JP7601394B2 - HTLV-I specific CTL activator - Google Patents

Description

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

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 lymphoma 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, there is a need to establish a safe and effective treatment method for aggressive ATL.

On the other hand, the smoldering and chronic types of ATL (both types are collectively referred to as 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 can actually hasten the progression of the disease, so the clinical principle is to observe the disease without treatment until signs of transformation to aggressive ATL (such as an increase in blood lactate dehydrogenase (LDH) concentration) appear and the disease actually transforms into an aggressive form. Although early diagnosis of ATL is possible, there is nothing that can be done about indolent ATL until the disease has progressed, and there is a need to establish a treatment method for indolent ATL that is safe, effective, and can be applied early.

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).

Furthermore, since 2010, the recommended level for HTLV-1 antibody testing in prenatal checkups has been raised, and those who test positive for antibodies are now being notified of their infection. This is to reduce the frequency of vertical transmission of HTLV-1 to infants via 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. Furthermore, because there are no established methods for reducing the risk of developing the disease in HTLV-1-infected individuals or for preventing the disease, the reality is that only counseling is provided to infected pregnant women themselves, and the psychological and social problems associated with being notified of the infection are immeasurable. The same problem applies to notifying those who are found to be HTLV-1-infected during blood donation. Therefore, it is urgent to establish a method for preventing the disease from developing in HTLV-1-infected individuals.

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 conducted 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 an 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 achieved favorable results of surviving 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 applicable patients are limited to those with human leukocyte antigen (HLA) types HLA-A0201, HLA-A2402, or HLA-A1101. In Japan, ATL patients with such 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 therapeutic method that can be applied to a larger number of ATL patients.

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, to activate the response of CD8+CTL, antigen-presenting cells (APCs) that have phagocytosed antigens must be activated to express costimulatory molecules (CD86, etc.) and produce cytokines such as IL-12, and present 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, adjuvants available for use in humans are limited, and the efficiency of inducing CD8+ CTL responses is uncertain.

The present inventors have previously found that a rat model of oral infection with HTLV-1 exhibits immune tolerance to HTLV-1-specific T cells, that the amount of HTLV-1 provirus in the aforementioned model rat is inversely correlated with the HTLV-1-specific T cell response, and that the inoculation of syngeneic HTLV-1-infected cells into the aforementioned model rat restored 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 the 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 approximately half of the ATL patients, as well as costimulatory molecules such as CD80 (Non-Patent Document 4). In addition, Non-Patent Document 4 describes that when peripheral blood lymphocytes derived from an ATL patient were cultured and then formalin-fixed, and then administered to healthy rats, an HTLV-1-specific T cell response was induced in a CD4-predominant manner.

However, it was not previously known that administering PBMCs obtained by culturing PBMCs collected from HTLV-1-infected individuals, such as ATL patients, to a subject and treating the PBMCs with an anticancer drug can efficiently activate HTLV-1-specific CD8-positive CTLs in the subject.

International Publication No. 2006/035681

Br J Haematol, 169: 356-367, 2015 J Virol 77: 2956-2963. 2003 J Virol 80:7375-7381. 2006 Int J Cancer 114:257-267. 2005

The object of the present invention is to provide an HTLV-I-specific CTL activator and a method for producing the same.

In order to solve the above problems, the present inventors have intensively conducted the following research.
First, CD8-positive Tax-specific CTL and antigen-presenting cells with MHC-I matching with the CTL were prepared. Next, HTLV-1-infected T cells with MHC-I not matching with both of these cells (CD8-positive Tax-specific CTL 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 CTL, whereby IFN-γ production was observed. 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 CTL recognized the Tax antigen and produced IFN-γ. In addition, when the "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 the "cultured infected T cells treated with anticancer drugs" can be used as an activator of HTLV-I-specific CTLs.

It has also been 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.

