JP2014133712A - Hla-dr1 restrictive tax specific cd4+ t cell epitope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an HTLV-I specific CD4T cell-inducing active peptide having an inducing activity of HTLV-I specific CD4T cells, an HTLV-I specific CTL inductive action enhancer containing the peptide, and an HTLV-I specific immune response-inducing vaccine and a diagnostic agent for immunological function test using them.SOLUTION: There is provided an HTLV-I specific CD4T cell-inducing activity peptide consisting of any of a specific amino acid sequence, an amino acid sequence consisting of 14-30 continuous amino acids in a specific amino acid sequence or an amino acid sequence made by binding the amino acid sequence and an amino acid sequence of an HTLV-I specific CTL-inducing activity peptide, and an amino acid sequence which has an identity of 80% or more to the above amino acid sequence, or in which one to some amino acids are deleted, substituted or added, wherein a peptide consisting of the amino acid sequence has HTLV-I specific CD4T cell-inducing activity.

Description

The present invention relates to an HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide having an activity to induce CD4 ⁺ T cells specific to human T-cell leukemia virus type I (HTLV-I), and an HTLV containing the peptide. The present invention relates to -I-specific CTL inducing action enhancer and the like.

Human T cell leukemia virus type I (HTLV-I) is a causative virus of adult T cell leukemia / lymphoma (ATL), which is an extremely refractory CD4 ⁺ T cell malignancy (non-patented) References 1 and 2). From 12,000 to 20 million people worldwide are infected with this virus, and there are many cases especially in the southern region of Japan, the Caribbean region, South America, Melanesia, and Equatorial Africa. About 5% of HTLV-I seropositive individuals develop ATL, and 2-3% of others have HTLV-I associated myelopathy / tropical spastic paraparesis (HAM / TSP: HTLV-I associated) It develops slowly progressive neurological disease known as myelopathy / tropical spastic paraparesis) and various chronic inflammatory diseases (Non-patent Document 3). The vast majority of HTLV-I infected individuals are in asymptomatic carriers throughout their lifetime.

  ATL is characterized by a very poor prognosis, mainly because ATL inherently has drug resistance to anticancer drugs. It has been reported that an improvement in the prognosis of ATL that could not be achieved by autologous hematopoietic stem cell transplantation was observed in allogeneic hematopoietic stem cell transplantation (non-patent documents 4 and 5). In the case of allogeneic hematopoietic stem cell transplantation that transplants stem cells of other people including relatives, in order to eradicate the patient's cancer cells and weaken the patient's own immunity, it is easy to engraft the donor cells in the patient's bone marrow, Prior to transplantation, a large amount of anticancer drug administration or whole body irradiation was performed, and pretreatment was conventionally performed to reduce the bone marrow cells of the patient to the limit. However, in recent years, donor lymphocytes that increase in the patient's body after transplantation attack the patient's cancer cells that remain unkilled by the pretreatment as a non-self foreign substance, and eventually lead to a complete cure. Once the GVL effect is known, mitigating the use of immunosuppressive agents to engraft donor hematopoietic stem cells instead of minimizing the amount of radiation and anticancer agents previously used as pretreatment Pretreatment (RIC: reduced intensity conditioning) has been performed. Allogeneic hematopoietic stem cell transplantation with palliative pretreatment, also called mini-transplantation or non-myeloablative transplantation, can be applied to elderly patients who have not been able to receive allogeneic hematopoietic stem cell transplantation until now and patients with organ disorders It is becoming widely recognized as a law.

In a clinical trial previously conducted by the ATL allo-HSCT study group, the overall survival rate within 3 years after allogeneic hematopoietic stem cell transplantation by palliative pretreatment was 36% (Non-patent Document 6). HTLV-I provirus is no longer detected in some ATL patients who have achieved complete remission after allogeneic hematopoietic stem cell transplantation and this undetected condition continues, which makes allogeneic hematopoietic stem cell transplantation an effective treatment for ATL (Non-patent documents 6 to 8). In connection with these studies, we are part of ATL patients who have achieved complete remission after allogeneic hematopoietic stem cell transplantation, with donor-derived HTLV-ITax-specific CD8 ⁺ cytotoxic T lymphocytes (CTL: reported that cytotoxic T lymphocytes were induced (Non-patent Document 9), and found HTLV-I-specific CTL-inducing active peptides (Tax-specific CTL epitopes) restricted by human HLA-A24 (patent) Reference 1). The aforementioned CTL was able to lyse autologous HTLV-I infected T cells in vitro. This suggested that CTL may contribute to the graft versus leukemia effect. In addition, the present inventors have also found an HTLV-I-specific CTL inducing active peptide (Tax-specific CTL epitope) restricted by human HLA-A11 (Patent Document 2). In general, CD8 ⁺ T cells, particularly CTL, play an important role in the control of viral replication in various infectious diseases involving HIV, hepatitis B virus (HBV), hepatitis C virus (HCV) and the like. In HTLV-I infection, it is considered that HTLV-I-specific CD8 ⁺ T cells recognize the Tax antigen (pX gene product) as a predominant and contribute to the control of infected cells (Non-Patent Documents 10 and 11). Functional Tax-specific CD8 ⁺ T cells could be detected with high frequency in HAM / TSP patients and some asymptomatic carriers (AC), but in the majority of ATL patients and in a small population of ACs The Tax-specific CD8 ⁺ T cell response was significantly reduced (Non-patent Documents 12 and 13). The mechanism by which the HTLV-I-specific CD8 ⁺ T cell response is suppressed in these patients is not yet fully understood.

In order to induce and maintain virus-specific CTL, a virus-specific CD4 ⁺ helper T cell response is required in many viral infections (Non-Patent Documents 14 to 18), but HTLV-I-specific helper T cell response There were few reports (Non-Patent Documents 19 to 22). This is probably because HTLV-I-specific helper T cells are sensitive to HTLV-I infection in vivo and in vitro (Non-Patent Document 23), and infected cells produce HTLV-I antigens within a few hours after culture. (Non-Patent Documents 24 and 25). On the other hand, in patients with HAM / TSP, CD4 ⁺ at the site of early progressive inflammatory spinal cord injury with spontaneous production of inflammatory neurotoxic cytokines such as IFN-γ and tumor necrosis factor (TNF) -α (Non-Patent Document 26). T cells are found in predominant (Non-Patent Documents 27 to 28), suggesting that this is involved in the development of HAM / TSP. However, the exact role of HTLV-I-specific CD4 ⁺ T cells in HTLV-I infection has not yet been elucidated.

International Publication No. 2004/092373 Pamphlet International Publication No. 2006/035681 Pamphlet

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Circulating CD8 + cytotoxic T lymphocytes specific for HTLV-I pX in patients with HTLV-I associated neurological disease.Nature. 1990; 348 (6298): 245-248. Kannagi M, Harada S, Maruyama I, et al. Predominant recognition of human T cell leukemia virus type I (HTLV-I) pX gene products by human CD8 + cytotoxic T cells directed against HTLV-I-infected cells.Int Immunol. 1991; 3 (8): 761-767. Takamori A, Hasegawa A, Utsunomiya A, et al. Functional impairment of Tax-specific but not cytomegalovirus-specific CD8 + T lymphocytes in a minor population of asymptomatic human T-cell leukemia virus type 1-carriers. Retrovirology. 2011; 8: 100 . Shimizu Y, Takamori A, Utsunomiya A, et al. Impaired Tax-specific T-cell responses with insufficient control of HTLV-1 in a subgroup of individuals at asymptomatic and smoldering stages.Cancer Sci. 2009; 100 (3): 481- 489. 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As described in the background art, several peptides having an activity of inducing CD8 ⁺ T cells (CTL) specific for HTLV-I have been found so far, and a vaccine for inducing HTLV-I specific CTL. It is expected to be put to practical use. However, in order to induce and maintain virus-specific CTL, a virus-specific CD4 ⁺ helper T cell response is required in many virus infections. A peptide having an activity of inducing CD4 ⁺ T cells specific for HTLV-I is required. However, since HTLV-I infection occurs preferentially in HTLV-I-specific CD4 ⁺ T cells, etc., analysis of such CD4 ⁺ T cells has not progressed much, and CD4 ⁺ T specific for HTLV-I has not progressed. No peptide having cell-inducing activity has been found so far.

An object of the present invention, and HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide having an activity to induce CD4 ⁺ T cells specific for HTLV-I, HTLV-I-specific CTL-inducing action containing the peptide An object of the present invention is to provide a potentiator, a vaccine for inducing an HTLV-I-specific immune response using these, a diagnostic agent for examining an immune function, and the like.

As a result of intensive studies under the above-mentioned background art, the present inventors have found the following findings (a) to (c) and completed the present invention.
(A) Investigating the cellular immune response of the same patient after allogeneic hematopoietic stem cell transplantation by palliative pretreatment against HTLV-I infected T cells derived from ATL patients before allogeneic hematopoietic stem cell transplantation by palliative pretreatment, Both Tax-specific CD4 ⁺ T cell responses and CD8 ⁺ T cell responses are induced in ATL patients after allogeneic hematopoietic stem cell transplantation with palliative pretreatment.
(B) Tax-specific CD4 ⁺ T cell minimal epitope restricted to human HLA-DRB1 ^* 0101 (hereinafter also referred to as “HLA-DR1” in this specification), ie, HTLV-I specific CD4 ⁺ Identified a T cell-inducing active peptide.
(C) When such an HTLV-I-specific CD4 ⁺ T cell-inducing active peptide and an HTLV-I-specific CTL-inducing active peptide are used in combination, the HTLV-I-specific CTL inducing active peptide is used alone. HTLV-I-specific CTLs proliferated significantly.

That is, the present invention
(1) HTLV-I specific CD4 ⁺ T cell-inducing peptide consisting of the amino acid sequence shown in any of the following (A) to (E):
(A) Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence induces HTLV-I-specific CD4 ⁺ T cells. Amino acid sequence with activity:
(D) In the amino acid sequence shown in the above (A) or (B), one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is HTLV-I specific Amino acid sequence having CD4 ⁺ T cell inducing activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL An amino acid sequence combined with the amino acid sequence of the inducing active peptide,
(2) The HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide according to (1) above, which is a Tax epitope restricted by HLA-DR1,
(3) The HTLV-I specific CD4 ⁺ T cell-inducing peptide according to (1) or (2) above, wherein the CD4 ⁺ T cell is a Th1-type helper T cell,
(4) a fusion peptide obtained by binding the HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide according to any one of (1) to (3) above to a marker protein and / or a peptide tag;
(5) a protein-peptide conjugate in which HLA-DR1 is bound to the HTLV-I-specific CD4 ⁺ T cell-inducing peptide according to any one of (1) to (3) above,
(6) a tetramer of a protein-peptide conjugate in which HLA-DR1 is bound to the HTLV-I-specific CD4 ⁺ T cell-inducing active peptide according to any one of (1) to (3) above,
(7) The present invention relates to a fusion protein in which the protein-peptide conjugate described in (5) above or the tetramer of the protein-peptide conjugate described in (6) above is bound to a marker protein and / or a peptide tag.

