasma was used as the plasma of the ALT patient.

[0107] The results of these experiments are shown in Figure 8. The horizontal axis represents the number of HTLV-1-infected T cells. The first to third bars from the left represent the number of HTLV-1-infected T cells in 10 5 The fourth to sixth bars from the left show the results for 5 × 10 HTLV-1-infected T cells (i.e., the ratio was 1:1). 4The first to third bars from the right show the results for a 2.5×10 4 8 shows the results when the "MMC-ILT / (+)plasma" bar represents the results when the "MMC-treated ILT-A cells" were contacted with the plasma of an HTLV-1-infected individual, the gray bar represents the results when the "MMC-treated ILT-A cells" were contacted with the plasma of an uninfected individual, and the white bar represents the results when the "MMC-ILT" bar represents the results when the "MMC-treated ILT-A cells" were not contacted with the plasma of an HTLV-1-infected individual.

[0108] From the results in Figure 8, it was found that when ILT-A cells were treated with plasma from HTLV-1-infected individuals, the CTL activation effect tended to increase to some extent. In addition, this tendency was observed when the number of target cells, "MMC-treated ILT-A cells," was relatively small compared to the number of antigen-presenting cells ("5 to 2.5 × 10 4 The increase in CTL activation effect when ILT-A cells were treated with plasma from HTLV-1-infected individuals was considered to be the result of an opsonization effect caused by the binding of anti-HTLV-1 antibodies in the plasma of infected individuals to ILT-A cells.

[0109] (3) Effects of reverse transcriptase inhibitors Zidovudine (AZT) (trade name Retrovir (registered trademark), manufactured by GlaxoSmithKline) was used as a reverse transcriptase inhibitor. THP1 cells, which are antigen-presenting cells, were precultured for 1 hour at 37°C in a medium containing 20 μM AZT. On the other hand, ILT-A cells were cultured in an IL-15-added medium for 2 days, and then treated with 50 μg / mL MMC (manufactured by Kyowa Hakko Kirin Co., Ltd.) (a type of anticancer drug) at 37°C for 30 minutes, and then washed to obtain "MMC-treated ILT-A cells (MMC-treated ILT-A cells)". MMC-treated ILT-A cells were added to the medium of the THP1 cells that had been precultured as described above, and cocultured at 37°C for 16 hours. The AZT concentration in the medium during coculture was 10 μM. All cells obtained by the co-culture were fixed with 1% by weight aqueous formalin solution at room temperature for 15 minutes, and then the washed cells were co-cultured with HLA-A2-restricted CD8-positive Tax-specific CTL (Tc-M1) for 16 hours. The supernatant of the culture solution obtained by the co-culture was collected, and the IFN-γ concentration in the supernatant was measured by ELISA (BD OptiEIA (registered trademark)). The IFN-γ concentration was used as an index of CTL activity.

[0110] The results of these IFN-γ concentration measurements are shown in FIG. 9. The first to third bars from the right show the results of HTLV-1-infected T cells (ILT-A cells) at 10 5 The fourth to sixth bars from the right show the results when HTLV-1-infected T cells (ILT-A cells) were 3 × 10 4 9 shows the results when HTLV-1-infected T cells (ILT-A cells) were not used (i.e., the aforementioned ratio was 0.3:1), and the first to third bars from the left show the results when HTLV-1-infected T cells (ILT-A cells) were not used. In addition, in FIG. 9, the black bar ("THP1-AZT") shows the results when AZT was added to the medium and THP1 cells were cultured, the gray bar ("THP1") shows the results when AZT was not added to the medium, and the white bar ("Medium") shows the results when THP1 cells were not used.

[0111] As can be seen from the results in Figure 9, AZT treatment did not significantly affect CTL activity. These results indicate that the induction of Tax antigen cross-presentation by HTLV-1-infected cells is not due to de novo infection with HTLV-1.

