Pharmaceutical composition for treating or preventing adult t-cell leukemia

Abstract

To provide pharmaceutical compositions for treating or preventing adult T-cell leukemia.SOLUTION: Provided is a pharmaceutical composition for treating or preventing adult T-cell leukemia, which comprises a peptide consisting of a specific amino acid sequence, as a technique available for cancer immunotherapy of adult T-cell leukemia (ATL).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for treating or preventing adult T-cell leukemia (ATL), more specifically to a pharmaceutical composition for treating or preventing ATL, which comprises a peptide consisting of a specific amino acid sequence. [Background technology]

[0002] Cancer immunotherapy using cancer antigen peptides is being developed. For example, Patent Document 1 discloses cancer immunotherapy using peptides derived from the cancer antigen glypican 3 (GPC3). It has been revealed that GPC3 is expressed in almost all hepatocellular carcinomas, but is hardly expressed in normal livers or liver cirrhosis. In addition to hepatocellular carcinoma, GPC3 is also known to be highly expressed in melanoma, ovarian cancer, and other cancers.

[0003] The elimination of cancer cells by cytotoxic T cells (CTLs) is induced by the specific recognition by T cell antigen receptors (TCRs) of cancer antigens consisting of 8-11 amino acids (HLA epitopes) presented on major histocompatibility antigen (HLA) class I molecules on the surface of cancer cells. HLA molecules are broadly divided into class I molecules (HLA-A, B, C) and class II molecules (HLA-DP, DQ, DR).

[0004] Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that primarily infects CD4+ T cells and causes adult T-cell leukemia (ATL).

[0005] ATL is a malignant hematologic tumor that infiltrates various organs throughout the body and is classified into four disease types: acute, lymphomatous, chronic, and smoldering. The acute and lymphomatous types are also called "aggressive ATL" and have the poorest prognosis of hematopoietic malignancies. The chronic and smoldering types are also called "indolent ATL," but the majority of cases progress to acute stage during the course of the disease and have a poor prognosis.

[0006] Treatment for ATL includes combination chemotherapy. However, even in the most recent reports, the median survival time for "aggressive ATL" is only 13 months, which is unsatisfactory. Patients with "indolent ATL" may survive for a long time without treatment, but they are often monitored without treatment until the disease worsens, such as when it becomes acute. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2016 / 143816 Summary of the Invention [Problem to be solved by the invention]

[0008] The main objective of the present disclosure is to provide techniques that can be used for cancer immunotherapy of ATL. [Means for solving the problem]

[0009] To solve the above problems, the present disclosure provides the following [1]-

[15] . [1] A pharmaceutical composition for treating or preventing adult T-cell leukemia (ATL), comprising a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1-68. [2] The pharmaceutical composition of [1], wherein the peptide binds to one or more types of major histocompatibility antigen (HLA) molecules. [3] The pharmaceutical composition of [2], wherein the peptide consists of any one of the amino acid sequences of SEQ ID NOs: 1-47 and binds to two or more types of HLA molecules. [4] The pharmaceutical composition of [3], wherein the peptide consists of any one of the amino acid sequences of SEQ ID NOs: 1-33 and binds to three or more types of HLA molecules. [5] The pharmaceutical composition according to any one of [1] to [4], wherein the peptide induces cytotoxic T lymphocytes (CTLs). [6] The pharmaceutical composition of [5], wherein the peptide consists of any one of the amino acid sequences of SEQ ID NOs: 16, 19, 20, 21, 47, and 66. [7] Any of the pharmaceutical compositions of [1]-[6] in the form of a vaccine.

[0010] [8] A peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1-68. [9] The peptide of [8], which binds to one or more types of HLA molecules.

[10] A peptide according to [9], which comprises any one of the amino acid sequences of SEQ ID NOs: 1-47 and binds to two or more types of HLA molecules.

[11] A peptide according to

[10] , which comprises any one of the amino acid sequences of SEQ ID NOs: 1-33 and binds to three or more types of HLA molecules.

[12] Any of the peptides [8]-

[11] that induce CTL.

[13] The peptide of

[12] , consisting of any one of the amino acid sequences of SEQ ID NOs: 16, 19, 20, 21, 47, and 66.

[0011]

[14] A method for producing antigen-presenting cells having cytotoxic T cell inducing activity, comprising a step of contacting any of the peptides [8]-

[13] with antigen-presenting cells in vitro.