The present invention also provides:
(8) An HTLV-I-specific CTL inducing action potentiator comprising, as an active ingredient, an HTLV-I-specific CD4 ⁺ T cell-inducing peptide consisting of any of the following amino acid sequences (A) to (E):
(A) Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence induces HTLV-I-specific CD4 ⁺ T cells. Amino acid sequence with activity:
(D) In the amino acid sequence shown in the above (A) or (B), one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is HTLV-I specific Amino acid sequence having CD4 ⁺ T cell inducing activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL An amino acid sequence combined with the amino acid sequence of the inducing active peptide,
(9) An expression vector comprising a promoter and a polynucleotide comprising the polynucleotide sequence shown in any of the following (a) to (e), wherein the polynucleotide is operably linked downstream of the promoter. HTLV-I specific CTL inducing action enhancer containing as an active ingredient:
(A) a polynucleotide sequence encoding the amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) a polynucleotide sequence encoding an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 32 and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) a polynucleotide sequence having 80% or more identity to the polynucleotide sequence shown in the above (a) or (b), wherein the peptide has HTLV-I-specific CD4 ⁺ T cell inducing activity Encoding polynucleotide sequence:
(D) a polynucleotide sequence obtained by deleting, substituting, or adding one or several nucleotides in the polynucleotide sequence shown in (a) or (b), wherein the HTLV-I specific CD4 ⁺ T cell Polynucleotide sequence encoding a peptide having inductive activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising at least an amino acid sequence of amino acid numbers 155 to 167, and an HTLV-I-specific CTL A polynucleotide sequence encoding an amino acid sequence in which the amino acid sequence of the inducing active peptide is linked,
(10) HTLV-I containing the HTLV-I-specific CTL inducing action potentiator according to (8) or (9) above, and an HTLV-I-specific CTL inducing active peptide or a polynucleotide encoding the peptide The present invention relates to a vaccine for inducing a specific immune response.

Furthermore, the present invention provides
(11) below (A) containing - a HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide comprising the amino acid sequence shown in any of (E) as an active ingredient, HTLV-I-specific CD4 ⁺ T cells Immune function test diagnostic agent to identify:
(A) Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence induces HTLV-I-specific CD4 ⁺ T cells. Amino acid sequence with activity:
(D) In the amino acid sequence shown in the above (A) or (B), one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is HTLV-I specific Amino acid sequence having CD4 ⁺ T cell inducing activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL An amino acid sequence combined with the amino acid sequence of the inducing active peptide,
(12) HTLV comprising, as an active ingredient, a protein-peptide conjugate in which HLA-DR1 is bound to the HTLV-I-specific CD4 ⁺ T cell-inducing active peptide according to any one of (1) to (3) above -Immune function test diagnostic agent for identifying I-specific CD4 ⁺ T cells,
(13) A tetramer of a protein-peptide conjugate in which HLA-DR1 and the HTLV-I-specific CD4 ⁺ T cell-inducing peptide according to any one of (1) to (3) above are bound as an active ingredient The present invention relates to a diagnostic agent for immune function test for identifying HTLV-I-specific CD4 ⁺ T cells.

The present invention also provides:
(14) HTLV-I-specific CD4 characterized in that HTLV-I-infected T cells derived from ATL patients before HSCT are used to stimulate PBMCs of the same patients after HSCT from allogeneic HLA-type donors ⁺ T cell induction method,
(15) HTLV-I-specific, characterized by stimulating PBMC of HLA-DR1-positive ATL patients using the HTLV-I specific CTL inducing action enhancer according to (8) or (9) above CD4 ⁺ T cell induction method,
(16) HTLV-I specific CD4 ⁺ T cell response and HTLV-I specific, characterized by stimulating PBMC of HLA-DR1-positive ATL patients using the following (X) and (Y) Methods for inducing HTLV-I specific immune responses, including CTL responses:
(X) The HTLV-I specific CTL inducing action enhancer according to (8) or (9) above:
(Y) an HTLV-I-specific CTL-inducing active peptide or polynucleotide encoding the peptide that matches the type of HLA-A locus of the ATL patient.

(1) According to the present invention, a minimal epitope of Tax-specific CD4 ⁺ T cells restricted by HLA-DR1 was found. Such an epitope has the activity of inducing CD4 ⁺ T cells specific for HTLV-I. Therefore, such epitope peptides are useful as HTLV-I specific CD4 ⁺ T cell-inducing active peptides.
(2) At present, epitopes can be predicted to some extent from amino acid anchor motifs having affinity for each HLA. However, the host immune response to pathogens in vivo is not necessarily consistent with this prediction. The epitope identified according to the present invention is obtained from an infected individual and is recognized with a much stronger selectivity than other epitopes.
(3) The present inventors have found a Tax-specific CTL epitope so far, and that such an epitope peptide has HTLV-I-specific CTL inducing activity and can be used as a vaccine for HTLV-I-recognizing CTL induction. I have found it. When the HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide found by the present inventors is used in combination with the aforementioned HTLV-I-specific CTL-inducing activity peptide, the HTLV-I-specific CTL-inducing activity peptide alone is used. HTLV-I specific CTLs were significantly induced compared to Therefore, the HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide of the present invention is useful as an agent for enhancing HTLV-I-specific CTL inducing action.
(4) The HTLV-I-specific CD4 ⁺ T cell-inducing active peptide of the present invention, the protein-peptide conjugate in which HLA-DR1 and the peptide of the present invention are bound, and the tetramer of the protein-peptide conjugate are It is also useful as a diagnostic reagent for immune function tests for identifying HTLV-I-specific CD4 ⁺ T cells. Development of a novel peptide vaccine strategy against ATL by observing Tax-specific CD4 ⁺ T cell responses in HTLV-I infected patients such as ATL patients and HAM / TSP patients using such diagnostic reagents for immune function tests, It is considered useful for the development of strategies for preventing progression or recurrence of ATL, and for elucidating the pathology of HAM / TSP.

FIG. 6 shows Tax-specific T cell immune responses in ATL patients after allogeneic hematopoietic stem cell transplantation with palliative pretreatment. (AB) PBMC obtained from 18 ATL patients 180 days after allogeneic hematopoietic stem cell transplantation with palliative pretreatment or 2 patients 540 days after allogeneic hematopoietic stem cell transplantation with palliative pretreatment (# 350 and # 341) total PBMC and CD8 ⁺ cell-depleted PBMC (B) in the absence of GST protein (white), in the presence of GST protein (grey), or in the presence of GST-Tax protein (black) After culturing for 4 days, the IFN-γ concentration in the supernatant was quantified by ELISA. The horizontal dotted line in (A) indicates the detection limit (23.5 pg / ml). The error range represents the standard deviation of duplicate measurements. ( ^* P <0.05) It is a figure which shows the phenotype and function of CD4 ⁺ T cell line (T4) produced from patient # 350. (A) The phenotype on the cell surface of T4 cells was analyzed by flow cytometry. (B) Total RNA was extracted from LCL- # 350 (lane 1), T4 cells (lane 2), ILT- # 350 (lane 3) and MT-2 (lane 4). Tax mRNA expression of each cell type was analyzed by RT-PCR. GAPDH was used as an internal standard. (C) T4 cells were stimulated for 24 hours in the presence / absence of ILT- # 350 cells or LCL- # 350 cells fixed with formaldehyde. The cytokine concentration in the supernatant was quantified with a cytometry bead array system. It is a figure which shows the identification process of the dominant Tax origin epitope recognized by the established T4 cell. (A) Donor-derived LCL- # 350 with GST, GST-Tax-A, GST-Tax-B, GST-Tax-C, or a mixture of GST-Tax-A, -B and -C (GST-TaxABC) Peptide stimulation was performed for 24 hours, and then co-culture with T4 cells was performed at a responder cell / stimulator cell (R / S) ratio of 3 for 24 hours. The amount of IFN-γ produced by T4 cells was quantified by ELISA. (B, C) 25 mer (B) or 15 mer (C) overlapping synthetic peptide (10 μg / ml) in the Tax-B region was used for peptide stimulation of LCL- # 350 for 1 hour, and responder cells (T4) / Stimulation cells (LCL- # 350) were co-cultured at an R / S ratio of 3 for 6 hours, and IFN-γ in the supernatant was measured by ELISA. (B) Formaldehyde-fixed ILT was used as a positive control stimulator cell. (D, E) 12-25mer overlapping synthetic peptides (100 ng / ml) were used, and the IFN-γ production ability of T4 cells was examined in the same manner as in (B, C). (F) IFN-γ production of T4 cells for 13-15mer peptides at various concentrations was measured in the same manner as in (B, C). Each figure shows one example of experiments repeated three times. The error range represents the standard deviation of triplicate measurements. Statistical significance was analyzed by independent t-test. “Ns” indicates no significant difference. It is a figure which shows HLA-DRB1 ^* 0101 restriction | limiting of Tax155-167 recognized by the established T4 cell. (A) T4 cells and ILT- # 350 in the presence / absence of blocking antibodies (10 μg / ml anti-human HLA-DR antibody, anti-human HLA-DQ antibody, anti-HLA-class I antibody or isotype control antibody) The IFN-γ produced by T4 cells released into the culture supernatant was quantified by ELISA. (B) Each LCL (# 350; self, # 307 # 341 Kan; allogeneic) pulsed for 1 hour under the conditions of Tax155-167 peptide stimulation (black) or no peptide stimulation (white), and T4 cells (# 350) Origin) for 6 hours. The HLA-DR allele of each LCL strain is shown in parentheses. The amount of IFN-γ produced by T4 cells was measured by ELISA. The error range indicates the standard deviation of triplicate measurements. Statistical significance was analyzed by independent t-test. (C) The amino acid sequence consisting of residues 155 to 167 of Tax contained a putative anchor motif of HLA-DRB1 ^* 0101. It is a figure which shows the proliferation enhancement of Tax-specific CD8 ^<+> T cell by costimulation with a CTL induction | guidance | derivation epitope and Tax155-167 peptide. From ATL patients (# 350, # 364 and # 341) who have undergone allogeneic hematopoietic stem cell transplantation with palliative pretreatment and whose Major Histocompatibility Complex (MHC) class I and class II are HLA-A24 / HLA-DRB1 ^* 0101, respectively The obtained PBMC was cultured for 13 days in the presence of 100 nM CTL epitope (Tax301-309) alone, a mixture of Tax301-309 peptide (100 nM) and Tax155-167 peptide (100 nM), or DMSO alone (negative control). did. Data shows the percentage of HLA-A ^* 2402 / Tax301-309 tetramer positive cells in CD3 ⁺ CD8 ⁺ T cells. It is a figure which shows the detection of Tax155-167 specific CD4 ^<+> T cell in a HLA-DRB1 ^* 0101 ⁺ HTLV-I infected person sample. (A) Using samples from two ATL patients (# 350 and # 364) who received allogeneic hematopoietic stem cell transplantation with palliative pretreatment and HLA-DRB1 ^* 0101 ⁺ seronegative donor (# 365) The frequency of CD4 ⁺ T cells binding to HLA-DRB1 ^* 0101 / Tax155-167 tetramer was analyzed in PBMC cultured for 13-14 days in the presence of Tax155-167 peptide (100 nM). The data shows the percentage of tetramer positive cells in CD3 ⁺ CD4 ⁺ T cells. (B) CD4 ⁺ T cells binding to HLA-DRB1 ^* 0101 / Tax155-167 tetramer in fresh PBMC obtained from asymptomatic carrier # 310 and HTLV-I related myelopathy / tropical spastic paraparesis patient # 294 Frequency was analyzed. The data shows the percentage of tetramer positive cells in CD3 ⁺ CD4 ⁺ T cells.