In addition, it was shown that peripheral blood mononuclear cells obtained by removing CD8-positive cells from peripheral blood mononuclear cells collected from subjects infected with HTLV-1 and culturing the resulting cells were treated with an anticancer drug, resulting in a higher proportion of cells expressing Tax protein, an antigen derived from HTLV-1.

The inventors discovered these things and completed the present invention.

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-I 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, 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 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 (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.

According to the present invention, it is possible to provide an HTLV-I-specific CTL activator and a method for producing the same. Furthermore, according to the present invention, it is expected that it will be 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. Furthermore, according to the present invention, it is expected that it will be possible to provide a treatment that can be applied at an early stage to smoldering and chronic ATL (indolent ATL), which is currently treated as a rule by observation without treatment due to the lack of a safe and effective treatment.

Fig. 1 shows the results of detecting HTLV-1 Tax protein in cells stained with a secondary antibody (A488-labeled anti-mouse IgG) after 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 mouse anti-IgG antibody as a control antibody). The left panel of Fig. 1 shows the results on Day 0, and the right panel of Fig. 1 shows the results on Day 3. FIG. 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 FIG. 2 (Whole day 1): shows the results when aATL PBMCs were cultured as is for one day. The right panel of FIG. 2 (CD8(-) day 1): shows the results when CD8-positive cells were removed from aATL PBMCs and then the PBMCs were cultured for one day. FIG. 3 shows the results of detecting intracellular Tax protein in PBMCs after removing CD8-positive cells from the PBMCs of a chronic ATL patient (cATL) and stimulating them with CD3 and CD28 antibodies and culturing the PBMCs for 24 days. 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. 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. 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 represents the 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 represents the 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 represents the 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, which is a CTL, was cultured). 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. 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 bar graph ("MMC-ILT/(+)plasma") represents the results when "MMC-treated ILT-A cells" were contacted with plasma from an HTLV-1-infected individual, the gray bar graph ("MMC-ILT/(-)plasma") represents the results when "MMC-treated ILT-A cells" were contacted with plasma from a non-infected individual, and the open bar graph ("MMC-ILT") represents the results when "MMC-treated ILT-A cells" were not contacted with plasma from an HTLV-1-infected individual. 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 an aqueous formalin solution, and co-culture with HLA-A2-restricted CD8-positive Tax-specific CTLs (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 in FIG. 9 shows the results when ILT-A cells were not used. In addition, in Figure 9, the black bar graph ("THP1-AZT") represents the results when AZT was added to the medium and THP1 cells, etc. were cultured, the gray bar graph ("THP1") represents the results when AZT was not added to the medium, and the open bar graph ("Medium") represents the results when THP1 cells were not used. 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 line for 24 hours and 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). The lower left panel of FIG. 10 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 line. The upper left panel of FIG. 10 shows the results of measuring the expression of CD83 in cells obtained by culturing healthy immature dendritic cells without using MMC-treated ILT-C line. The lower right panel of FIG. 10 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 line. FIG. 10, upper right panel: shows the results of measuring the expression of CD86 in cells obtained by culturing immature dendritic cells from healthy donors without using the MMC-treated ILT-C line. FIG. 11 shows the results of measuring the IL-12 concentration (pg/mL) in the culture supernatant of ILT-A cells 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 (n.d.). 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), which was 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 bar graph ("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 (n.d.). 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 represents the results when cells obtained by culturing ILT-B cells in a DMSO-containing medium and treating the cells with MMC were used. "Medium" on the horizontal axis of FIG. 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 bar graph ("DC")), and the left side of each item represents the results when dendritic cells were not used (open white bar graph ("Medium"). Note that "n.d." represents below the detection limit.

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 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 (or an HTLV-I-specific CTL activator or composition for activating HTLV-I) for administration to a subject, comprising:
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.

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 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) 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 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) 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 the 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 a disease 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 agent" 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 above aspects are also included.

(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").

Types of peripheral blood mononuclear cells (PBMCs) in the present invention include peripheral blood-derived T cells, B cells, NK cells, and monocytes, of which peripheral blood-derived T cells are particularly preferred.

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 developing 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.