[0112] Study 4. [Enhancement of costimulatory molecule expression in antigen-presenting cells by HTLV-1-infected T cells] In order for antigen-presenting cells such as dendritic cells to activate T cells, they need to express costimulatory signals such as CD83 and CD86 (e.g., Leukemia & lymphoma 38:247-263). Therefore, to examine whether MMC-treated ILT-C cells (HTLV-1-infected cell line obtained by long-term culture of PBMCs from ATL patients) enhance the expression of costimulatory molecules on antigen-presenting cells, we performed the following experiment.

[0113] (1) Immature dendritic cells derived from PBMCs of healthy individuals Immature dendritic cells that still retain the ability to take up antigens were used as antigen-presenting cells. These immature dendritic cells were obtained by placing RPMI1640 medium containing 10% FCS in a plastic plate, culturing PBMCs from a healthy individual in the medium for 2 hours, adding rhGM-CSF (manufactured by Miltenyi Biotec) (final concentration in the medium: 1000 u / mL) and rhIL-4 (manufactured by Miltenyi Biotec) (final concentration in the medium: 500 u / mL) to the monocytes attached to the plastic plate, and culturing for 5 days (hereinafter, also referred to as "healthy individual immature dendritic cells").

[0114] (2) Evaluation of the expression of costimulatory molecules ILT-C22 cells (HLA-A2 positive) were cultured in IL-2-supplemented medium for 2 days, then treated with 50 μg / mL MMC (Kyowa Hakko Kirin Co., Ltd.) (a type of anticancer drug) at 37°C for 30 minutes and washed to obtain "MMC-treated ILT-C cells." Next, the above-mentioned healthy donor immature dendritic cells (HLA-A2 negative) and the MMC-treated ILT-C line were co-cultured for 24 hours in RPMI1640 medium supplemented with 10% FCS, and then stained for 30 minutes on ice with an FITC-labeled HLA-A2 antibody (BD 551285; Becton Dickinson and Company), a PerCP / Cy5.5-labeled anti-human CD83 antibody (305320; Biolegend) or a PerCP / Cy5.5-labeled anti-human CD86 antibody (BD 561129; Becton Dickinson and Company). After washing, the expression intensities of CD83 and CD86 in the HLA-A2 negative dendritic cell fraction were measured by flow cytometry using a flow cytometer MACSQuant (registered trademark) (Miltenyi Biotec). In addition, the expression intensities of CD83 and CD86 were measured using the same method except that MMC-treated ILT-C cells were not used (i.e., using healthy immature dendritic cells not co-cultured with MMC-treated ILT-C cells). Dendritic cells and ILT-C cells were distinguished based on the presence or absence of HLA-A2 expression.

[0115] These measurement results are shown in Figure 10. Compared with healthy donor immature dendritic cells without the MMC-treated ILT-C line, healthy donor immature dendritic cells cocultured with the MMC-treated ILT-C line showed a mild increase in CD83 expression (upper left and lower left panels in Figure 10), and a significant increase in CD86 expression (upper right and lower right panels in Figure 10). These results demonstrated that when MMC-treated HTLV-1-infected T cells and immature dendritic cells were cocultured, the MMC-treated HTLV-1-infected T cells stimulated the immature dendritic cells and enhanced the expression of costimulatory molecules such as CD83 and CD86.

[0116] Study 5. [Enhancement of IL-12 expression in antigen-presenting cells by HTLV-1-infected T cells] It is believed that the production of IL-12 from antigen-presenting cells is important for inclining the immune environment to Th1 type during antigen presentation and inducing CTL (Therapeutic immunology 1:187-196.) Therefore, the following experiment was performed to examine the effect of co-culturing antigen-presenting cells with MMC-treated ILT-A or ILT-B cells on IL-12 production in antigen-presenting cells.

[0117] (1) Effect of drug treatment of HTLV-1-infected T cells on IL-12 expression in antigen-presenting cells A SAHA-containing medium containing 0.5 μM SAHA was prepared by adding suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, to RPMI 1640 medium supplemented with 10% FCS (fetal calf serum) containing IL-15 or IL-2. Dimethyl sulfoxide (DMSO) was also added to RPMI 1640 medium supplemented with 10% FCS (fetal calf serum) to prepare a DMSO-containing medium (control medium) containing 0.001 wt% DMSO.