The HTLV-I-specific CD4 ⁺ T cell-inducing peptide of the present invention, that is, a peptide having an activity to induce CD4 ⁺ T cells specific to HTLV-I, is any of the following (A) to (E) The peptide is not particularly limited as long as it is an HTLV-I-specific CD4 ⁺ T cell-inducing peptide (hereinafter also referred to as “the peptide of the present invention”) having the amino acid sequence shown above.
(A) Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence induces HTLV-I-specific CD4 ⁺ T cells. Amino acid sequence with activity:
(D) In the amino acid sequence shown in the above (A) or (B), one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is HTLV-I specific Amino acid sequence having CD4 ⁺ T cell inducing activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL Amino acid sequence combined with the amino acid sequence of the inducing active peptide:

All of the amino acid sequences of SEQ ID NOs listed in (A) above are HLA-DRB1 ^* 0101-restricted Tax-specific CD4 ⁺ T cell epitopes, and SEQ ID NO: 4 is 154 to 178 of HTLV-I Tax. The amino acid sequence consisting of the amino acid (Tax154-178), SEQ ID NO: 10 is the amino acid sequence consisting of the 151st to 165th amino acids of Tax of HTLV-I (Tax151-165), and SEQ ID NO: 11 is the HTLV- It is an amino acid sequence consisting of amino acids 154 to 168 of Tax of I (Tax154-168), and SEQ ID NO: 12 is an amino acid sequence consisting of amino acids 155 to 169 of Tax of HTLV-I (Tax155-169) SEQ ID NO: 13 is an amino acid sequence consisting of amino acids 156 to 170 of Tax of HTLV-I (Tax 15 6-170), SEQ ID NO: 16 is an amino acid sequence consisting of amino acids 155 to 168 of Tax of HTLV-I (Tax155-168), and SEQ ID NO: 17 is from 155th to 167 of Tax of HTLV-I. The amino acid sequence (Tax155-167) is the amino acid sequence (Tax155-167), and SEQ ID NO: 19 is the amino acid sequence (Tax151-178) consisting of the 151st to 178th amino acids of Tax of HTLV-I. Peptides consisting of these amino acid sequences can be suitably exemplified among the peptides of the present invention in that they are excellent in HTLV-I-specific CD4 ⁺ T cell inducing activity. Among them, SEQ ID NO: 11 (Tax154-168 ), SEQ ID NO: 12 (Tax155-169), SEQ ID NO: 16 (Tax155-168), SEQ ID NO: 17 (Tax155-167), SEQ ID NO: 4 (Tax154-178), SEQ ID NO: 13 (Tax156-170) Among them, SEQ ID NO: 11 (Tax154-168), SEQ ID NO: 12 (Tax155-169), SEQ ID NO: 16 (Tax155-168), and SEQ ID NO: 17 (Tax155-167) are more preferably exemplified. SEQ ID NO: 17 (Tax155-167) is particularly preferably exemplified in that it is a minimal epitope of a HLA-HLA-DR1-restricted Tax-specific CD4 ⁺ T cell epitope (hereinafter also simply referred to as “minimal epitope”). can do. Incidentally, HTLV-I-specific CD4 ⁺ from the viewpoint of obtaining a suitable induction of T cell, HTLV-I-specific CD4 ⁺ T cell-inducing activity peptides of the present invention, HLA-HLA-DRB1 type is ^HLA-DRB1 * 0101 It is preferable to use for the object which is.

The amino acid sequence of (B) above is 14 to 30, preferably 14 to 25, more preferably 14 to 20, and more preferably 14 to 15 amino acids in the amino acid sequence of Tax (SEQ ID NO: 32). An amino acid sequence comprising at least amino acid sequences of amino acid numbers 155 to 167. Since such an amino acid sequence contains a minimal epitope, it is an HTLV-I-specific CD4 ⁺ T cell-inducing active peptide.

The amino acid sequence of (C) above is 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, even more than the amino acid sequence shown in (A) or (B) above. Preferably, the amino acid sequence has 98% or more identity, and the peptide comprising the amino acid sequence is an amino acid sequence having HTLV-I-specific CD4 ⁺ T cell inducing activity. Such an amino acid sequence is highly likely to be an HTLV-I-specific CD4 ⁺ T cell-inducing active peptide.

The amino acid sequence (D) is an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in (A) or (B) above, and consists of the amino acid sequence. The peptide is an amino acid sequence having HTLV-I-specific CD4 ⁺ T cell inducing activity. Here, the “amino acid sequence in which one or several amino acids are deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and more preferably 1 Means an amino acid sequence in which an arbitrary number of amino acids of ~ 5, more preferably 1 to 3, more preferably 1 to 2 are deleted, substituted or added, and amino acid "substitution, deletion or addition" As for the degree and position thereof, the modified peptide should be a synergistic compound having HTLV-I-specific CD4 ⁺ T cell inducing activity similar to the peptide consisting of the amino acid sequence listed in (A) above. There is no particular limitation, and the alteration (mutation) of the amino acid sequence may be caused by, for example, mutation or post-translational modification, but may be artificially altered. In the present invention, all modified peptides having the above characteristics are included regardless of the cause and means of such modification / mutation.

  The amino acid sequence of (E) above is an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least an amino acid sequence of amino acid numbers 155 to 167, It is an amino acid sequence in which an amino acid sequence of an HTLV-I-specific CTL-inducing active peptide is bound. The number of consecutive 14 to 30 amino acids in the amino acid sequence (E) is preferably 18 to 30, more preferably 22 to 30.

The “HTLV-I-specific CTL-inducing activity peptide” in the present specification is not particularly limited as long as it is a peptide having an activity to induce CTL specific to HTLV-I, and among them, Tax-specific CTL-inducing activity. Peptides can be preferably exemplified, and among these, Tax 301-309 (SEQ ID NO: 34) (see Patent Document 1) which is an HLA-A ^* 2402-restricted CTL epitope can be particularly preferably exemplified. In recent years, a fusion peptide (Helper / Killer-Hybrid Epitope Long Peptide) in which a cancer antigen epitope for CD4 ⁺ T cells, which are helper T cells, and a cancer antigen epitope for CD8 ⁺ T cells, which are CTLs, are administered is administered to a subject. Thus, it is known to effectively enhance immunity against cancer cells of a test subject (for example, Cancer Science (2012) 103, 150-153, etc.). Also in the present invention, by using the peptide having the amino acid sequence of E, HTLV-I-specific CTL can be induced together with HTLV-I-specific CD4 ⁺ T cells, and immunity against HTLV-I can be effectively enhanced. It is considered possible.

In addition, as a CD4 ^<+> T cell in this specification, a helper T cell can be illustrated suitably, Th1 type helper T cell can be illustrated more suitably especially.

  The peptide of the present invention can be produced by chemical or genetic engineering techniques. The chemical method includes peptide synthesis methods by ordinary liquid phase methods and solid phase methods. More specifically, the peptide synthesis method is based on the amino acid sequence information, and a stepwise erosion method in which each amino acid is sequentially linked one by one to extend the chain, and a fragment consisting of several amino acids is synthesized in advance. Then, a fragment condensation method in which each fragment is subjected to a coupling reaction is included. The peptide of the present invention can be synthesized by any of them.

  The condensation methods employed for the peptide synthesis can also follow various known methods. Specific examples thereof include, for example, azide method, mixed acid anhydride method, DCC method, active ester method, redox method, DPPA (diphenylphosphoryl azide) method, DCC + additive (1-hydroxybenzotriazole, N-hydroxysuccinamide) N-hydroxy-5-norbornene-2,3-dicarboximide, etc.), Woodward method and the like. Solvents that can be used in each of these methods can be appropriately selected from general solvents that are well known to be used in this type of peptide condensation reaction. Examples thereof include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and mixed solvents thereof.

  In the peptide synthesis reaction, amino acids that do not participate in the reaction and carboxyl groups in the peptide are generally esterified, for example, lower alkyl esters such as methyl ester, ethyl ester, tertiary butyl ester, such as benzyl ester, p- It can be protected as methoxybenzyl ester, p-nitrobenzyl ester aralkyl ester and the like. An amino acid having a functional group in the side chain, for example, a hydroxyl group of Tyr may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, or the like, but such protection is not necessarily required. . Further, for example, the guanidino group of Arg is a suitable protecting group such as nitro group, tosyl group, 2-methoxybenzenesulfonyl group, methylene-2-sulfonyl group, benzyloxycarbonyl group, isobornyloxycarbonyl group, adamantyloxycarbonyl group and the like. Can be protected. The deprotection reaction of these protecting groups in the amino acids having the above-mentioned protecting groups, peptides and finally obtained peptides of the present invention is also carried out by conventional methods such as catalytic reduction, liquid ammonia / sodium, hydrogen fluoride, odor. It can be carried out according to a method using hydrogen fluoride, hydrogen chloride, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid or the like.

  In addition to being obtained by chemical synthesis as described above, the peptides of the present invention can also be produced by conventional methods using genetic engineering techniques. The peptide of the present invention thus obtained can be obtained according to a conventional method, for example, peptide such as ion exchange resin, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution method and the like. The purification can be appropriately performed according to a method widely used in the chemical field.

  The fusion peptide of the present invention may be any peptide as long as the peptide of the present invention is bound to the marker protein and / or peptide tag, and the marker protein is a conventionally known marker protein. It is not particularly limited as long as it is, for example, alkaline phosphatase, Fc region of antibody, HRP, GFP and the like can be specifically mentioned, and peptide tags include epitope tags such as HA, FLAG, Myc, Specific examples of conventionally known peptide tags such as affinity tags such as GST, maltose-binding protein, biotinylated peptide, oligohistidine and the like can be exemplified. Such a fusion peptide can be prepared by a conventional method. Purification of the peptide of the present invention utilizing the affinity between Ni-NTA and His tag, detection of the peptide of the present invention, and quantification of an antibody against the peptide of the present invention It is also useful as a research reagent in this field.