[15] Use of any of the peptides [8]-

[13] for the manufacture of a pharmaceutical composition for the treatment or prevention of ATL. [Effects of the Invention]

[0012] The present disclosure provides techniques that can be used for cancer immunotherapy of ATL. [Brief explanation of the drawings]

[0013] [Figure 1] The results of stimulating peripheral blood mononuclear cells (PBMC) from an ATL patient (sample 1) with peptides and measuring the amount of IFN-γ produced are shown. [Figure 2] PBMCs from an ATL patient (sample 3) were stimulated with peptides, and the amount of IFN-γ produced was measured. [Figure 3] PBMCs from an ATL patient (sample 4) were stimulated with peptides, and the amount of IFN-γ produced was measured. [Figure 4] PBMCs from an ATL patient (sample 12) were stimulated with peptides, and the amount of IFN-γ produced was measured. [Figure 5] The PBMCs of an ATL patient (sample 21) were stimulated with peptides, and the amount of IFN-γ produced was measured. [Figure 6] The PBMCs of an ATL patient (sample 24) were stimulated with peptides, and the amount of IFN-γ produced was measured. [Figure 7] The PBMCs of an ATL patient (sample 29) were stimulated with peptides, and the amount of IFN-γ produced was measured. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1. Immunogenic peptides The peptides of the present disclosure consist of any of the amino acid sequences of SEQ ID NOs: 1-68. The peptides disclosed herein are derived from six proteins (Casp-8, c-Myb, Fas, Helios, Wnt5a, gp46) that have been found to have abnormal amino acid sequences or four proteins (c-Myb, CADM1, EVC1, EVC2, p19, tax) that have been found to be highly expressed in patients with adult T-cell leukemia (ATL).

[0015] When a peptide according to the present disclosure is derived from a protein whose amino acid sequence is abnormal in an ATL patient, the amino acid sequence of the peptide according to the present disclosure may include the abnormal amino acid sequence of the protein. The abnormal amino acid sequence of the protein may be any sequence different from the wild-type amino acid sequence, and may be, for example, an amino acid sequence that includes a fusion resulting from deletion of all or part of the amino acid sequence of an exon, a substitution of all or part of the amino acid sequence of an exon (alternative usage of exon), an insertion of an amino acid sequence derived from all or part of an intron, or a substitution, deletion, or insertion of one or more amino acid sequences of an exon.

[0016] When the peptide according to the present disclosure is derived from a protein that is found to be highly expressed in ATL patients, the amino acid sequence of the peptide according to the present disclosure may consist of a portion of the wild-type amino acid sequence of the protein.

[0017] Details of the Casp-8, c-Myb, Fas, Helios, Wnt5a, CADM1, EVC1, EVC2, p19, tax, and gp46 genes are shown below.

[0018] [Table 1]

[0019] Based on the abnormal or wild-type amino acid sequences of each gene described above, a 9-amino acid sequence predicted to exhibit high binding affinity to major histocompatibility antigen (HLA) molecules was selected using a hypothesis derived from an active learning experiment (Japanese Patent Laid-Open Publication No. 8-151396). Peptides consisting of the selected amino acid sequences were synthesized, and their ability to bind to HLA molecules and to induce cytotoxic T lymphocytes (CTLs) was evaluated in vitro using interferon-γ (IFN-γ) production as an indicator.

[0020] The amino acid sequences of the peptides and their binding abilities to HLA molecules are shown in Table 1. The binding ability to HLA molecules is indicated by the dissociation constant Kd value (-logKd value). In the table, ">-3.0" indicates that the Kd value is greater than -3.0 and there is no significant binding ability. Furthermore, for example, the description ">-3.37" as seen in the measurement value for "vs. A*02:06" in SEQ ID NO: 26 indicates that the Kd value was smaller than -3.0 and significant binding ability was confirmed, but the Kd value could not be determined definitively and was between -3.37 and -3.0.