Known methods can be used to determine whether a subject is infected with HTLV-1. Such known methods include the Western blot method or line blot method, which examines whether anti-HTLV-1 antibodies are present in the subject's blood, and nucleic acid detection methods (PCR methods) that specifically detect HTLV-1 viral DNA (proviral DNA) in the genome of the subject's PBMC.

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 above density gradient centrifugation method.

A preferred example of peripheral blood mononuclear cells collected from a subject infected with HTLV-1 ("PBMCs derived from an infected subject") is PBMCs infected with HTLV-1. A cell population of PBMCs collected from a subject infected with HTLV-1 needs only to contain PBMCs infected with HTLV-1, and it is not necessary for all individual cells in the cell population to be infected with HTLV-1, but the higher the proportion of infected cells, the better, and examples of cells in the PBMCs of such a cell population that are infected with HTLV-1 include 5% or more, 15% or more, and 40% or more of the cells.

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

As a preferred example of PBMCs derived from an infected subject, there can be mentioned 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, is increased when the PBMCs are cultured, which is preferable.

(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 RPMI 1640 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.

The animal cell culture medium of the present invention may be a serum-free medium, but may also be 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 basic animal cell culture medium.

In addition, 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 PBMCs derived from infected subjects are 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.

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 PBMCs derived from an infected subject, when such PBMCs are co-cultured with antigen-presenting cells (APCs), IL-12 production from the APCs increases, and the cross-presentation efficiency of the APCs is also improved, which is preferable.

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., of which SAHA and VPA are preferred. Two or more types of HDAC inhibitors may be used in combination. Commercially available HDAC inhibitors can be used.

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

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 antibodies 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 antibodies and CD28 antibodies attached 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, to the animal cell culture medium of the present invention.

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

The aforementioned temperature conditions can be, for example, 30 to 39°C, preferably 35 to 39°C.

Examples of the aforementioned carbon dioxide concentration conditions include 3 to 7%, 4 to 6%, etc.

The lower limit of the aforementioned culture period can be, for example, 1 day or more, and from the viewpoint of increasing the proportion 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 specific examples of the aforementioned culture period can include 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.

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 mL of medium used at the start of culture can be, for example, ¹⁰ to ¹⁰ cells/mL, 5 x ¹⁰ to 5 x ¹⁰ cells/mL, etc.

(Anticancer drug)
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 believed that HTLV-1 neutralizing antibodies already present in the body fluids of the infected person will act, and new HTLV-I infection based on the cultured infected PBMCs will not essentially occur. Furthermore, in the present invention, the cultured infected PBMCs themselves are almost completely killed by treating the cultured infected PBMCs with an anticancer drug, so that there is almost no possibility of new HTLV-I infection occurring, and even if there is any, it is believed to be extremely limited and transient.

In this specification, the term "anticancer agent" 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 normally used as anticancer agents, but anticancer agents are preferred.

Such anticancer agents include DNA replication inhibitors such as antitumor antibiotics, alkylating agents, platinum preparations, and 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.

Examples of the above-mentioned antitumor antibiotics include mitomycin C (MMC), doxorubicin, epirubicin, daunorubicin, bleomycin, sarkomycin, etc., of which mitomycin C is preferred.

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

Examples of platinum agents include cisplatin, carboplatin, oxaliplatin, and nedaplatin.

Examples of the above topoisomerase inhibitors include irinotecan, etoposide, doxorubicin, epirubicin, ofloxacin, ciprofloxacin, and levofloxacin.

Examples of the above-mentioned folate metabolic antagonists include dihydropteroate synthase inhibitors such as sulfadiazine, sulfamethoxazole, and diaphenylsulfone; dihydrofolate reductase inhibitors such as methotrexate, trimethoprim, and pyrimethamine; and the like.

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

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.

Examples of the above-mentioned microtubule polymerization inhibitors include vinblastine, vincristine, vindesine, and fordesine.

Examples of the above-mentioned microtubule depolymerization inhibitors include paclitaxel and docetaxel.