The protein-peptide conjugate of the present invention is not particularly limited as long as it is a conjugate of HLA-DR1 and the peptide of the present invention. For example, HLA-DR1 molecules and the above (A) to (E) Preferred is a form capable of binding to a CD4 ⁺ T cell that recognizes such a conjugate, such as a conjugate with a peptide consisting of any of the amino acid sequences shown. The tetramer of the protein-peptide conjugate of the present invention is not particularly limited as long as it is a tetramer of a protein-peptide conjugate in which HLA-DR1 and the peptide of the present invention are bound. The protein-peptide conjugate can be exemplified by a tetramer having streptavidin as a nucleus. For example, a substrate of the enzyme Bir-A is expressed at the C-terminus of HLA-DR1, and Bir- It can be obtained by mixing HLA-DR1 biotinylated by the A-dependent biotinilation method and phycoerythrin (PE) -labeled deglycosylated avidin at a ratio of 4: 1 (Altman, JD, et al .: Science 274, 94- 96, 1996). These protein-peptide conjugates and tetramers thereof are prepared by conventional methods using chemically synthesized peptides of the present invention and genetic engineering techniques utilizing the HLA-DR1 gene (GenBank Accession Number AF142457). -By combining the α chain and β chain of DR1 in vitro in a refolding buffer, or using genetic engineering techniques using the polynucleotide encoding the peptide of the present invention and the HLA-DR1 gene, respectively. It can be produced by co-expressing the peptide of the present invention and the HLA-DR1 α chain and β chain in the same host cell by a conventional method, and binding them after purification.

The fusion protein of the present invention may be any protein as long as the protein-peptide conjugate or protein-peptide conjugate tetramer is bound to the marker protein and / or peptide tag. The protein is not particularly limited as long as it is a conventionally known marker protein. For example, fluorescent dyes, alkaline phosphatase, antibody Fc region, HRP, GFP and the like can be specifically mentioned. Specific examples of the tag include epitope tags such as HA, FLAG, and Myc, and conventionally known peptide tags such as affinity tags such as GST, maltose-binding protein, biotinylated peptide, and oligohistidine. . Such a fusion protein can be prepared by a conventional method, such as purification of a protein-peptide conjugate utilizing the affinity between Ni-NTA and His tag, detection of HTLV-I-specific CD4 ⁺ T cells, and the like. It is also useful as a research reagent in the field.

Specific examples of antibodies that specifically bind to the peptide of the present invention include immunospecific antibodies such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and the like. The peptide of the present invention can be prepared by an ordinary method using the antigen as an antigen. Among them, a monoclonal antibody is more preferable in terms of its specificity. Such an antibody that specifically binds to the peptide of the present invention such as a monoclonal antibody is not only useful for diagnosis of, for example, ATL and HAM / TSP, but also HTLV-I specific CD4 ⁺ T cells of the peptide of the present invention. It is useful for clarifying the activity mechanism and molecular mechanism of induction.

  The antibody against the peptide of the present invention can be obtained by subjecting an animal (preferably non-human) to a peptide of the present invention, a complex of the peptide of the present invention and an immunogenic protein, For example, for the preparation of monoclonal antibodies, the hybridoma method (Nature 256, 495-497, 1975) results in antibodies produced by continuous cell line cultures. ), Trioma method, human B cell hybridoma method (Immunology Today 4, 72, 1983) and EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) The method can be used.

The polynucleotide of the present invention is a polynucleotide encoding an HTLV-I specific CD4 ⁺ T cell inducing peptide, that is, a polynucleotide comprising the polynucleotide sequence shown in any of the following (a) to (d): The nucleotide is not particularly limited as long as it is a nucleotide (hereinafter also referred to as “polynucleotide of the present invention”).
(A) a polynucleotide sequence encoding the amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) a polynucleotide sequence encoding an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 32 and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) a polynucleotide sequence having 80% or more identity to the polynucleotide sequence shown in the above (a) or (b), wherein the peptide has HTLV-I-specific CD4 ⁺ T cell inducing activity Encoding polynucleotide sequence:
(D) a polynucleotide sequence obtained by deleting, substituting, or adding one or several nucleotides in the polynucleotide sequence shown in (a) or (b), wherein the HTLV-I specific CD4 ⁺ T cell Polynucleotide sequence encoding a peptide having inductive activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising at least an amino acid sequence of amino acid numbers 155 to 167, and an HTLV-I-specific CTL Polynucleotide sequence encoding an amino acid sequence linked to the amino acid sequence of an inducing active peptide:

  The polynucleotide sequence of (a) is not particularly limited as long as it encodes any of the listed amino acid sequences, but is shown in SEQ ID NO: 24 as a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4. The polynucleotide sequence shown in SEQ ID NO: 25 can be preferably exemplified as the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 10. The polynucleotide sequence shown in SEQ ID NO: 26 can be preferably exemplified as the polynucleotide sequence encoding the amino acid sequence shown, and is shown in SEQ ID NO: 27 as the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 12. Suitable examples of polynucleotide sequences A polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 16 can be preferably exemplified as the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 13, and the polynucleotide encoding the amino acid sequence shown in SEQ ID NO: 16 The polynucleotide sequence shown in SEQ ID NO: 29 can be preferably exemplified as the sequence, and the polynucleotide sequence shown in SEQ ID NO: 30 is preferably exemplified as the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 17. Can do.

  The polynucleotide sequence of (b) above is an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising an amino acid sequence comprising at least amino acid numbers 155 to 167. The encoding polynucleotide sequence. Such a polynucleotide sequence is not particularly limited as long as it encodes the above-mentioned amino acid sequence. However, in the Tax polynucleotide sequence shown in SEQ ID NO: 33, a polynucleotide sequence corresponding to the above-mentioned amino acid sequence is preferably exemplified. be able to. Such a polynucleotide sequence can be grasped by comparing SEQ ID NO: 32 showing the amino acid sequence of the entire Tax and SEQ ID NO: 33 showing the polynucleotide sequence of the entire Tax.

The polynucleotide sequence (c) is 80% or more, preferably 85% or more, more preferably 90% or more, and still more preferably 95% or more with respect to the polynucleotide sequence shown in (a) or (b). Even more preferably, it is a polynucleotide sequence having 98% or more identity, and encoding a peptide having HTLV-I specific CD4 ⁺ T cell inducing activity.

The polynucleotide sequence of (d) is a polynucleotide sequence in which one or several nucleotides are deleted, substituted or added in the polynucleotide shown in (a) or (b) above, and the HTLV-I A polynucleotide sequence encoding a peptide having specific CD4 ⁺ T cell inducing activity. Here, the “polynucleotide sequence in which one or several nucleotides are deleted, substituted or added” is, for example, 1 to 20, preferably 1 to 15, more preferably 1 to 10, and still more preferably. It means a polynucleotide sequence in which any number of nucleotides of 1 to 5, more preferably 1 to 3, more preferably 1 to 2 are deleted, substituted or added, and the nucleotide “substitution, deletion or The degree of “addition” and the position thereof are such that the peptide encoded by the modified polynucleotide sequence induces HTLV-I-specific CD4 ⁺ T cells in the same manner as the peptide consisting of the amino acid sequence listed in (A) above. The nucleotide is not particularly limited as long as it has the same activity, and such nucleotide alteration (mutation) is caused by, for example, mutation or post-translational modification. There is also a, but can also be artificially altered. In the present invention, all modified polynucleotide sequences having the above characteristics are included regardless of the cause and means of such modification / mutation.

  The polynucleotide sequence of (e) above is an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in column number 32, and comprising at least an amino acid sequence of amino acid numbers 155 to 167; The polynucleotide sequence which codes the amino acid sequence which combined the amino acid sequence of HTLV-I specific CTL induction | guidance | derivation active peptide. The polynucleotide sequence (e) is a polynucleotide sequence corresponding to the amino acid sequence (E).

  In addition, the polynucleotide of the present invention can be advantageously used when preparing the peptide of the present invention by a conventional method using genetic engineering techniques. In particular, the antisense strand of the polynucleotide of the present invention is ATL or the like. It is useful as a diagnostic probe for HTLV-I tumors.

The HTLV-I-specific CTL inducing action enhancer or the HTLV-I-specific CTL inducing action-enhancing composition of the present invention (preferably a pharmaceutical composition) includes “an amino acid of any one of (A) to (E) above” HTLV-I-specific CD4 ⁺ T cell-inducing peptide consisting of a sequence ”or“ a promoter and a polynucleotide consisting of a polynucleotide sequence shown in any of the following (a) to (e): There is no particular limitation as long as it contains an “expression vector in which nucleotides are operably linked downstream of a promoter” as an active ingredient.

The peptide of the present invention or the peptide of the present invention expressed from the expression vector of the present invention induces HTLV-I specific CD4 ⁺ T cells, and such CD4 ⁺ T cells induce HTLV-I specific CTL inducing action. To strengthen. In the present specification, the phrase “enhancing the HTLV-I-specific CTL inducing action” means an HTLV-I-specific CTL-inducing peptide (HTLV-I-specific peptide) through induction of HTLV-I-specific CD4 ⁺ T cells. Which has the ability to enhance the HTLV-I-specific CTL inducing action exerted by a peptide having an activity of inducing a typical CTL). Whether or not a substance having the ability to enhance the HTLV-I-specific CTL-inducing action exhibited by the HTLV-I-specific CTL-inducing active peptide is determined by, for example, determining the substance and the HTLV-I-specific CTL-inducing active peptide. When used in combination, it can be easily confirmed by examining whether HTLV-I-specific CTLs are induced more markedly than when HTLV-I-specific CTL-inducing activity peptide alone is used. . If HTLV-I-specific CTL is more significantly induced when used in combination, the substance has the ability to enhance the HTLV-I-specific CTL inducing action exhibited by the HTLV-I-specific CTL-inducing active peptide. It can be said that it has.

  The expression vector includes a promoter and a polynucleotide comprising the polynucleotide sequence shown in any of (a) to (e) above, and the polynucleotide is operably linked downstream of the promoter. Any expression vector may be used, and plasmids derived from Escherichia coli (eg, pET28, pGEX4T, pUC118, pUC119, pUC18, pUC19, and other plasmid DNAs), plasmids derived from Bacillus subtilis (Eg, pUB110, pTP5, and other plasmid DNA), yeast-derived plasmids (eg, YEp13, YEp24, YCp50, and other plasmid DNAs), λ phage (λgt11, λZAP, etc.), mammalian plasmids (pCMV, pSV40) Virus vectors (adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors) Terpolymers, animal virus vectors such as vaccinia virus vectors, and insect virus vectors such as baculovirus vectors, etc.), plant vector (binary vector pBI system, etc.), etc. cosmid vector can be used.