[0021] [Table 2] TIFF2025172927000004.tif240170

[0022] The peptides of SEQ ID NOs: 1-4, 23, and 48 are derived from CADM1. The peptides of SEQ ID NOs: 5, 6, 24, 25, 36, 37, 52-60, and 76-78 are derived from c-Myb. The peptides of SEQ ID NOs: 7-10, 26-28, 38, and 39 are derived from EVC1. The peptides of SEQ ID NOs: 11-15, 40-43, 61, and 79 are derived from EVC2. The peptides of SEQ ID NOs: 16, 17, 29, 30, 44-46, 62, and 63 are derived from Fas. The peptides of SEQ ID NOs: 18 and 31 are derived from gp46. The peptides of SEQ ID NOs: 19, 478, 64, 65, 80, and 81 are derived from Helios. The peptide of SEQ ID NO: 20 is derived from p19. The peptides of SEQ ID NOs: 21, 22, 32, and 33 are derived from tax. The peptides of SEQ ID NOs: 34, 35, 49-51, 69-75 are derived from Casp-8. The peptides of SEQ ID NOs: 66-68 are derived from Wnt5a.

[0023] The peptides according to the present disclosure bind to one or more types (subtypes) of HLA molecules, preferably two or more types, and more preferably three or more types of HLA molecules. The peptides of SEQ ID NOs: 1-33 exhibit binding affinity to all of the products of the HLA-A*24:02 gene (HLA-A*24:02 molecule), the HLA-A*02:01 gene (HLA-A*02:01 molecule), and the HLA-A*02:06 gene (HLA-A*02:06 molecule). The peptides of SEQ ID NOs: 34-47 exhibit binding affinity to two or more of HLA-A*24:02 molecules, HLA-A*02:01 molecules, and HLA-A*02:06 molecules. The peptides of SEQ ID NOs: 48-68 exhibit binding to any one of HLA-A*24:02 molecules, HLA-A*02:01 molecules, and HLA-A*02:06 molecules.

[0024] The HLA subtypes to which the peptides of the present disclosure can bind are not limited to HLA-A*24:02, HLA-A*02:01, and HLA-A*02:06. However, these HLA subtypes cover approximately 85% of Asians, including Japanese, and approximately 55% of Westerners, and therefore the multi-HLA peptides of the present invention are considered to have a wide patient coverage rate in immunotherapy and the like.

[0025] Furthermore, the peptides according to the present disclosure induce CTLs. In particular, the peptides of SEQ ID NOs: 16, 19, 20, 21, 47, and 66 exhibit CTL-inducing activity in ATL patients.

[0026] The peptides disclosed herein have HLA binding activity and are immunogenic. As used herein, "immunogenicity" refers to the ability to induce an immune response (immune induction ability), for example, to increase the cytotoxic T cell (CTL)-inducing activity of antigen-presenting cells and further to increase the cytotoxic activity of CTLs against cancer cells. Furthermore, as used herein, "CTL induction" means in vitro or in vivo, inducing or proliferating CTLs that specifically recognize a certain antigen by presenting a peptide according to the present disclosure on the surface of an antigen-presenting cell, differentiating naive T cells into effector cells that have the ability to kill target cells such as cancer cells (cytotoxic activity), and / or increasing the cytotoxic activity of CTLs. CTL-inducing activity can be measured by assessing cytokine (e.g., IFN-γ) production by CTLs. For example, CTL-inducing activity may be measured by assessing the increase in cytokine-producing cells induced from precursor cells by antigen-presenting cells such as peripheral blood mononuclear cells stimulated with a peptide according to the present disclosure, using a known highly sensitive immunoassay such as ELISPOT (Enzyme-Linked ImmunoSpot) or ELISA (Enzyme-Linked ImmunoSorbent Assay). The cytotoxic activity of CTLs 51 It can be measured by known methods such as the Cr release method. When the above activity is significantly increased compared to the control, for example, when it is increased by 5% or more, 10% or more, 20% or more, preferably 50% or more, it can be evaluated that immunity or CTLs have been induced.

[0027] The peptides disclosed herein have high HLA binding affinity and high CTL induction capacity, and are therefore expected to be useful as cancer vaccines. They are also expected to be applied to various immunotherapies, particularly dendritic cell therapy. Furthermore, the peptides disclosed herein can bind to multiple HLA types. Therefore, the peptides disclosed herein can provide cancer vaccines and dendritic cell therapy that cover an extremely wide range of ATL patients.