[0118] ILT-A cells or ILT-B cells were cultured in SAHA-containing medium or DMSO-containing medium (control medium) for 24 hours at 37°C in a CO2 incubator. The cultured cells were treated with 50 μg / mL MMC (Kyowa Hakko Kirin) for 30 minutes at 37°C and then washed. These MMC-treated ILT cells (i.e., MMC-treated ILT-A cells or ILT-B cells) were co-cultured with HLA-A2-positive healthy immature dendritic cells (DCs) for 20 hours at 37°C. The supernatant of the medium after the co-culture was collected, and the IL-12 concentration in the supernatant was measured by ELISA (BD OptiEIA (registered trademark)). These results are shown in "DC" in Figure 11. As a control, the above-mentioned MMC-treated ILT cells (i.e., MMC-treated ILT-A cells or ILT-B cells) were cultured at 37°C for 20 hours, not together with healthy donor immature dendritic cells (DCs). The supernatant of the culture medium after such culture was collected, and the IL-12 concentration (pg / mL) in the supernatant was measured by ELISA (BD OptiEIA (registered trademark)). These results are shown in "Medium" in Figure 11.

[0119] When ILT-B cells cultured in DMSO-containing medium were treated with MMC, washed, and co-cultured with dendritic cells, approximately 120 pg / mL of IL-12 production was detected in the culture medium supernatant (black bar ("DC") of "ILT-B" in Figure 11), whereas when ILT-A cells cultured in DMSO-containing medium were treated with MMC, washed, and co-cultured with dendritic cells, the IL-12 concentration in the culture medium supernatant was below the sensitivity (right side of "DMSO" of "ILT-A" in Figure 11). On the other hand, when ILT cells (ILT-A cells or ILT-B cells) cultured in SAHA-containing medium containing 0.5 μM SAHA (a type of HDAC inhibitor) were used, IL-12 production into the medium was significantly increased compared to the case of using ILT cells (ILT-A cells or ILT-B cells) cultured in DMSO-containing medium (filled bar graph of "SAHA" for "ILT-A" ("DC") and filled bar graph of "SAHA" for "ILT-B" ("DC") in Figure 11).

[0120] The results when another HDAC inhibitor, valproic acid (VPA), was used instead of SAHA are shown in FIG. 12. ILT-B cells were cultured in VPA-containing medium or DMSO-containing medium (control medium) for 24 hours. The cells were treated with MMC, washed, and co-cultured with dendritic cells, and the IL-12 concentration in the supernatant was measured. As shown in "DC" in FIG. 12, when ILT-B cells were cultured in a medium containing 1 mM VPA, IL-12 production into the medium was significantly increased compared to when ILT-B cells were cultured in a DMSO-containing medium (solid bars in FIG. 12 ("DC")).

[0121] (2) Effect of drug treatment of HTLV-1-infected T cells on antigen cross-presentation by antigen-presenting cells After treating each of the cells after co-culture in Figure 11 with 1% by weight formalin for 15 minutes, the washed cells were co-cultured with HLA-A2-restricted CD8-positive Tax-specific CTL (Tc-M1) for 16 hours. The supernatant of the culture solution obtained by co-culture was collected, and the IFN-γ concentration (pg / mL) in the supernatant was measured by ELISA (BD OptiEIA (registered trademark)). These results are shown in Figure 13.

[0122] As shown in Figure 13, when SAHA-treated ILT cells were used, the amount of IFN-γ produced by CTLs was higher than when non-SAHA-treated ILT cells were used. These results indicate that treatment of HTLV-1-infected T cells with HDAC inhibitors such as SAHA also improves the efficiency of antigen-presenting cells cross-presenting antigens to Tax-specific CTLs. [Industrial Applicability]

[0123] According to the present invention, it is possible to provide an HTLV-I-specific CTL activator and a method for producing the same. It is also expected that according to the present invention, it is possible to provide a relatively inexpensive and effective immunotherapy to many patients, regardless of the HLA type of the patient suffering from a disease caused by HTLV-1 infection. It is also expected that according to the present invention, it is possible to provide a therapy that can be applied in an early stage to smoldering or chronic ATL (indolent ATL), which is currently observed without treatment as a rule due to the lack of a safe and effective therapy.