  The promoter is not particularly limited, and a suitable promoter may be selected depending on the host, and any of a constitutive promoter and an inducible promoter known in the art may be used. Specifically, as the promoter, CMV promoter, SV40 promoter, CAG promoter, synapsin promoter, rhodopsin promoter, CaMV promoter, glycolytic enzyme promoter, lac promoter, trp promoter, tac promoter, GAPDH promoter, GAL1 promoter, PH05 promoter, PGK promoter etc. can be mentioned. The expression vector may further contain a terminator downstream of the target polynucleotide.

  The above “polynucleotide is operably linked downstream of the promoter” means that the promoter and the polynucleotide are operably linked so that the promoter can initiate transcription of the polynucleotide sequence. Means that

  The host cell used when the peptide of the present invention is expressed using the above expression vector may be any host cell as long as the expression vector can express the peptide of the present invention, such as Escherichia coli and Streptomyces. Bacterial prokaryotic cells such as Bacillus subtilis, Streptococcus and Staphylococcus, eukaryotic cells such as yeast and Aspergillus, insect cells such as Drosophila S2 and Spodoptera Sf9, L cells, CHO cells, COS cells, HeLa cells, C127 Examples include cells, BALB / c3T3 cells (including mutants lacking dihydrofolate reductase and thymidine kinase), BHK21 cells, HEK293 cells, Bowes melanoma cells, and oocyte and plant cells. In addition, the introduction of an expression vector capable of expressing the peptide of the present invention into a host cell is described in Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), as described in many standard laboratory manuals such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, Cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection and the like.

  As an optional component other than the peptide of the present invention and the above expression vector contained in the HTLV-I specific CTL inducing action enhancer or HTLV-I specific CTL inducing action enhancing composition (preferably pharmaceutical composition) of the present invention, For example, it may contain a pharmaceutically acceptable carrier or diluent, an immunostimulant, an additive, etc. Examples of such a carrier or diluent include a stabilizer such as SPGA, sorbitol, mannitol, starch. Specific examples include carbohydrates such as sucrose, glucose and dextran, proteins such as albumin and casein, protein-containing substances such as bovine serum and skim milk, and buffers such as phosphate buffer, physiological saline and water. be able to. Specific examples of immunostimulators include cytokines such as interleukin-2 (IL-2), interleukin-12 (IL-12), and tumor necrosis factor α (THF-α). Examples include low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates containing glucose or dextran, chelating agents such as EDTA, protein stabilizers, microbial growth inhibitors or inhibitors, and the like. However, it is not limited to these.

  The HTLV-I-specific CTL inducing action enhancer or HTLV-I-specific CTL inducing action enhancing composition of the present invention (preferably a pharmaceutical composition) is orally, intravenously, intraperitoneally, intranasally, intradermally. A form that can be administered subcutaneously, intramuscularly, and the like is preferable. The effective amount to be administered can be appropriately determined in consideration of the type / composition of the pharmaceutical or pharmaceutical composition, the administration method, the age or weight of the patient, etc., and these can be administered one to several times per day. preferable. When administered orally, it is usually administered in the form of a preparation prepared by mixing with a pharmaceutical carrier. In this case, as a carrier that can be used in the preparation, a substance that is commonly used in the preparation field and does not react with the peptide of the present invention is used. Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations can be prepared according to conventional methods, and in the case of liquid preparations in particular, they can be dissolved or suspended in water or other suitable medium at the time of use. Tablets and granules may be coated by a known method. In the case of injection, it is prepared by dissolving the peptide of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good. These preparations may also contain other components having therapeutic value.

The vaccine for inducing an HTLV-I-specific immune response of the present invention includes the aforementioned HTLV-I-specific CTL inducing action enhancer of the present invention, an HTLV-I-specific CTL-inducing active peptide, or a polynucleotide encoding the peptide If it contains, it will not restrict | limit in particular. The HTLV-I specific CTL inducing peptide is as described above. According to such a vaccine, both HTLV-I-specific CD4 ⁺ T cells and HTLV-I-specific CTL can be induced, and the HTLV-I-specific CTL can be significantly induced. The vaccine for inducing immune response of the present invention can be used for the treatment of HTLV-I tumors such as ATL. From the viewpoint of obtaining a suitable induction of HTLV-I-specific CTL, it is preferable to use a peptide suitable for the type of HLA-A to be administered as the HTLV-I-specific CTL-inducing active peptide. An HTLV-I specific CD4 ⁺ T cell-inducing active peptide consisting of the amino acid sequence of (E) above or an expression vector containing the polynucleotide of (e) above as an HTLV-I specific CTL inducing action enhancer When it is contained as an active ingredient, it contains an HTLV-I-specific CTL-inducing active peptide or a polynucleotide encoding the peptide, and need not be separately included. In the present specification, the term “containing the HTLV-I-specific CTL inducing action enhancer of the present invention and an HTLV-I-specific CTL-inducing peptide or a polynucleotide encoding the peptide” includes the above (E The aspect of the HTLV-I specific CD4 ⁺ T cell-inducing activity peptide consisting of the amino acid sequence of ()) and the aspect of the expression vector containing the polynucleotide (e) are also included for convenience.

The vaccine for inducing immune response according to the present invention is more preferably one containing various adjuvants that enhance cellular or local immunity. Examples of such adjuvants include peptide-specific CD4 ⁺ efficiently. Dendritic cells that can induce T cells and CTLs, ISS-ODN (Immunostimulatory DNA sequences-oligodeoxynucleotides; Nat. Med. 3, 849-854, 1997) containing CpG motifs, QS21 that stimulates cytotoxic T cells ( Specific examples thereof include Quil1aia saponaria, commercially available from Cambridge Biotech, Worcester, MA), aluminum hydroxide, aluminum phosphate, aluminum oxide, oil emulsion, saponin, vitamin E lysate, and the like. When an adjuvant is used, it should be used as a recombinant fusion protein or recombinant fusion peptide prepared from a polynucleotide that continuously encodes various bacterial cell components and toxins that serve as adjuvants and the peptide of the present invention. You can also.

The immunological function test diagnostic agent of the present invention includes the peptide of the present invention, the expression vector of the present invention, a protein-peptide conjugate in which HLA-DR1 and the peptide of the present invention are bound, or 4 of the protein-peptide conjugate. It is not particularly limited as long as it can test and diagnose immune function, particularly immune function against HTLV-I, with a monomer as an active ingredient. An expression vector capable of expressing the peptide of the present invention to be a label, a label of a protein-peptide conjugate in which HLA-DR1 and the peptide of the present invention are bound, or a tetramer of the protein-peptide conjugate It is preferable to use a label. As a labeling substance used for forming a label, a radioisotope can be used in addition to the marker protein and the peptide tag. As an immune function test diagnostic agent of the present invention, an immune function test diagnostic agent for identifying HTLV-I-specific CD4 ⁺ T cells can be preferably exemplified. In the immune function test diagnosis using the immune function test diagnostic agent of the present invention, the immune function test diagnostic agent of the present invention is brought into contact with peripheral blood leukocytes (lymphocytes) of a subject subject to recognize an epitope in the peptide or the like of the present invention. by binding to CD4 ⁺ T cells can be identified HTLV-I Tax-specific CD4 ⁺ T cells. Among immunodiagnostic diagnostic agents, fluorescent labels such as tetramer PE of the protein-peptide conjugate described above enable detection and quantification of CD4 ⁺ T cells by flow cytometry. In addition, it is particularly useful for determining the effect of inducing HTLV-I-specific CD4 ⁺ T cells. For example, a mononuclear cell fraction is separated from a heparin peripheral blood sample, and a PE-labeled tetramer (a tetramer of a protein-peptide conjugate) and an activation marker antibody such as a CD4 antibody labeled with FITC or PE-Cy5 are used. By double-staining and calculating the number of CD4-positive tetramer-positive cells with a flow cytometer, the immune function of the subject can be examined and diagnosed. In addition, since the number of tetramer positive cells is often very small in a fresh blood sample, not only a fresh blood sample but also a peptide of the present invention or a cell expressing the same is used for several days to 1 week. The same staining analysis can be performed after the culture.

As a method for inducing HTLV-I-specific CD4 ⁺ T cells of the present invention, HTLV-I-infected T cells derived from an ATL patient before HSCT are used to derive from an allogeneic HLA type, that is, an HLA-DR1 type donor. A method for inducing HTLV-I-specific CD4 ⁺ T cells, characterized by stimulating PBMCs of the same patient after HSCT in vitro, in vivo or ex vivo, and using the peptide of the present invention, HLA-DR1 positive A method for inducing HTLV-I-specific CD4 ⁺ T cells characterized by stimulating PBMCs of ATL patients in vitro, in vivo or ex vivo, and using the expression vector of the present invention, for example, for antigen-presenting cells in PBMCs In vitro expression of PBMCs from HLA-DR1-positive ATL patients, such as genetically expressing peptides It is not particularly limited as long as it is a HTLV-I-specific CD4 ⁺ T method for inducing cells, characterized by stimulating in vivo or ex vivo, HTLV-I-specific CD4 ⁺ T cells obtained by such induction method , addition can be used as HTLV-I-specific CTL-inducing effect enhancer, or to reveal the active mechanism or the molecular mechanism of HTLV-I-specific CD4 ⁺ T cell induction, and HTLV-I-specific CD4 ⁺ T cells induced It is also useful for examining the relationship with ATL and HAM / TSP.

As a method for inducing an HTLV-I-specific immune response including an HTLV-I-specific CD4 ⁺ T cell response and an HTLV-I-specific CTL response of the present invention, PBMCs of HLA-DR1-positive ATL patients are used as follows: The method is not particularly limited as long as it is a method characterized by stimulation using (X) and (Y).
(X) HTLV-I-specific CTL inducing action enhancer of the present invention:
(Y) an HTLV-I-specific CTL-inducing active peptide that matches the type of HLA-A locus of the ATL patient or a polynucleotide encoding the peptide:

As an HTLV-I-specific CTL inducing action potentiator, an HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide consisting of the amino acid sequence of (E) above or an expression vector containing the polynucleotide of (e) above When it is contained as an active ingredient, it contains an HTLV-I-specific CTL-inducing active peptide or a polynucleotide encoding the peptide, so that it is not necessary to prepare it separately to stimulate PBMC. Stimulating the PBMC using the above (X) and (Y) includes stimulating PBMC with an HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide consisting of the amino acid sequence of (E), Stimulating PBMC with an expression vector containing the polynucleotide of (e) above is also included for convenience.

Other aspects of the present invention include the use of the peptide of the present invention for enhancing the HTLV-I-specific CTL inducing action, and the peptide of the present invention and HTLV-I for inducing an HTLV-I-specific immune response. ATL characterized by administering HTLV-I-specific CD4 ⁺ T cells obtained by the use of a specific CTL-inducing active peptide or the HTLV-I-specific CD4 ⁺ T cell induction method of the present invention to a mammal Administration of HTLV-I-specific CD4 ⁺ T cells and HTLV-I-specific CTL obtained by a method for preventing, treating or ameliorating HTLV-I or a method for inducing an HTLV-I-specific immune response of the present invention to a mammal A characteristic ATL prevention, treatment or amelioration method is also included.