[0028] The peptides of the present disclosure may be modified in the amino acid residues constituting the amino acid sequence of SEQ ID NO: 1-68 or portions thereof, so long as they retain immunogenicity. The amino acid sequence represented by SEQ ID NO: 1-68 is intended to be the amino acid sequence in the state presented on antigen-presenting cells; however, when the peptide is administered directly into the body, depending on the route of administration, the peptide termini may be subject to alteration, such as digestion by the digestive organs. Therefore, the peptides of the present disclosure may exist in the form of a precursor in which one or more amino acid residues have been added to the N-terminus and / or C-terminus before being taken up by antigen-presenting cells, so that the amino acid residues represented by SEQ ID NO: 1-68 are retained when binding to a specific HLA class I molecule on antigen-presenting cells.

[0029] The peptides of the present disclosure may have one or more amino acid residues substituted, inserted, deleted, or added, and / or may be modified by glycosylation, side chain oxidation, and / or phosphorylation, as long as they have the desired immunogenicity. As used herein, the term "amino acid" is used in its broadest sense and includes not only naturally occurring amino acids but also artificial amino acid variants and derivatives. As used herein, amino acids include naturally occurring proteinaceous L-amino acids; D-amino acids; chemically modified amino acids such as amino acid variants and derivatives; naturally occurring non-proteinaceous amino acids such as norleucine, β-alanine, and ornithine; and chemically synthesized compounds having properties known in the art that are characteristic of amino acids. Examples of unnatural amino acids include α-methylamino acids (such as α-methylalanine), D-amino acids, histidine-like amino acids (such as β-hydroxyhistidine, homohistidine, α-fluoromethylhistidine, and α-methylhistidine), amino acids with an extra methylene in the side chain ("homo" amino acids), and amino acids in which the carboxylic acid functional group in the side chain is replaced with a sulfonic acid group (such as cysteic acid).

[0030] With regard to substitution of amino acid residues, those skilled in the art can appropriately substitute amino acid residues constituting the peptides of the present disclosure, taking into consideration the regularity of peptide sequences that exhibit HLA binding (J. Immunol., 152: p. 3913, 1994; Immunogenetics, 41: p. 178, 1995; J. Immunol., 155: p. 4307, 1994).

[0031] More specifically, in the case of a peptide that binds to an HLA-A*24:02 molecule, the second amino acid constituting the peptide may be substituted with tyrosine, phenylalanine, methionine, or tryptophan, and / or the C-terminal amino acid may be substituted with phenylalanine, leucine, isoleucine, tryptophan, or methionine. Furthermore, in the case of peptides that bind to HLA-A*02:01 molecules, the amino acid at position 2 may be substituted with leucine or methionine, and / or the C-terminal amino acid may be substituted with valine or leucine. Furthermore, for peptides that bind to HLA-A*02:06 molecules, the amino acid at position 2 may be substituted with valine or glutamine, and / or the C-terminal amino acid may be substituted with valine or leucine.

[0032] The peptides of SEQ ID NO: 3 and 4 derived from CADM1 are peptides of SEQ ID NO: 2 in which an amino acid substitution has been introduced at the second position. The peptides of SEQ ID NO:6 and SEQ ID NO:25 derived from c-Myb are peptides of SEQ ID NO:5 in which an amino acid substitution has been introduced at the second position. The peptides of SEQ ID NOs: 10, 27, 28, and 39 derived from EVC1 are peptides of SEQ ID NOs: 7 and 9 in which an amino acid substitution has been introduced at the second position. The peptides of SEQ ID NOs: 40-43, 61, and 79 derived from EVC2 are peptides of SEQ ID NO: 13 in which an amino acid substitution has been introduced at the second position. The peptides of SEQ ID NO: 17, 29, 30, 45, 46, and 63 derived from Fas are peptides of SEQ ID NO: 16 with an amino acid substitution at the second position.

[0033] The peptides according to the present disclosure can be produced using techniques known to those skilled in the art. For example, they may be artificially synthesized by solid-phase or liquid-phase methods such as the Fmoc method or the tBoc method. Alternatively, desired peptides may be produced by expressing a polynucleotide encoding a peptide according to the present disclosure or a recombinant vector containing the polynucleotide. Furthermore, any peptides thus obtained can be identified using techniques known to those skilled in the art. For example, they can be identified using the Edman degradation method or mass spectrometry.

[0034] 2. Pharmaceutical Compositions A pharmaceutical composition for treating or preventing ATL according to the present disclosure comprises, as an active ingredient, for example, a peptide consisting of amino acid residues of SEQ ID NOs: 1-68.

