WO2014098012A1 - ヘルパーt細胞の活性化方法 - Google Patents
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- WO2014098012A1 WO2014098012A1 PCT/JP2013/083580 JP2013083580W WO2014098012A1 WO 2014098012 A1 WO2014098012 A1 WO 2014098012A1 JP 2013083580 W JP2013083580 W JP 2013083580W WO 2014098012 A1 WO2014098012 A1 WO 2014098012A1
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- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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- A61K39/001152—Transcription factors, e.g. SOX or c-MYC
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- A61K39/461—Cellular immunotherapy characterised by the cell type used
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- A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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Definitions
- the present invention relates to a method for activating helper T cells, comprising adding a WT1 peptide to an antigen-presenting cell and activating helper T cells, wherein the WT1 peptide is an HLA-DRB1 * 08: 02 molecule , HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule, and HLA-DQB1 * 04: 01 molecule
- Method capable of binding to any MHC class II molecule, composition therefor, method for activating cytotoxic T cells, cytotoxic T cell (CTL) activation inducer, and helper T The present invention relates to a pharmaceutical composition for treating and / or preventing cancer by activating cells and / or cytotoxic T cells.
- the WT1 gene (Wilms' tumor 1 gene) has been identified as a causative gene for Wilms tumor, a childhood renal cancer, and encodes a transcription factor having a zinc finger structure (Non-patent Documents 1 and 2, cited herein by reference). included). Subsequent studies have shown that it acts as an oncogene in hematopoietic tumors and solid cancers (Non-Patent Documents 3-6, incorporated herein by reference).
- CTLs cytotoxic T cells
- helper T cells are induced and activated by recognizing complexes of MHC class II molecules of antigen-presenting cells and antigenic peptides. Activated helper T cells assist B cell proliferation, differentiation, and maturation by producing cytokines such as IL-2, IL-4, IL-5, IL-6, or interferon.
- cytokines such as IL-2, IL-4, IL-5, IL-6, or interferon.
- helper T cells have a function of activating the immune system by promoting the proliferation and activation of B cells and T cells, so that helper T cells are mediated through MHC class II-binding antigen peptides in cancer immunotherapy. It is suggested that it is useful to enhance the function of cells and enhance the effect of cancer vaccine (Non-patent Document 9, incorporated herein by reference).
- WT1 peptide a specific peptide having a part of an amino acid sequence encoding a WT1 protein (hereinafter also referred to as a WT1 peptide in this specification) can bind to a plurality of MHC class II molecules and activate helper T cells. It was shown that helper peptides are present (US Pat. However, it has been extremely difficult to verify whether this WT1 peptide has an effect on other MHC class II molecules because there are many types of MHC class II molecules.
- the problem to be solved by the present invention is to use a specific WT1 peptide to apply a wide range of MHC class II molecule-positive subjects to activate helper T cells, to activate cytotoxic T cells, It is intended to provide a cytotoxic T cell activation inducer, a pharmaceutical composition for treating / preventing cancer, and the like.
- the present invention (1) A method for activating helper T cells comprising the step of activating helper T cells by adding WT1 peptide to antigen-presenting cells, wherein the WT1 peptide comprises HLA-DRB1 * 08: 02 molecules, Any selected from HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule, and HLA-DQB1 * 04: 01 molecule
- WT1 peptide is HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 A molecule, and a method according to (1), having the ability to bind to at least two MHC class II molecules of HLA
- the WT1 peptide is a peptide comprising the amino acid sequence: Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His (SEQ ID NO: 2), a variant or modification thereof, (1) to (4 ) (6)
- a composition comprising a WT1 peptide for activating a helper T cell by adding a WT1 peptide to an antigen-presenting cell, wherein the WT1 peptide comprises an HLA-DRB1 * 08: 02 molecule, an HLA- One selected from DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule, and HLA-DQB1 * 04: 01 molecule
- WT1 peptide is HLA-DRB1 * 08: 02 molecule, HLA-DRB
- the WT1 peptide is a peptide comprising the amino acid sequence: Lys Arg Tyr Phe Lys Leu His His Leu Gln Met His Ser Arg Lys His (SEQ ID NO: 2), a mutant or a modified form thereof (6) to (9 )
- the composition according to any one of (11) An antigen-presenting cell in which a complex of an antigen peptide containing a WT1 peptide and a MHC class II molecule is presented, wherein the MHC class II molecule is an HLA-DRB1 * 08: 02 molecule, an HLA-DRB1 * Any MHC class selected from 13:02 molecules, HLA-DRB1 * 14: 03 molecules, HLA-DRB1 * 14: 05 molecules, HLA-DQB1 * 03: 02 molecules, and HLA-DQB1 * 04: 01 molecules
- Antigen-presenting cells which are II molecules
- (12) A helper T cell that recognizes a complex of an antigen peptide
- HLA-DRB1 * 08 02 molecule
- HLA-DRB1 * 13 02 molecule
- HLA-DRB1 * 14 03 molecule
- HLA-DRB1 * 14 05 molecule
- HLA -DQB1 * 03 02 molecule
- composition therefor, method for activating cytotoxic T cells therefore Composition, a cytotoxic T cell activation inducer, and a pharmaceutical composition for treating and / or preventing cancer by activating helper T cells and cytotoxic T cells.