[Reference example]
We have previously induced Tax-specific CD8 ⁺ T cells in some ATL patients who received allogeneic hematopoietic stem cell transplantation (mini-transplantation) with palliative pretreatment using siblings with the same HLA type as donors. (Non-Patent Document 9). In the present invention, Tax-specific T cell responses were examined in a number of ATL patients who received allogeneic hematopoietic stem cell transplantation with palliative pretreatment. Table 1 shows that peripheral blood was collected from 18 ATL patients 180 days after allogeneic hematopoietic stem cell transplantation with palliative pretreatment, and Tax-specific CD8 ⁺ T cells (CTL) were obtained by flow cytometry using Tax / HLA tetramer. The detected results are shown together with clinical information. During the period, all patients were fully chimeric with donor-derived hematopoietic stem cells> 95%. Uses four available tetramers (HLA-A ^* 0201 / Tax11-19, HLA-A ^* 2402 / Tax301-309, HLA-A ^* 1101 / Tax88-96, HLA-A ^* 1101 / Tax272-280) In 14 patients, Tax-specific CD8 ⁺ T cells were observed (see “Tetramer-positive cells (%)” in Table 1 below). Since in most cases the donor was uninfected (see “-(donor serum anti-HTLV-I antibody response (−)” in Table 1), Tax-specific donor-derived CD8 ⁺ T cells were induced in the recipient. This indicates that a new immune response to the HTLV-I has occurred in the recipient, and such evidence reinforces our previous findings (Non-Patent Document 9 and Harashima N , Tanosaki R, Shimizu Y, et al. Identification of two new HLA-A * 1101-restricted tax epitopes recognized by cytotoxic T lymphocytes in an adult T-cell leukemia patient after hematopoietic stem cell transplantation.J Virol. 2005; 79 (15 ): 10088-10092).

  EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

A [Method and materials]
A-1 Subjects The specimens of ATL patients used in this example were 18 cases that received allogeneic hematopoietic stem cell transplantation by palliative pretreatment, 1 anti-HTLV-I antibody seronegative person (# 365), and HLA -2 anti-HTLV-I antibody seropositive donors carrying DRB1 ^* 0101 (1 asymptomatic carrier: # 360 and HTLV-I related myelopathy / tropical spastic paraparesis (HAM / TSP) patient 1 Example: Obtained written informed consent from # 294) and received peripheral blood.

  In Table 1 above, “chimerism (%)” indicates the proportion of patient-derived T cell chimerism (indicating the engraftment rate of transplanted cells), and “tetramer-positive cells (%)” are the CD8-positive T cells. The ratio of tetramer positive cells (Tax antigen-specific CTL) is shown. “Infected cell rate” shows the number of infected cells per 1000 peripheral blood mononuclear cells (PBMC) determined quantitatively by nucleic acid. “M” "F" represents female, "NT" represents untested, "r-PB" represents a peripheral blood stem cell transplant between relatives, and "ur-BM" represents unrelated It shows that the bone marrow cell transplantation between persons was performed.

  About half of the ATL patients in Table 1 above have received allogeneic peripheral blood stem cell transplants from sibling donors with matching HLA-A-, B- and DR-, and the other half are HLA-A-, He has undergone allogeneic bone marrow cell transplantation by an unrelated donor who is seronegative for anti-HTLV-I antibody with matched B- and DR- (see Table 1). These patients are participants of a clinical trial conducted by the ATL Allogeneic Hematopoietic Stem Cell Transplantation Research Group with support from the Ministry of Health, Labor and Welfare in Japan. Have gained.

A-2 Establishment of patient and donor cell lines
PBMCs were isolated by density gradient centrifugation using Ficoll-Paque (registered trademark) PLUS (GE Healthcare UK) and suspended in Bambanker stock solution (manufactured by Nippon Genetics). Until stored in liquid nitrogen. Some of these stock cells were used to establish IL-2-dependent HTLV-I infected T cell line (ILT) and Epstein-Barr virus (EBV) transformed lymphoblast B cell line (LCL). ILT- # 350 spontaneously immortalized (cancerous) by long-term culture of PBMC obtained from patient # 350 before allogeneic hematopoietic stem cell transplantation was treated with 20% fetal calf serum (FCS; manufactured by Sigma Aldrich). The cells were subcultured with RPMI 1640 (Life Technologies, Inc.) containing 30 U / ml of recombinant human interleukin-2 (rhIL-2; manufactured by Shionogi & Co., Ltd.). LCL- # 307, LCL- # 341 and LCL- # 350 were established from PBMCs obtained from ATL patients # 307, # 341 and # 350 after allogeneic hematopoietic stem cell transplantation, respectively. These PBMCs were subcultured with RPMI1640 containing 20% FCS, and then infected with B95-8 cell line culture supernatant containing EBV. Similarly, LCL-Kan was established from PBMC derived from healthy individuals.

A-3 Synthetic peptides In the central region of Tax antigen (103 to 246 residues), a total of 18 overlapping peptides of 12 to 25 mer in length were purchased from Scrum Inc. (see Table 2) for epitope mapping. used. HLA-A ^* 2402 restricted CTL epitope (Tax301-309) (SEQ ID NO: 34) (Non-Patent Document 9) was used for in vitro peptide stimulation of Tax-specific CTL (Hokudo Co., Ltd.).

A-4 Immunoassay using GST-Tax fusion protein Glutathione-S-transferase (GST) fusion proteins (GST-Tax-A and -B, respectively) in the N-terminal, central, and C-terminal regions of Tax of HTLV-I And -C) (Non-Patent Document 13, Kurihara K, Shimizu Y, Takamori A, et al. Human T-cell leukemia virus type-I (HTLV-I) -specific T-cell responses detected using three-divided glutathione-S -Transfer (GST) -Tax fusion proteins. J Immunol Methods. 2006; 313 (1-2): 61-73.), and the immune response of T cells specific for HTLV-ITax was evaluated.

<ELISA>
PBMC (1 × 10 ⁶ cells / ml) containing 10% FCS in the presence / absence (presence or absence) of a mixture of GST-Tax-A, -B and -C proteins (GST-TaxABC) Incubated with RPMI 1640 (200 μl). After 4 days, the supernatant was collected, and the IFN-γ concentration in the supernatant was measured using OptiEIA Human IFN-γ ELISA Kit (BD Biosciences). The minimum detectable amount of IFN-γ in this assay was 23.5 pg / ml.

<Quantitative measurement of cytokines>
CD8 ⁺ cells were removed from the cultured PBMC by Dynabeads M-450 CD8 (manufactured by Invitrogen) by the negative selection method according to the manufacturer's protocol. To perform cytokine profiling of the HTLV-I specific CD4 ⁺ T cell line, the isolated cells were stimulated with ILT- # 350 fixed with formaldehyde for 48 hours. The culture supernatant was collected, and various cytokines were measured using a human Th1 / Th2 / Th17 cytokine kit (BD Biosciecnes) of Cytokine Beads Array.

Induction of A-5 HTLV-I specific CD4 + T cell line (T4 cells) Patient # 350 PBMC (1 × 10 ⁶ cells / ml) who achieved complete remission at day 180 after allogeneic hematopoietic stem cell transplantation with palliative pretreatment ) Was cultured for 2 weeks in the presence of Tax301-309 peptide (100 nM). Cultured PBMCs were then isolated from CD4 ⁺ cells by negative selection using Human CD4 T lymphocyte Enrichment Set-DM (BD Biosciecnes) and contained 20% FCS and rhIL-2 (100 U / ml). Subcultured with RPMI 1640. Every 2-3 weeks thereafter, isolated CD4 ⁺ cells were stimulated with ILT- # 350 fixed with formaldehyde.

A-6 Reverse transcription polymerase chain reaction (RT-PCR)
Total RNA was isolated from the cells using ISOGEN (Nippon Gene Co., Ltd.) and TURBO DNA-free (Life Technologies, Inc.). First-strand complementary DNA (cDNA) was prepared from 0.5 μg of RNA using ReverTra Ace primer and Oligo (dt) ₂₀ primer contained in ReverTra Ace-α-kit (manufactured by TOYOBO). ReverTra Dash (manufactured by TOYOBO), HTLV-IpX specific primer (pX1: 5'-CCA CTT CCC AGG GTT TAG ACA GAT CTT C-3 '(SEQ ID NO: 20) and pX4: 5'-TTC CTT ATC CCT CGA CTC CCC TCC TTC CCC-3 ′ (SEQ ID NO: 21)) PCR was performed in 50 μl of a reaction mixture containing 0.5 μM of each and 2 μl of cDNA. As internal standards, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specific primers (GAPDH5 ′: 5′-ACC ACA GTC CAT GCC ATC AC-3 ′ (SEQ ID NO: 22), and GAPDH3 ′: 5′-TCC ACC ACC CTG TTG CTG TA-3 ′ (SEQ ID NO: 23)) was used. As thermal cycle conditions, an initial activation step was performed at 94 ° C. for 1 minute, followed by 30 cycles consisting of denaturation (98 ° C., 10 seconds), annealing (60 ° C., 2 minutes) and extension (74 ° C., 30 seconds). Cycled. After electrophoresis on a 2% (w / v) agarose gel, PCR products were visualized with ethidium bromide.

A-7 Flow cytometry The following fluorescent dye-labeled mouse anti-human monoclonal antibody (mAb) was used for cell surface staining. CD3-FITC (UCHT1, manufactured by BioLegend); CD4-FITC (RPA-T4, manufactured by BioLegend); CD8-FITC (RPA-T8, manufactured by BioLegend) and CD8-PE-Cy5 (HIT8a, manufactured by BD Biosciences) . For tetramer staining, labeled with phycoerythrin (PE) HLA-A ^* 0201 / Tax11-19, HLA-A ^* 1101 / Tax88-96, HLA-A ^* 1101 / Tax272-280, HLA-A ^* 2402 / Each tetramer of Tax301-309 was purchased from MBL (Medical & Biological Laboratories Co. Ltd., Medical and Biological Laboratories). PE-labeled HLA-DRB1 ^* 0101 / Tax155-167 tetramer was newly made by special order from MBL. The PE-labeled Tax / HLA tetramer stained whole blood or cultured cells with a combination of CD3-FITC and CD8-PE-Cy5 or CD4-PE-Cy5. The whole blood sample was lysed with red blood cells, fixed with a BD FACS solution (BD Biosciences), and then washed. Samples were analyzed using FACS Calibur (Becton Dickinson), and FlowJo software (Tree Star, Inc.) was used for data analysis.

A-8 Epitope Mapping T4 cells (3 × 10 ⁵ cells / ml) were treated with LCL- # 350 peptide-stimulated for 1 hour at 37 ° C. under various synthetic peptides and concentration conditions, responder cells (T4) / Stimulation cells (LCL- # 350) were co-cultured for 6 hours at an R / S ratio of 3. ELISA was performed using the collected culture supernatant, and IFN-γ produced by T4 cells by various peptide-specific stimuli was quantified.