Examples of the above molecular targeted therapeutic drugs include mogamulizumab.

Two or more of these anticancer drugs may be used in combination. Commercially available anticancer drugs may also be used.

(Method of treating 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.

The concentration of the anticancer agent in a solution such as a medium is not particularly limited as long as it is capable of inhibiting 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 agent 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.

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.

After treating the cultured infected PBMCs with the anticancer drug, it is preferable to wash the cultured infected PBMCs with a medium or the like in order to remove the anticancer drug from the cultured infected PBMCs. After such washing, the cultured infected PBMCs can be collected by centrifugation or the like.

(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 enhance phagocytosis by antigen-presenting cells, it is preferable to further contact them with an antibody capable of binding to HTLV-1-infected cells.

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

An example of a method for contacting cultured, infected PBMCs treated with an anticancer drug with an antibody capable of binding to HTLV-1-infected cells is to place the cultured, infected PBMCs treated with an anticancer drug 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.

(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 agent (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 agent), or may further contain other components (i.e., optional components).

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

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

The content of "cultured infected PBMCs treated with an anticancer agent" 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. Furthermore, the content of "cultured infected PBMCs treated with an anticancer agent" in the HTLV-I-specific CTL activator of the present invention can be, for example, ¹⁰ to ¹⁰ cells/mL, 5 x ¹⁰ to 5 x ¹⁰ cells/mL, etc., expressed in terms of the number of "cultured infected PBMCs treated with an anticancer agent" per mL of HTLV-I-specific CTL activator.

(Dosage)
The dose of the HTLV-I-specific CTL activator of the present invention is adjusted appropriately depending on the state of the disease, the age, weight, etc. of each patient, but the number of "cultured infected PBMCs treated with an anticancer agent" in a single administration to a subject may be, for example, ¹⁰ to ¹⁰ , preferably ⁵ x 10 to 5 x ^10. It is preferable to repeatedly administer such an administration once every few days, weeks, or months, for a total of two or more times (e.g., 2 to 10 times).

(Administration Method)
The method of administration of the HTLV-I-specific CTL activator of the present invention is not particularly limited as long as the HTLV-I-specific CTL activating effect can be obtained, and examples thereof include subcutaneous administration, intradermal administration, and intramuscular administration, with subcutaneous administration being preferred.

(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 this case, the HTLV-I-specific CTL activator can activate HTLV-I-specific CTLs in the subject without causing a rejection reaction.

(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, an effect of preventing recurrence after chemotherapy, etc. Furthermore, 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 who have not developed HTLV-1 disease but are infected with HTLV-I, by activating HTLV-I-specific CTL.

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 diseases caused by HTLV-1 infection (or a pharmaceutical composition for improving), an agent for suppressing the progression of diseases caused by HTLV-1 infection (or a pharmaceutical composition for suppressing the progression), an agent for suppressing the onset of diseases caused by HTLV-1 infection (or a pharmaceutical composition for suppressing the onset), an agent for preventing or treating diseases caused by HTLV-1 infection (or a pharmaceutical composition for preventing or treating), etc.

(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, improving the cytotoxic activity of HTLV-I-specific CTL against HTLV-1 in the body of a subject to which the HTLV-I-specific CTL activator of the present invention or "cultured infected PBMC treated with an anticancer agent" is administered, by inducing cross-presentation of antigen-presenting cells (preferably dendritic cells) in the body of the subject, and then inducing HTLV-I-specific CTL from naive CD8-positive T cells that have no cytotoxic activity.

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 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.

(Method for 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 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;
There is no particular limitation as long as it has the above structure.

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 with both CD3 and CD28 antibodies, and then culturing the resulting 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, more preferably 0.8 to 1.2 times) the number of PBMCs (peripheral blood mononuclear cells from which CD8-positive cells have been removed).

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 step (a) with an anticancer drug and then harvesting the peripheral blood mononuclear cells. The method of treatment with the anticancer drug and the method of harvesting are as described above.