[0035] The peptides disclosed herein induce CTLs by being presented on antigen-presenting cells, and the induced CTLs then damage cancer cells. Therefore, the active ingredient of the pharmaceutical composition disclosed herein is not limited to the peptides disclosed herein, but may also be a component capable of directly or indirectly inducing CTLs, such as a polynucleotide encoding the peptide or a vector containing the same, or an antigen-presenting cell or exosome secreted by the antigen-presenting cell that presents a complex of the peptide and an HLA molecule on its surface, or a combination thereof. Examples of antigen-presenting cells that can be used include macrophages and dendritic cells, but it is preferable to use dendritic cells, which have a high CTL inducibility. Other components known to be used in cancer treatment, such as chemokines, cytokines, tumor necrosis factors, chemotherapeutic agents, etc., may also be included in the pharmaceutical compositions of the present disclosure. The dose of the peptide may be, for example, about 1 to 10 mg per day for an adult patient. However, the dose varies depending on the patient's age, weight, administration method, etc., and can be appropriately determined by one skilled in the art.

[0036] Although not intended to be limiting, the pharmaceutical composition according to the present disclosure is believed to be useful for killing cancer cells, etc., through the following mechanism of action. When the pharmaceutical composition according to the present disclosure is administered to an ATL patient, the peptides in the pharmaceutical composition are presented on the surface of antigen-presenting cells in a bound state to HLA molecules. When CTLs recognize peptides on such antigen-presenting cells, they become activated, proliferate, and circulate in the systemic circulation. When peptide-specific CTLs invade cancer tissue, they recognize the same peptide derived from a specific cancer antigen that is naturally bound to HLA molecules on the surface of cancer cells, and kill those cancer cells. This mechanism can contribute to the treatment of cancer.

[0037] The pharmaceutical composition according to the present disclosure can be used not only for cancer treatment but also for cancer prevention. For example, by administering the pharmaceutical composition according to the present disclosure to a healthy human body, CTLs are induced, and the induced CTLs remain in the body, so that when cancer cells appear, they can damage the cancer cells. Similarly, by administering the pharmaceutical composition according to the present disclosure to a human body after cancer treatment, the recurrence of cancer can be prevented.

[0038] The pharmaceutical composition according to the present disclosure can be dissolved in a water-soluble solvent, formulated into a pharmaceutically acceptable salt form, and administered to a patient. Such pharmaceutically acceptable salt forms include physiologically acceptable water-soluble salts, such as salts of sodium, potassium, magnesium, calcium, etc., buffered at physiological pH. In addition to water-soluble solvents, water-insoluble solvents can also be used, and examples of such water-insoluble solvents include alcohols such as ethanol and propylene glycol.

[0039] Formulations containing the pharmaceutical composition of this embodiment may also contain agents for various purposes, such as preservatives and buffers. Preservatives include sodium bisulfite, sodium bisulfate, sodium thiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate, methylparaben, polyvinyl alcohol, phenylethyl alcohol, ammonia, dithiothreitol, and beta-mercaptoethanol. Buffers include sodium carbonate, sodium borate, sodium phosphate, sodium acetate, and sodium bicarbonate. These agents can be present in an amount sufficient to maintain the pH of the system between 2 and 9, preferably between 4 and 8.

[0040] The dosage form of the pharmaceutical composition according to the present disclosure is not particularly limited, and when used in the form of a vaccine, its dosage form can be, for example, an injection (intramuscular, subcutaneous, or intradermal), an oral preparation, or a nasal drop preparation. When the pharmaceutical composition according to the present disclosure is in the form of a vaccine, it can be a mixed cocktail vaccine containing multiple active ingredients. For example, such a vaccine can contain multiple active ingredients in combination with any two or more of the peptides of SEQ ID NOs: 1-68, or with other active ingredients.

[0041] The vaccine may also be a vaccine containing an inactive component, which is a component other than the pharmaceutical composition that is inactive in itself but has the effect of further enhancing the efficacy of the pharmaceutical composition as a vaccine. Examples of inactive components include adjuvants, toxoids, etc. Examples of adjuvants include, but are not limited to, precipitating types such as aluminum hydroxide, aluminum phosphate, and calcium phosphate, and oil-based types such as Freund's complete adjuvant and Freund's incomplete adjuvant.