- helper T cells and cytotoxic T cells in subjects with class II molecules, as well as cancer treatment and prevention, etc. are possible.
- Data are mean ⁇ SD (Triplicate). $: Data including extrapolated values. It is a figure which shows the HLA restraint property of Clone (R) R143-1. The horizontal axis represents the amount of IFN- ⁇ produced (pg / mL) by adding WT1-332 (black column) or solvent (white column). The vertical axis shows the type of antigen-presenting B-LCL cells. Data are mean ⁇ SD (Triplicate). $: Data including extrapolated values. It is a figure which shows the HLA restraint property of Clone (R) R145-2. The horizontal axis represents the amount of IFN- ⁇ produced (pg / mL) by adding WT1-332 (black column) or solvent (white column).
- the vertical axis shows the type of antigen-presenting B-LCL cells. Data are mean ⁇ SD (Triplicate). $: Data including extrapolated values. It is a figure which shows the HLA restraint property of Clone
- the horizontal axis represents the amount of IFN- ⁇ produced (pg / mL) by adding WT1-332 (black column) or solvent (white column).
- the vertical axis shows the type of antigen-presenting B-LCL cells. Data are mean ⁇ SD (Triplicate). $: Data including extrapolated values. It is a figure which shows the HLA restraint property of Clone
- the horizontal axis represents the amount of IFN- ⁇ produced (pg / mL) by adding WT1-332 (black column) or solvent (white column).
- the vertical axis shows the type of antigen-presenting B-LCL cells. Data are mean ⁇ SD (Triplicate). $: Data including extrapolated values.
- the present invention provides a method for activating a helper T cell or cytotoxic T cell comprising the step of adding a WT1 peptide to an antigen-presenting cell and activating the helper T cell or cytotoxic T cell.
- a method is provided wherein the WT1 peptide has the ability to bind to MHC class II molecules.
- the step of activating cytotoxic T cells may be performed through a step of activating helper T cells.
- the WT1 peptide used in the present invention includes HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 It has the ability to bind to any MHC class II molecule selected from * 03: 02 molecules and HLA-DQB1 * 04: 01 molecules.
- the WT1 peptide used in the present invention may also have the ability to bind to at least two or more of the MHC class II molecules.
- the WT1 peptide used in the present invention may have the ability to bind to any MHC class II molecule among, for example, HLA-DR molecule, HLA-DQ and HLA-DP molecule.
- the WT1 peptide may be a peptide having a part of the amino acid sequence of the human WT1 protein represented by SEQ ID NO: 1.
- the amino acid sequence and length are not specifically limited, However, When a peptide is too long, it will become easy to receive the effect
- the length of the peptide of the present invention is preferably 10 to 25 amino acids, more preferably 15 to 21 amino acids, still more preferably 16 to 20 amino acids, for example, 16 amino acids, 17 amino acids, 18 amino acids, or 19 amino acids.
- a specific example of the peptide of the present invention includes the amino acid sequence: Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His (SEQ ID NO: 2).
- the WT1 peptide used in the present invention includes a variant of the above peptide.