A preferred example of step (b) above 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 harvesting the peripheral blood mononuclear cells.

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 step (b) with a pharma- ceutically acceptable carrier to prepare a formulation. Such a method is as described above.

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

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, it is necessary to express the HTLV-1 Tax antigen. Therefore, the following test was carried out to investigate how the Tax antigen is expressed by culturing HTLV-1-infected peripheral blood mononuclear cells.

(1) Method of culturing PBMC 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. The PBMCs were added directly at 5 x 105 cells/mL to RPMI 1640 medium (hereinafter referred to as "IL-2-added medium") containing 10% FCS (fetal calf serum) and rhIL-2 (Immunace (registered trademark), manufactured by Shionogi Pharmaceutical Co., Ltd.) at ¹⁰ u/mL, and cultured at 37°C in a _CO2 incubator for several days to several weeks.

(2) Method for culturing PBMCs from which CD8-positive cells have been removed Instead of adding the above-mentioned PBMCs directly to an IL-2-supplemented medium, the above-mentioned PBMCs from which CD8-positive cells have been removed were added to an IL-2-supplemented medium and cultured in a _CO2 incubator at 37° C. for several days to several weeks. For the removal of CD8-positive cells from the PBMCs, immunobeads (Dynabeads (registered trademark), ThermoFisher) to which CD8 antibodies have been attached were used.

(3) Confirmation of Tax antigen expression in PBMCs after culture After culturing PBMCs derived from aATL patients for 3 days using the method described in (1) above, 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.

After 3 days of culture, the 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 the PBMCs were stained for 30 minutes. After washing these PBMCs, HTLV-1 Tax protein in the PBMCs was detected using a cell analyzer MACSQuant (Miltenyi Biotec) (right panel of Figure 1 (Day 3)).

On the other hand, as a control, similar flow cytometry was performed on pre-culture PBMCs 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)).

As can be seen from the results in Figure 1, Tax protein was not detectable at all in the PBMCs before culture, but after 3 days of culture, Tax protein was detectable in more than half of the PBMCs. These results indicate that culturing PBMCs from aATL patients induces intracellular expression of the Tax antigen.

(4) Effect of removing CD8 positive cells from PBMCs After culturing PBMCs from aATL patients for 1 day using the method described in (1) above, 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.

In addition, after removing CD8 positive cells from PBMCs derived from aATL patients using the method described in (2) above, the PBMCs were cultured for one day using the method described in (1) above. 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 right panel of Figure 2.

As can be seen from the results in Figure 2, the percentage of cells in which Tax protein was detected (7.15%) when CD8-positive cells were removed from PBMCs and then cultured for one day was higher than the percentage (5.33%) when PBMCs were cultured as is for one day. These results demonstrate that removing CD8-positive cells from infected PBMCs before culturing is preferable in that it increases the percentage of cells expressing Tax protein, an antigen derived from HTLV-1.

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

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

(6) Effect 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 PBMCs from an acute ATL patient, and the ILT-B line is an IL-2-dependent cell line derived from PBMCs from 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 cells or ILT-B cells are cultured.

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.

ILT-A cells were cultured in a SAHA-containing medium and a control medium for 2 days each at 37°C in a _CO2 incubator. The ILT-A cells were subjected to flow cytometry by 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 when the PBMC cell line was cultured in the presence of an HDAC inhibitor, the efficiency of induction of Tax protein expression was improved compared to when the PBMC cell line was cultured in the absence of an HDAC inhibitor.

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 to 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 CTL recognizes the presented Tax antigen and produces IFN-γ in the culture medium. The level of activation of CTL by presentation can be evaluated based on the level of IFN-γ concentration in the culture medium supernatant. Evaluation of cross-presentation using this evaluation system was specifically performed by the following method.