[0042] When the pharmaceutical composition according to the present disclosure is in the form of a vaccine, it is preferably administered into the body by injection or infusion, such as intradermal, subcutaneous, intravenous, or intramuscular administration, or by transdermal administration or inhalation through mucous membranes such as the nose or pharynx. The single dose can be set between an amount that can significantly induce cytotoxic T cells and an amount that does not damage a significant number of non-cancer cells.

[0043] The pharmaceutical compositions disclosed herein are intended for administration to the human body as well as for ex vivo use. More specifically, the pharmaceutical compositions disclosed herein may be used to stimulate antigen-presenting cells in vitro or ex vivo to enhance CTL induction activity. For example, in the case of cancer dendritic cell therapy, the pharmaceutical compositions disclosed herein may be administered to a patient by contacting them with antigen-presenting cells, such as dendritic cells, derived from a patient in need of cancer treatment or prevention, and then returning the antigen-presenting cells to the patient. The peptides contained in the pharmaceutical compositions can be introduced into antigen-presenting cells by, for example, lipofection or injection. When a polynucleotide encoding a peptide disclosed herein is used in such applications, the polynucleotide can be introduced into antigen-presenting cells by techniques known in the art. For example, antigen-presenting cells derived from a patient may be transformed in vitro with a target polynucleotide or a vector encoding the polynucleotide by lipofection, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate, or the like.

[0044] 3. Method for producing antigen-presenting cells The method for producing antigen-presenting cells according to the present disclosure includes, for example, a step of contacting antigen-presenting cells in vitro with a peptide consisting of the amino acid sequence of SEQ ID NO: 1-68.

[0045] The peptides used in the production methods of the present disclosure are thought to bind to HLA class I molecules on the surface of antigen-presenting cells and be presented to CTLs as antigenic peptides, thereby inducing the CTL activity of the antigen-presenting cells. Therefore, the component contacted with antigen-presenting cells is not limited to the peptides of the present disclosure; it may also be a component capable of directly or indirectly inducing CTLs, such as a polynucleotide encoding the peptide or a vector containing the same, or an antigen-presenting cell or exosome secreted by the antigen-presenting cell that presents a complex of the peptide and an HLA molecule on its surface, or a combination thereof. Examples of antigen-presenting cells that can be used include macrophages and dendritic cells, but it is preferable to use dendritic cells, which have high CTL inducibility.

[0046] Antigen-presenting cells produced by the production method of the present disclosure are intended to be used not only as the active ingredient of the pharmaceutical composition or immune inducer, but also for immunotherapy and other purposes. For example, in the case of cancer dendritic cell therapy, the produced antigen-presenting cells can be administered to a patient by contacting them with antigen-presenting cells, such as dendritic cells with low CTL inducibility, derived from a patient in need of immune induction, and then returning the antigen-presenting cells to the patient. Peptides of the present disclosure can be introduced into antigen-presenting cells by, for example, liposome-mediated transfection (lipofection method), injection, or the like. When a polynucleotide encoding a peptide of the present disclosure is used in such applications, the polynucleotide can be introduced into antigen-presenting cells by techniques known in the art. For example, antigen-presenting cells derived from a patient may be transformed in vitro with a polynucleotide of interest or a vector encoding the polynucleotide by lipofection, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate, or the like. [Example]

[0047] 1. Extraction of amino acid sequences The prediction, experiment, and evaluation procedures in this example were carried out based on the active learning experiment design described in WO 2006 / 004182, and rules were constructed by repeating the following steps as a whole.

[0048] (1) Run one trial of the lower-level learning algorithm described below. That is, generate multiple hypotheses from random resampling of the accumulated data, and select the point with the largest variance of the predicted values ​​for the randomly generated query candidate points (peptides) as the query point to be tested.

[0049] (2) The selected peptides are prepared using the synthesis and purification methods described below, and their actual binding ability is measured using the experiments described below and added to the accumulated data.

[0050] By using this active learning method, it would be possible to identify 500 billion (=20) candidates for HLA-binding peptides, which would normally consist of 9 amino acid residues. 9 ) reducing the number of binding experiments that need to be performed.

[0051] According to the rules described above, the amino acid sequences shown in SEQ ID NOs: 1 to 81 were extracted. Specifically, amino acid sequences with predicted binding scores of 3 or more, calculated as a -logKd value, to the HLA-A*24:02 gene product (HLA-A*24:02 molecule), the HLA-A*02:01 gene product (HLA-A*02:01 molecule), and the HLA-A*02:06 gene product (HLA-A*02:06 molecule) were selected.