A-9 Analysis of restriction to HLA class II Anti-human HLA-DR antibody (10 μg / ml; L423, manufactured by BioLegend), anti-human HLA-DQ antibody (10 μg / ml; SPVL3, manufactured by Beckman Coulter, Inc.) or T4 cells (5 × 10 ⁵ cells / ml) and ILT- # 350 (1 × 10 ⁵ cells / ml) in the presence / absence of anti-HLA-ABC antibody (10 μg / ml; W6 / 32, manufactured by BioLegend) For 6 hours. IFN-γ in the supernatant was quantified by ELISA.
To identify HLA class II molecules involved in antigen presentation to T4 cells, Tax155-167 peptide was used using various HLA type LCLs (LCL- # 350, LCL- # 341, LCL- # 307 and LCL-Kan). Specific IFN-γ production response was evaluated. These LCLs (1 × 10 ⁵ cells / ml) were stimulated with 100 ng / ml Tax155-167 peptide for 1 hour, fixed with 2% formaldehyde and then in the presence of T4 cells (3 × 10 ⁵ cells / ml). Cultured for 6 hours. The culture supernatant was collected, and IFN-γ in the supernatant was measured by ELISA.

A-10 Analysis of proliferative ability PBMC (1.0 × 10 ⁶ cells / ml) was cultured for 13 days or 14 days in the presence / absence of 100 nM antigenic peptide. Cells were stained with a combination of HLA / Tax tetramer-PE, CD3-FITC, CD8-PE-Cy5 or CD4-PE-Cy5 and subjected to flow cytometry analysis.

A-11 Statistical analysis Statistical significance was evaluated by Graphpad Prism 5 (Graphpad Prism Software, Inc.) using an independent t-test. In all cases, a bilateral P value of less than 0.05 was considered significant.

B [Results and Discussion]
B-1 Tax-specific T cell immune response in ATL patients after allogeneic hematopoietic stem cell transplantation with palliative pretreatment To assess Tax-specific T cell response, an immunoassay using a GST-Tax fusion protein (A above) -4). The immunological function measurement method using the GST-Tax fusion protein can analyze both CD4 ⁺ T cell responses and CD8 ⁺ T cell responses regardless of the HLA type. The results of such an assay are shown in FIG. From the results of 16 patient PBMCs in FIG. 1A, it was revealed that the degree of immune response to Tax protein stimulation based on IFN-γ production was diverse. In addition, by this assay, 11 out of 16 patients (# 239, # 241, # 301, # 317, # 341, # 344, # 350, # 351, # 352, # 358, # 364), that is, 68 It has been found that as many as 8% of patients can detect Tax-specific T cell responses. Further, among the IFN-γ produced by T cells, in order to clarify the contribution of Tax-specific CD4 ⁺ T cells, analysis was made in comparison with CD8 ⁺ cell-depleted PBMC (“CD8 (−)”). Although the amount of IFN-γ produced is lower than that of (“whole”), both the # 350 and # 341 specimens stimulated Tax protein stimulation with T cells mainly composed of CD8 ⁺ cell-depleted PBMC, that is, CD4 ⁺ It was shown that IFN-γ production was significantly performed in response to (FIG. 1B).

B-2 Induction of HTLV-I specific CD4 ⁺ T cell line from patient # 350 Allogeneic hematopoietic stem cell transplantation using HTLV-I infected T cell line (ILT- # 350) as antigen-presenting cells and palliative pretreatment HTLV-I specific CD4 ⁺ T cells were induced from the same patient # 350 PBMC 180 days later. Peptide stimulation of freshly isolated patient PBMC with Tax 301-309 (CTL dominant epitope presented in HLA-A ^* 2402) for 2 weeks induces Tax-specific CTL and is originally present in patient PBMC HTLV-I infected cells were removed. Next, CD4 ⁺ cells were isolated from cultured cells and stimulated with ILT- # 350 fixed with formaldehyde every 2-3 weeks. When the phenotype on the cell surface of the established cell line was analyzed by flow cytometry (see A-7 above), the cells expressed CD3 and CD4 but not CD8 (FIG. 2A), CD4 ⁺ T cell line was confirmed. Hereinafter, this is called a T4 cell.

HTLV-I is known to preferentially infect CD4 ⁺ T cells both in vivo and in vitro (Non-patent Document 23). When the HTLV-I infection in the T4 cells described above was examined by RT-PCR (see A-7 above), no band was confirmed at the position indicated by pX (FIG. 2B), indicating that HTLV-I was infected. No HTLV-I specific CD4 ⁺ T cells were shown. Tax is a pX gene product, and absence of Tax in the cell indicates that HTLV-I is uninfected. EB virus-transformed B cell line LCL- # 350 was used as a negative control, HTLV-I-infected T cell line ILT- # 350, and ATL cell line MT-2 was used as a positive control.

Next, to examine the production of various cytokines in T4 cells, T4 cells were stimulated for 24 hours in the presence / absence of ILT- # 350 cells or LCL- # 350 cells fixed with formaldehyde, The cytokine concentration was quantified by a cytometry bead array system. As a result, T4 cells produced large amounts of IFN-γ and TNF-α in response to ILT- # 350 and produced low amounts of IL-2, IL-4 and IL-10, but LCL- # 350 Production was not observed (FIG. 2C). Thus, it was shown that T4 cells are HTLV-I specific and are a Th1-type CD4 ⁺ cell line.

B-3 Determination of minimum epitope recognized by T4 cells To examine the antigenic epitope of Tax protein recognized by T4 cells, LCL- # 350 stimulated with GST-Tax protein was used as an antigen presenting cell, The responsiveness of T4 cells was examined (see A-4 and A-8 above). The result is shown in FIG.

  T4 cells produced significantly higher IFN-γ for GST-TaxABC and GST-TaxB (TaxB: amino acid sequence of amino acid numbers 113 to 237 of SEQ ID NO: 32) than GST protein (control). However, for GST-TaxA (TaxA: amino acid sequence of amino acid numbers 1 to 127 of SEQ ID NO: 32) and GST-TaxC (TaxC: amino acid sequence of amino acid numbers 224 to 353 of SEQ ID NO: 32), production is significantly higher. Was not seen (FIG. 3A). This indicates that T4 cells recognized mainly the central region of Tax protein as an antigen.

  Next, 8 types of 25mer overlapping peptides having the central part of Tax (residues 103 to 246) as a region were synthesized (SEQ ID NOs: 1 to 8; see A-3 and Table 2 above), and T4 of these peptides was synthesized. The ability to stimulate cells was analyzed. As a result, only when peptide stimulation was performed with Tax154-178 (SEQ ID NO: 4), IFN-γ production of the T4 cell line showed a high value (FIG. 3B).

  Next, the epitope region was further narrowed, and four types of 15-mer overlapping peptides were prepared from the region of 154-178 residues of Tax (SEQ ID NOs: 10, 13, 14, 15; see A-3 and Table 2 above) ), The ability of T4 cells to produce IFN-γ for each epitope peptide was examined. As a result, Tax151-165 (SEQ ID NO: 10) and Tax156-170 (SEQ ID NO: 13) significantly induced IFN-γ production of T4 cells, although not at levels comparable to Tax154-178 (SEQ ID NO: 4). (FIG. 3C). From these results, it was suggested that the epitope recognized by T4 cells may be present in the N-terminal half of Tax154-178.

  Thus, epitope peptides of Tax154-168 (SEQ ID NO: 11), Tax155-169 (SEQ ID NO: 12), Tax156-170 (SEQ ID NO: 13) were prepared or prepared (see A-3 and Table 2 above) and examined in the same manner. As a result, Tax156-170 produced IFN-γ comparable to Tax154-178, and Tax154-168 and Tax155-169 induced significantly higher IFN-γ production than Tax156-170. These results suggested that the minimal epitope may exist in residues 155 to 168 of Tax (FIG. 3D).

  Next, in order to identify the minimum epitope recognized by T4 cells, three types of overlapping peptides starting from 155 residues of Tax and having a length of 12 to 14 mer were synthesized (SEQ ID NOs: 16 to 18; A-3 above) And Table 2). Tax155-167 induced the same level of IFN-γ production as Tax155-169 and Tax155-168, but Tax155-166 did not reach the same level (FIG. 3E).

  Furthermore, with respect to Tax155-169, Tax155-168, Tax155-167, Tax146-160 (negative control), IFN-γ production was examined by changing the concentration conditions. It was found that the amount of IFN-γ produced in T4 cells in a concentration-dependent manner with respect to Tax155-167 was comparable to that for Tax155-169 and Tax155-168 (FIG. 3F).

  The series of experimental results in B-3 clearly showed that the minimum epitope recognized by T4 cells was Tax155-167.

B-4 HLA-DRB1 ^* 0101 restriction of Tax-specific T4 cells To examine the types of HLA class II molecules involved in antigen presentation of the minimal epitope (Tax155-167) found in B-3 above, anti-HLA-DR In the presence / absence of anti-HLA-DQ and anti-HLA class I blocking antibodies, the immune response of T4 cells stimulated with ILT- # 350 was analyzed (see A-9 above). This epitope was shown to be HLA-DR restricted because blocking of HLA-DR suppressed IFN-γ production of T4 cells in response to stimulation from ILT- # 350 (FIG. 4A).

Furthermore, four types of HLA-type LCLs that display different HLA-DRs were used to examine HLA-DR alleles involved in the presentation of minimal epitopes (see A-9 above). When Tax155-167 was presented by autologous LCL- # 350 (DR1 / 14) and allogeneic LCL- # 341 (DR1 / 15), production of IFN-γ by T4 cells was confirmed (FIG. 4B). This result clearly shows that this epitope is presented on antigen-presenting cells by HLA-DRB1 ^* 0101. In the identified Tax Tax155-167, the known HLA-DRB1 ^* 0101 motif (Rammensee HG, Friede T, Stevanoviic S. MHC ligands and peptide motifs: first listing. Immunogenetics. 1995; 41 (4): 178-228.) As a result of searching for the presence or absence, it was found that this epitope contains the HLA-DRB1 ^* 0101 motif (FIG. 4C).