The Tc-M1 line, which is an HLA-A2-restricted CD8-positive Tax-specific CTL, was used as the CD8-positive Tax-specific CTL. The Tc-M1 line was derived by culturing PBMCs from a HAM/TSP patient in the presence of 100 u/mL rhIL-2 and repeatedly stimulating them with autologous HTLV-1-infected cells at two-week intervals (International immunology 3:761-767). The human monocyte-derived THP1 cell line (Virology 318:17-23) was used as the HLA-A2-positive antigen-presenting cell. The antigen cells used were "ILT-A cells (HTLV-1-infected T cell line derived from an ATL patient) not having HLA-A2 cultured in an IL-15-supplemented medium for 2 days, 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, also referred to as "MMC-treated ILT-A cells"), or "ILT-A cells (HTLV-1-infected T cell line derived from an ATL patient) not having HLA-A2 cultured in an IL-15-supplemented medium for 2 days, treated with 1% by weight formalin for 15 minutes, and then washed."

The aforementioned antigen cells not bearing HLA-A2 were co-cultured with the 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 the washed cells were then co-cultured with the aforementioned HLA-A2-restricted CD8-positive Tax-specific CTL (Tc-M1) for 16 hours in RPMI1640 medium containing 25 u/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.

As a control experiment, the antigen cells were not co-cultured with the antigen-presenting cells, but were 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.

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 CTLs were cultured).

The results in FIG. 6 indicate the following.
CD8-positive Tax-specific CTL (Tc-M1) is hardly activated even when co-cultured only with MMC-treated ILT-A cells. Also, CD8-positive Tax-specific CTL (Tc-M1) is hardly activated even when co-cultured only with THP1 cells (antigen-presenting cells). However, CD8-positive Tax-specific CTL (Tc-M1) is activated (produces 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) are co-cultured with antigen-presenting cells (e.g., THP1 cells), the antigen-presenting cells take up the HTLV-1-infected T cells and present the Tax antigen derived from HTLV-1 on MHC-I. In other words, it was considered that the antigen-presenting cells cross-present the Tax antigen.
As can be seen from the results of "ILT-Formalin+THP1" in Figure 6, even when CTLs were co-cultured with "cells co-cultured with formalin-fixed HTLV-1-infected T cells and antigen-presenting cells," the CTLs were hardly activated.

Test 3. [Effects of HTLV-1 neutralizing antibodies and reverse transcriptase inhibitors]
In the above-mentioned Test 2, it was shown that 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". In order to confirm that this phenomenon in Test 2 is not the result of HTLV-1 antigens generated from newly infected cells infected with HTLV-1 from ILT-A cells, which are HTLV-1-infected cells, newly infected with HTLV-1 were 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 experiment was performed to examine the effect of HTLV-1 neutralizing antibodies and reverse transcriptase inhibitors on the efficiency of cross-presentation induction of Tax antigen by HTLV-1-infected cells.

(1) Effect of HTLV-1 neutralizing antibodies High concentrations of HTLV-1 neutralizing antibodies are contained in the blood of HTLV-1 infected individuals, and it is expected that these antibodies will bind to cells expressing HTLV-1 antigens. Mogamulizumab (trade name Poteligeo (registered trademark); manufactured by Kyowa Hakko Kirin Co., Ltd.), an anti-CCR4 antibody, also binds to HTLV-1 infected cells. The following experiment was carried out to examine the effect of these antibodies binding to HTLV-1 infected cells.

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 ⁵ cells of the aforementioned "MMC-treated ILT-A cells" were added to the plasma aqueous solutions to bring the antibodies in the plasma into contact with the ILT-A cells. After washing, the MMC-treated ILT-A cells were co-cultured with approximately 10 ⁵ cells of THP1 cells. All cells obtained by this 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.

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 an aqueous solution containing 0.5% by weight of patient plasma, and the IFN-γ concentration in the supernatant was measured.