[0052] [Table 3] TIFF2025172927000006.tif237170

[0053] 2. Peptide synthesis and purification Peptides having the amino acid sequences of SEQ ID NOs: 1-81 were manually synthesized using Fmoc amino acids by Merrifield solid-phase synthesis. After deprotection, the peptides were purified by reverse-phase HPLC using a C18 column to a purity of 95% or higher. Peptide identity and purity were confirmed by MALDI-TOF mass spectrometry (AB SCIEX MALDI-TOF / TOF5800). Peptide quantification was performed using a Micro BCA assay (Thermo Scientific) using BSA as a standard protein.

[0054] 3. Peptide binding experiment to HLA-A molecules (1)HLA-A*24:02 molecule The binding ability of peptides to the HLA-A*24:02 molecule, a product of the HLA-A*24:02 gene, was measured using C1R-A24 cells expressing the HLA-A*24:02 molecule (created by Professor Masafumi Takiguchi of Kumamoto University and provided with permission by Assistant Professor Masataka Yasukawa of Ehime University).

[0055] First, C1R-A24 cells were exposed to an acidic pH of 3.3 for 30 seconds to dissociate and remove the endogenous peptide originally bound to HLA-A*24:02 molecules and the light chain β2m commonly associated with HLA class I molecules. After neutralization, purified β2m was added to C1R-A24 cells, followed by a dilution series of peptides and incubation on ice for 4 hours. The resulting triple complex (MHC-pep) of HLA-A*24:02, peptide, and β2m reassembled during this incubation was stained with fluorescently labeled monoclonal antibody 17A12.

[0056] The number of MHC-pep molecules per C1R-A24 cell (proportional to the fluorescence intensity of the fluorescent antibody) was then quantified using a FACScan (Becton-Dickinson) fluorescent cell analyzer. The binding dissociation constant Kd between the HLA-A*24:02 molecule and the peptide was calculated from the mean fluorescence intensity per cell using the method published by the present inventors in their paper (Udaka et al., Immunogenetics, 51, 816-828, 2000).

[0057] (2)HLA-A*02:01 molecule The binding ability of the peptides to the HLA-A*02:01 molecule, which is the product of the HLA-A*02:01 gene, was measured using the cell line T2 (purchased from ATCC) that expresses the HLA-A*02:01 molecule.

[0058] T2 cells and purified β2m were added to a serial dilution of the peptide to be measured for binding activity, and then incubated at 37°C for 4 hours. HLA-A*02:01 molecules, whose expression level increased in a peptide concentration-dependent manner up to this point, were stained with the interaction-specific fluorescently labeled monoclonal antibody BB7.2.

[0059] Thereafter, the amount of fluorescence per cell was measured using a flow cytometer, and the dissociation constant Kd value was calculated using the method published by the present inventors in their paper (Udaka et al., Immunogenetics, 51, 816-828, 2000).

[0060] (3)HLA-A*02:06 molecule The binding ability of peptides to the HLA-A*02:06 molecule, the product of the HLA-A*02:06 gene, was measured using RA2.6 cells (a newly created cell line at Kochi University) in which the cDNA of the HLA-A*02:06 gene was introduced into RMAS, a mouse TAP (transporter associated with antigen processing)-deficient cell line.

[0061] First, RA2.6 cells were cultured overnight at 26°C, and when HLA-A*02:06 molecules not binding to the peptide accumulated on the cell surface, a dilution series of the peptide was added and allowed to bind at 26°C for 60 minutes.

[0062] After incubation at 35°C for 4 hours, the unbound HLA-A*02:06 molecules were denatured and lost their three-dimensional structure. The fluorescently labeled monoclonal antibody BB7.2, which specifically recognizes peptide-bound HLA-A*02:06 molecules, was added and incubated on ice for 20 minutes to stain the cells.

[0063] Thereafter, the amount of fluorescence per cell was measured using a flow cytometer, and the dissociation constant Kd value was calculated according to the method described in the literature (Udaka et al., Immunogenetics, 51, 816-828, 2000).

[0064] (4) Results The dissociation constant Kd values ​​are shown in Table 3. When the Kd value (-logKd value) is smaller than -3, it can be determined that there is binding.