Enhancement of Tax-specific CD8 ⁺ T cell proliferation with help of B-5 Tax155-167 specific CD4 ⁺ T cells Next, Tax155-167 specific CD4 ⁺ T against Tax specific CTL proliferation in fresh PBMC of patient # 350 Cell helper function was evaluated. PBMCs (A24 / 26, DR1 / 14) that were just isolated from patient # 350 540 days after allogeneic hematopoietic stem cell transplantation with palliative pretreatment were replaced with HLA-A24 restricted CTL epitope Tax301-309 peptide and / or The HLA-DRB1 ^* 0101-restricted Th1-type epitope Tax155-167 peptide was stimulated for 13 days, and the proliferation of Tax-specific CD8 ⁺ T cells was measured using the HLA-A ^* 2402 / Tax301-309 tetramer (A above) -10). This utilizes the property that such tetramers bind to Tax-specific CD8 ⁺ T cells. The result of this measurement is shown in FIG. Tax301-309-specific CD8 ⁺ T cells proliferated to 9.26% when stimulated with Tax301-309 alone (FIG. 5, top middle panel), whereas both Tax301-309 and Tax155-167 were When stimulated in vitro, Tax-specific CD8 ⁺ T cells were significantly increased to 62.3% (upper right panel of FIG. 5). Measurement was performed in the same manner for HLA-DRB1 ^* 0101 ⁺ HTLV-I-infected patients who received allogeneic hematopoietic stem cell transplantation by two other palliative pretreatments. Patient # 364 used PBMC (A24 / 26, DR1 /-) that had just been isolated from the patient 180 days after transplantation, and patient # 341 had PBMC that had just been isolated from the patient 360 days after transplantation. (A24 / 33, DR1 / 15) was used. When PBMC was stimulated with Tax301-309 alone, an HLA-A24-restricted CTL epitope, Tax301-309 specific CD8 ⁺ T cells were 0.85% (patient # 364) and 7.7% (patient # 341). While proliferated (middle middle panel, lower middle panel in FIG. 5), when PBMC were co-stimulated with both Tax 301-309 and Tax 155-167, Tax 301-309 specific CD8 ⁺ T cells were 15.5. % (Patient # 364) and 59.6% (patient # 341) (Fig. 5, middle right panel, lower right panel). In addition, the PBMCs of 3 patients (# 350, # 364, # 341) had a detectable level of Tax-specific CD8 ⁺ T cells that bind to the tetramer described above, prior to stimulation with the Tax epitope. Were present.
Based on the above results, Tax155-167 specific CD4 ⁺ T cells are present in HLA-DRB1 ^* 0101 ⁺ HTLV-I infected patients who have undergone allogeneic hematopoietic stem cell transplantation by palliative pretreatment, Tax301-309 / Tax155-167 It was shown that the CD8 ⁺ T cell response was significantly enhanced by stimulation with mixed epitopes compared to Tax301-309 alone.

B-6 HLA-DRB1 ^* 0101 ⁺ HTLV-I infected individuals maintain Tax155-167 specific CD4 ⁺ T cells Next, HLA-DRB1 ^* 0101 / Tax155-167 tetramers were generated and Tax155-167 specific by detecting the CD4 ⁺ T cells directly, from the ^HLA-DRB1 * 0101 ⁺ two patients who received allogeneic hematopoietic stem cell transplantation in the PBMC of freshly isolated, whether there is Tax155-167-specific CD4 ⁺ T cells It was confirmed. Patient # 350 used PBMCs that had just been isolated from the patient 540 days after transplantation (A24 / 26, DR1 / 14), and patient # 364 had PBMCs that had been isolated from the patient 180 days after transplantation. (A24 / 26, DR1 /-) was used, and for patient # 341, PBMC (A24 / 33, DR1 / 15) just isolated from the same patient 360 days after transplantation was used. In patient # 350, Tax155-167 specific CD4 ⁺ T cells were present at levels detectable (0.11%) without culture or stimulation (ex vivo) (FIG. 6A, upper left panel), Tax155-167. 13 days after stimulation with the peptide, it grew to 11.6% (FIG. 6A, upper right panel). In patient # 364, no tax-specific CD4 ⁺ T cells were detected ex vivo (FIG. 6A, middle left panel), but proliferated to 0.37% when stimulated in vitro with Tax155-167 peptide ( FIG. 6A middle right panel). In HLA-DRB1 ^* 0101 ⁺ seronegative donor # 365 (negative control), no Tax-specific CD4 ⁺ T cells were detected ex vivo (lower left panel in FIG. 6A), 13 days after stimulation with Tax155-167 peptide However, it was not detectable (the right panel at the bottom of FIG. 6A).

In addition, Tax155- in 2 HTLV-I infected individuals carrying HLA-DRB1 ^* 0101 (asymptomatic carrier (AC) # 310 and HTLV-I associated myelopathy / tropical spastic paraparesis patient # 294). When the presence or absence of 167-specific CD4 ⁺ T cells was examined, 0.18% and 0.31% tetramer positive cells were detected in peripheral CD4 ⁺ T cells, respectively (FIG. 6B).

The above results showed that Tax155-167 specific CD4 ⁺ T cells were maintained in HTLV-I infected individuals expressing the HLA-DRB1 ^* 0101 allele regardless of the presence or absence of hematopoietic stem cell transplantation. .

The present invention can be used in fields related to the induction of CD4 ⁺ T cells specific for human T cell leukemia virus type I (HTLV-I). More specifically, an HTLV-I-specific CTL inducing action enhancer, an HTLV-I-specific immune response inducing vaccine, an immune function test diagnostic agent for identifying HTLV-I-specific CD4 ⁺ T cells, It can be suitably used in the field relating to the method for inducing HTLV-I-specific CD4 ⁺ T cells.

Claims (16)

HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide consisting of the amino acid sequence shown in any of (A) to (E) below:
(A) Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence induces HTLV-I-specific CD4 ⁺ T cells. Amino acid sequence with activity:
(D) In the amino acid sequence shown in the above (A) or (B), one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is HTLV-I specific Amino acid sequence having CD4 ⁺ T cell inducing activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL An amino acid sequence obtained by combining the amino acid sequence of the inducing active peptide. The HTLV-I-specific CD4 ⁺ T cell inducing peptide according to claim 1, which is a Tax epitope restricted by HLA-DR1. The HTLV-I-specific CD4 ⁺ T cell-inducing peptide according to claim 1 or 2, wherein the CD4 ⁺ T cell is a Th1-type helper T cell. A fusion peptide in which the HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide according to any one of claims 1 to 3 is bound to a marker protein and / or a peptide tag. A protein-peptide conjugate in which HLA-DR1 and the HTLV-I-specific CD4 ⁺ T cell-inducing active peptide according to any one of claims 1 to 3 are bound. A tetramer of a protein-peptide conjugate in which HLA-DR1 and the HTLV-I-specific CD4 ⁺ T cell-inducing peptide according to any one of claims 1 to 3 are bound. A fusion protein comprising the protein-peptide conjugate according to claim 5 or the tetramer of the protein-peptide conjugate according to claim 6 and a marker protein and / or a peptide tag. HTLV-I-specific CTL inducing action potentiator comprising, as an active ingredient, an HTLV-I-specific CD4 ⁺ T cell inducing peptide consisting of any of the following amino acid sequences (A) to (E): (A) Amino acid sequence shown in any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence induces HTLV-I-specific CD4 ⁺ T cells. Amino acid sequence with activity:
(D) In the amino acid sequence shown in the above (A) or (B), one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is HTLV-I specific Amino acid sequence having CD4 ⁺ T cell inducing activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL An amino acid sequence obtained by combining the amino acid sequence of the inducing active peptide. An expression vector comprising a promoter and a polynucleotide comprising the polynucleotide sequence shown in any of the following (a) to (e), wherein the polynucleotide is operably linked downstream of the promoter, as an active ingredient HTLV-I specific CTL inducing action enhancer containing:
(A) a polynucleotide sequence encoding the amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) a polynucleotide sequence encoding an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence represented by SEQ ID NO: 32 and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) a polynucleotide sequence having 80% or more identity to the polynucleotide sequence shown in the above (a) or (b), wherein the peptide has HTLV-I-specific CD4 ⁺ T cell inducing activity Encoding polynucleotide sequence:
(D) a polynucleotide sequence obtained by deleting, substituting, or adding one or several nucleotides in the polynucleotide sequence shown in (a) or (b), wherein the HTLV-I specific CD4 ⁺ T cell Polynucleotide sequence encoding a peptide having inductive activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising at least an amino acid sequence of amino acid numbers 155 to 167, and an HTLV-I-specific CTL A polynucleotide sequence encoding an amino acid sequence linked to an amino acid sequence of an inducing active peptide. An HTLV-I-specific immune response induction comprising the HTLV-I-specific CTL inducing action-enhancing agent according to claim 8 or 9, and an HTLV-I-specific CTL-inducing activity peptide or a polynucleotide encoding the peptide. vaccine. Containing the following (A) ~ one to HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide comprising the amino acid sequence shown in (E) as an active ingredient, identifying the HTLV-I-specific CD4 ⁺ T cells Immune function test diagnostic agent for:
(A) Amino acid sequence represented by any of SEQ ID NOs: 4, 10, 11, 12, 13, 16, 17, 19:
(B) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, and comprising at least the amino acid sequences of amino acid numbers 155 to 167:
(C) An amino acid sequence having 80% or more identity to the amino acid sequence shown in (A) or (B) above, wherein the peptide comprising the amino acid sequence induces HTLV-I-specific CD4 ⁺ T cells. Amino acid sequence with activity:
(D) In the amino acid sequence shown in the above (A) or (B), one or several amino acids are deleted, substituted or added, and the peptide comprising the amino acid sequence is HTLV-I specific Amino acid sequence having CD4 ⁺ T cell inducing activity:
(E) an amino acid sequence consisting of 14 to 30 consecutive amino acids in the amino acid sequence shown in SEQ ID NO: 32, comprising an amino acid sequence of at least amino acid numbers 155 to 167, and an HTLV-I-specific CTL An amino acid sequence obtained by combining the amino acid sequence of the inducing active peptide. HLA-DR1 and claims 1 to 3 or the HTLV-I-specific CD4 ⁺ T cell-inducing activity peptide is bound to proteins according - containing peptide conjugate as an active ingredient, HTLV-I-specific CD4 ⁺ A diagnostic reagent for immune function tests for identifying T cells. HTLV-I comprising, as an active ingredient, a tetramer of a protein-peptide conjugate in which HLA-DR1 and the HTLV-I-specific CD4 ⁺ T cell-inducing peptide according to any one of claims 1 to 3 are bound An immunological function test diagnostic agent for identifying specific CD4 ⁺ T cells. HTLV-I specific CD4 ⁺ T cells characterized in that HTLV-I infected T cells derived from ATL patients before HSCT are used to stimulate PBMCs of the same patients after HSCT from allogeneic HLA type donors Guidance method. 10. Induction of HTLV-I-specific CD4 ⁺ T cells, characterized by stimulating PBMC of HLA-DR1-positive ATL patients using the HTLV-I-specific CTL inducing action enhancer according to claim 8 or 9. Method. HTLV-I-specific CD4 ⁺ T cell response and HTLV-I-specific CTL response characterized by stimulating PBMC of HLA-DR1-positive ATL patients with the following (X) and (Y) Methods of inducing HTLV-I specific immune responses comprising:
(X) The HTLV-I-specific CTL inducing action potentiator according to claim 8 or 9:
(Y) an HTLV-I-specific CTL-inducing active peptide that matches the type of HLA-A locus of the ATL patient or a polynucleotide encoding the peptide.