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, IFN-γ concentrations were not significantly reduced when cells were treated with plasma from HTLV-1-infected individuals ("ATL-1," "ATL-2," "ATL-3," "HAM-1," "HAM-2," and "HAM-3" in Figure 7) or when cells were 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. Furthermore, no significant differences were observed between treatment with plasma from HTLV-1-infected individuals ("ATL-1," "ATL-2," "ATL-3," "HAM-1," "HAM-2," and "HAM-3" in Figure 7), treatment with plasma from non-infected individuals ("SN-1" and "SN-2" in Figure 7), and treatment with anti-CCR4 antibody ("Poteligeo" in Figure 7).

(2) Effect of the ratio of the number of cells between “MMC-treated ILT-A cells” and THP1 cells
The ILT-A cells were cultured in IL-15-supplemented medium for 2 days (as described above), 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."
When "MMC-treated ILT-A cells" and THP1 cells were co-cultured, the ratio of the number of ILT-A cells to that of THP1 cells was set to 1:1, 0.5:1, or 0.25:1, and the IFN-γ concentration was measured in the same manner as in "(1) Effect of HTLV-1 neutralizing antibody" above. In this case, ATL-1 plasma was used as the plasma of the ALT patient.

The results of these experiments are shown in Figure 8. The horizontal axis represents the number of HTLV-1-infected T cells, and the first to third bars from the left represent the results when the number of HTLV-1-infected T cells was ¹⁰⁵ (i.e., the aforementioned ratio was 1:1), the fourth to sixth bars from the left represent the results when the number of HTLV-1-infected T cells was ^5x104 (i.e., the aforementioned ratio was 0.5:1), and the first to third bars from the right represent the results when the number of HTLV-1-infected T cells was ^2.5x104 (i.e., the aforementioned ratio was 0.25:1). In addition, in Figure 8, the black bar graph ("MMC-ILT/(+)plasma") represents the results when "MMC-treated ILT-A cells" were contacted with plasma from an HTLV-1-infected individual, the gray bar graph ("MMC-ILT/(-)plasma") represents the results when "MMC-treated ILT-A cells" were contacted with plasma from a non-infected individual, and the open bar graph ("MMC-ILT") represents the results when "MMC-treated ILT-A cells" were not contacted with plasma from an HTLV-1-infected individual.

From the results in Figure 8, it was observed that the CTL activation effect tends to increase to some extent when ILT-A cells are treated with plasma from HTLV-1-infected individuals. This tendency was more pronounced when the number of target cells, "MMC-treated ILT-A cells," was relatively small compared to the number of antigen-presenting cells ("5-2.5 x 10 ⁴ " in Figure 8). The increase in the CTL activation effect when ILT-A cells are 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.

(3) Effect of reverse transcriptase inhibitor 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 pre-cultured in a medium containing 20 μM AZT at 37° C. for 1 hour. Meanwhile, 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 pre-cultured as described above, and co-cultured at 37° C. for 16 hours. The AZT concentration in the medium during co-culture was 10 μM. All cells obtained by this 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 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.

The measurement results of these IFN-γ concentrations are shown in FIG. 9. The first to third bars from the right show the results when HTLV-1-infected T cells (ILT-A cells) were 10 ⁵ cells (i.e., the aforementioned ratio was 1:1), the fourth to sixth bars from the right show the results when HTLV-1-infected T cells (ILT-A cells) were 3×10 ⁴ cells (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 FIG. 9, the black bars ("THP1-AZT") show the results when AZT was added to the medium and THP1 cells or the like were cultured, the gray bars ("THP1") show the results when AZT was not added to the medium, and the open bars ("Medium") show the results when THP1 cells were not used.

As can be seen from the results in Figure 9, AZT treatment had no significant effect on CTL activity. 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.

Test 4. [Enhancement of expression of costimulatory molecules in antigen-presenting cells by HTLV-1-infected T cells]
In order for antigen-presenting cells such as dendritic cells to activate T cells, it is necessary for them 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, the following experiment was performed.

(1) Immature dendritic cells derived from PBMCs of healthy subjects 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 supplemented with 10% FCS in a plastic plate, culturing PBMCs of a healthy subject 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 monocytes attached to the plastic plate, and culturing for 5 days (hereinafter, also referred to as "healthy subject immature dendritic cells").