[0065] [Table 4] TIFF2025172927000008.tif240170

[0066] The peptides of SEQ ID NOs: 1-33 exhibited binding to all of the HLA-A*24:02 molecule, HLA-A*02:01 molecule, and HLA-A*02:06 molecule. The peptides of SEQ ID NOs: 34-47 exhibited binding to two or more of HLA-A*24:02 molecules, HLA-A*02:01 molecules, and HLA-A*02:06 molecules. The peptides of SEQ ID NOs: 48-68 exhibited binding to any one of HLA-A*24:02 molecules, HLA-A*02:01 molecules, and HLA-A*02:06 molecules. Contrary to expectations, the peptides of SEQ ID NOs: 69-81 did not exhibit binding to any of the HLA-A*24:02, HLA-A*02:01 and HLA-A*02:06 molecules.

[0067] 4. Peptide immune induction test Peripheral blood mononuclear cells (PBMCs) isolated from ATL patients were cultured and stimulated with peptides, and the amount of IFN-γ produced was measured by ELISA.

[0068] A suspension of PBMCs (2 x 10 6The cells were seeded into 96-well plates (100-200 μl / well) and cultured for 2-3 days in a medium (ASF-104, 2% autologous serum, 20 U / ml IL-2) containing 5 μg / ml of peptide. After washing, the cells were resuspended in the same medium, and 5 μg / ml of peptide was added, followed by further culture for 3 to 5 days. PBMCs not stimulated with peptide served as a control. The culture supernatant was collected and subjected to measurement of the amount of IFN-γ produced by ELISA (Human IFN-γ ELISA MAX Deluxe Set, BioLegend).

[0069] Representative ELISA assay results for seven patients are shown in Figures 1-7. FIG. 1 shows the results for a patient (sample 1) who has HLA-A*24:02 as one of the alleles. FIG. 2 shows the results for a patient (sample 3) who has HLA-A*02:06 as one of the alleles. FIG. 3 shows the results for a patient (sample 4) who has HLA-A*02:06 as one of the alleles. FIG. 4 shows the results for a patient (sample 12) who has HLA-A*02:01 as one of the alleles. FIG. 5 shows the results for a patient (sample 21) who has HLA-A*02:01 as one of the alleles. FIG. 6 shows the results for a patient (sample 24) who has HLA-A*24:02 as one of the alleles. FIG. 7 shows the results for a patient (sample 29) with HLA-A*24:02 / *02:06. In the figure, "H1" means the peptide of SEQ ID NO: 47, "A15" means the peptide of SEQ ID NO: 66, "A10" means the peptide of SEQ ID NO: 16, "A19-1" means the peptide of SEQ ID NO: 20, "at-1" means the peptide of SEQ ID NO: 21, and "A13" means the peptide of SEQ ID NO: 19.

[0070] The peptides of sequence numbers 16, 19, 20, and 21, which show binding to all of the HLA-A*24:02, HLA-A*02:01, and HLA-A*02:06 molecules, showed significant IFN-γ production, confirming their immune induction ability. Furthermore, while many of the peptides that did not bind to any of the HLA-A*24:02, HLA-A*02:01, and HLA-A*02:06 molecules did not produce significant IFN-γ, the peptides of sequence numbers 47 and 66 produced significant IFN-γ, confirming their immune induction ability.

Claims

1. A pharmaceutical composition for treating or preventing adult T-cell leukemia, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 21 derived from tax.

2. The pharmaceutical composition of claim 1 , wherein the peptide binds to one or more types of major histocompatibility antigen (HLA) molecules.

3. The pharmaceutical composition of claim 2 , wherein the peptide binds to two or more types of HLA molecules.

4. The pharmaceutical composition according to claim 3 , wherein the peptide binds to three or more types of HLA molecules.

5. The pharmaceutical composition of claim 1 , wherein the peptide induces cytotoxic T cells.

6. The pharmaceutical composition of claim 1 in the form of a vaccine.

7. A peptide consisting of the amino acid sequence of SEQ ID NO:

21.

8. Use of the peptide according to claim 7 for the manufacture of a pharmaceutical composition for the treatment or prevention of ATL.

9. A method for producing antigen-presenting cells having cytotoxic T cell inducing activity, comprising a step of contacting the peptide according to claim 7 with antigen-presenting cells in vitro.

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