Claims

1. A HTLV-I-specific cytotoxic T cell (CTL) activator for administration to a subject, comprising peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from the subject infected with human T-cell leukemia virus type I (HTLV-1) in an animal cell culture medium, and treating the peripheral blood mononuclear cells with an anticancer agent.

2. The HTLV-I-specific CTL activator according to claim 1, wherein the peripheral blood mononuclear cells are obtained by culturing peripheral blood mononuclear cells collected from a subject infected with HTLV-1 for one day or more.

3. The HTLV-I-specific CTL activator according to claim 1 or 2, wherein the animal cell culture medium contains IL-2, IL-15 or both.

4. The HTLV-I-specific CTL activator according to any one of claims 1 to 3, wherein the animal cell culture medium contains a histone deacetylase inhibitor.

5. The HTLV-I-specific CTL activator according to any one of claims 1 to 4, wherein the peripheral blood mononuclear cells collected from a subject infected with HTLV-1 are peripheral blood mononuclear cells obtained by removing CD8 positive cells from peripheral blood mononuclear cells collected from a subject infected with HTLV-1.

6. The HTLV-I-specific CTL activator according to any one of claims 1 to 5, wherein the peripheral blood mononuclear cells collected from a subject infected with HTLV-1 are peripheral blood mononuclear cells activated with a CD3 antibody, or with both a CD3 antibody and a CD28 antibody, after removing CD8 positive cells from the peripheral blood mononuclear cells collected from the subject infected with HTLV-1.

7. The HTLV-I-specific CTL activator according to any one of claims 1 to 6, wherein the peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from a subject infected with HTLV-1 in an animal cell culture medium and treating the peripheral blood mononuclear cells with an anticancer drug are peripheral blood mononuclear cells obtained by culturing peripheral blood mononuclear cells collected from a subject infected with HTLV-1 in an animal cell culture medium, treating the peripheral blood mononuclear cells with an anticancer drug, and then contacting the peripheral blood mononuclear cells with an antibody capable of binding to HTLV-1-infected cells.

8. (a) culturing peripheral blood mononuclear cells collected from a subject infected with human T-cell leukemia virus type I (HTLV-1) in an animal cell culture medium; (b) treating the peripheral blood mononuclear cells obtained by the culture in the step (a) with an anticancer drug and then collecting the peripheral blood mononuclear cells; and (c) mixing the peripheral blood mononuclear cells collected in step (b) with a pharma- ceutically acceptable carrier to prepare a formulation; A method for producing an HTLV-I-specific CTL activator for administration to a subject, comprising the steps of:

9. The method for producing an HTLV-I-specific CTL activator according to claim 8, wherein the animal cell culture medium contains IL-2, IL-15 or both.

10. The method for producing an HTLV-I-specific CTL activator according to claim 8 or 9, wherein the animal cell culture medium contains a histone deacetylase inhibitor.

11. The method for producing an HTLV-I-specific CTL activator according to any one of claims 8 to 10, wherein the step (a) is a step of culturing peripheral blood mononuclear cells obtained by removing CD8 positive cells from peripheral blood mononuclear cells collected from a subject infected with HTLV-1 in an animal cell culture medium.

12. The method for producing an HTLV-I-specific CTL activator according to any one of claims 8 to 11, wherein the step (a) is a step of removing CD8 positive cells from peripheral blood mononuclear cells collected from a subject infected with HTLV-1, and then culturing the peripheral blood mononuclear cells activated with a CD3 antibody, or with both CD3 and CD28 antibodies, in an animal cell culture medium.

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