(2) Evaluation of expression of costimulatory molecules ILT-C22 cells (HLA-A2 positive) were cultured in IL-2-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 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 RPMI 1640 medium supplemented with 10% FCS, and then stained for 30 minutes on ice using 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 the MMC-treated ILT-C line was not used (i.e., using healthy immature dendritic cells not co-cultured with the MMC-treated ILT-C line). Dendritic cells and ILT-C cells were distinguished based on the presence or absence of HLA-A2 expression.

These measurement results are shown in Figure 10. Compared to healthy donor immature dendritic cells without the MMC-treated ILT-C line, healthy donor immature dendritic cells co-cultured with the MMC-treated ILT-C line showed a mild increase in CD83 expression (upper left and lower left panels of Figure 10) and a significant increase in CD86 expression (upper right and lower right panels of Figure 10). These results demonstrated that when MMC-treated HTLV-1-infected T cells and immature dendritic cells were co-cultured, 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.

Test 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 cells or ILT-B cells on IL-12 production in antigen-presenting cells.

(1) Effect of drug treatment of HTLV-1-infected T cells on IL-12 expression in antigen-presenting cells Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, was added to RPMI 1640 medium supplemented with 10% FCS (fetal calf serum) containing IL-15 or IL-2 to prepare a SAHA-containing medium containing 0.5 μM SAHA. 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.

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). After the culture, the supernatant of the medium was separated, 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 FIG. 11.

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 (solid bars ("DC") in "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 level (right side of "DMSO" in "ILT-A" in Figure 11). On the other hand, when ILT cells (ILT-A cells or ILT-B cells) cultured in a 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 when ILT cells (ILT-A cells or ILT-B cells) cultured in a DMSO-containing medium were used (solid bars for "SAHA" in "ILT-A" ("DC") and solid bars for "SAHA" in "ILT-B" ("DC") in Figure 11).

Figure 12 shows the results when another HDAC inhibitor, valproic acid (VPA), was used instead of SAHA. 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 by "DC" in Figure 12, when ILT-B cells were cultured in medium containing 1 mM VPA, IL-12 production into the medium was significantly increased compared to when ILT-B cells were cultured in DMSO-containing medium (solid bars in Figure 12 ("DC")).

(2) Effect of drug treatment of HTLV-1-infected T cells on antigen cross-presentation by antigen-presenting cells After each cell was treated with 1% formalin by weight for 15 minutes after co-culture in FIG. 11, 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®). These results are shown in FIG. 13.

As can be seen from 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 treating HTLV-1-infected T cells with an HDAC inhibitor such as SAHA also improves the efficiency of antigen-presenting cells cross-presenting antigens to Tax-specific CTLs.

According to the present invention, it is possible to provide an HTLV-I-specific CTL activator and a method for producing the same. Furthermore, according to the present invention, it is expected that it will be 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. Furthermore, according to the present invention, it is expected that it will be possible to provide a treatment that can be applied at an early stage to smoldering and chronic ATL (indolent ATL), which is currently treated as a rule by observation without treatment due to the lack of a safe and effective treatment.

Claims (12)

An HTLV-I-specific cytotoxic T cell (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 resulting peripheral blood mononuclear cells with an anticancer agent. 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. An HTLV-I-specific CTL activator according to claim 1 or 2, wherein the animal cell culture medium contains IL-2, IL-15 or both. An 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. An 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. An HTLV-I-specific CTL activator according to any one of claims 1 to 5, wherein 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. 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. (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: A method for producing an HTLV-I-specific CTL activator as described in claim 8, wherein the animal cell culture medium contains IL-2, IL-15 or both. A 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. A method for producing an HTLV-I-specific CTL activator according to any one of claims 8 to 10, wherein 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. A method for producing an HTLV-I-specific CTL activator according to any one of claims 8 to 11, wherein 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 peripheral blood mononuclear cells activated with a CD3 antibody or both CD3 and CD28 antibodies in an animal cell culture medium.

Images