- There are several variants for example, 1 to 9, preferably 1 to 5, 1 to 4, 1 to 3, and more preferably 1 to 2 in the amino acid sequence shown in SEQ ID NO: 2.
- one amino acid may comprise a peptide selected from the group consisting of peptides having an amino acid sequence substituted, deleted or added.
- Amino acid substitution in the peptide may be performed with any kind of amino acid at any position, and conservative amino acid substitution is preferred. Examples of conservative amino acid substitutions include Glu residues as Asp residues, Phe residues as Tyr residues, Leu residues as Ile residues, Ala residues as Ser residues, and His residues.
- Substitution with an Arg residue can be mentioned.
- the addition or deletion of amino acids is preferably performed at the N-terminal and C-terminal in the peptide, but may be performed within the sequence.
- a preferred specific example of the peptide of the present invention is that having SEQ ID NO: 2, but any of the above peptides can be HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA- Ability to bind to any MHC class II molecule selected from DRB1 * 14: 03, HLA-DRB1 * 14: 05, HLA-DQB1 * 03: 02, and HLA-DQB1 * 04: 01 It is necessary to activate helper T cells or cytotoxic T cells (also referred to herein as CTL).
- helper T cells or cytotoxic T cells also referred to herein as CTL.
- CTL helper T cells
- the peptide of the present invention may also be a modified form of the above amino acid sequence.
- An amino acid residue in the amino acid sequence can be modified by a known method.
- Such a modified product may be, for example, a functional group in the side chain of an amino acid residue subjected to esterification, alkylation, halogenation, phosphorylation, or the like.
- various substances can be bound to the N-terminus and / or C-terminus of the peptide containing the amino acid sequence.
- amino acids, peptides, analogs thereof and the like may be bound.
- a histidine tag may be added, and it may be a fusion protein together with a protein such as thioredoxin.
- a detectable label may be bound to the WT1 peptide.
- these substances are bound to the peptide of the present invention, these substances are treated with, for example, an in vivo enzyme or a process such as intracellular processing, and finally a peptide having the above amino acid sequence is generated. It is possible to obtain a helper T cell and / or cytotoxic T cell inducing effect by being presented on the cell surface as a complex with an MHC class II molecule.
- These substances may regulate the solubility of the peptide of the present invention, may improve the stability such as protease resistance, and are specific for a given tissue / organ, for example.
- the peptide of the present invention may be delivered, or it may have an action of enhancing the uptake efficiency of antigen-presenting cells.
- These substances may also be those that increase the ability to induce CTL, for example, helper peptides other than the peptides of the present invention.
- the WT1 peptide used in the present invention can be synthesized using a method usually used in the art or a modification thereof. Such synthetic methods include, for example, Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol 2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Maruzen Co., Ltd., 1985; Drug Development, Vol. 14, Peptide Synthesis, Hirokawa Shoten, 1991, etc. (these documents are incorporated herein by reference).
- the peptide used in the present invention can also be produced using genetic engineering techniques based on the nucleotide sequence information encoding the peptide. Such genetic engineering techniques are well known to those skilled in the art.
- the present invention also relates to a polynucleotide sequence encoding the WT1 peptide described above.
- the polynucleotide sequence encoding the WT1 peptide may be a DNA sequence or an RNA sequence. In the present invention, these polynucleotide sequences may be used instead of using the WT1 peptide. These polynucleotide sequences may be used by being incorporated into an appropriate vector. Examples of the vector include plasmids, phage vectors, virus vectors, etc. A, ⁇ ZAPII, ⁇ gt11, and the like.
- the vector may appropriately have factors such as a promoter capable of inducing expression, a gene encoding a signal sequence, a marker gene for selection, and a terminator. Methods for introducing these genes into cells and living organisms, expression methods, etc. are known to those skilled in the art.
- the antigen-presenting cell used in the present invention is a cell that can present an antigen peptide containing the WT1 peptide together with an MHC class II molecule to a helper T cell, such as dendritic cells, peripheral blood mononuclear cells, etc. means. Therefore, the subject from which the antigen-presenting cell used in the present invention is derived is the same molecule (for example, HLA-DRB1 * 08: 02 molecule, HLA-DRB1) as the MHC class II molecule to which the added WT1 peptide can bind.
- a helper T cell such as dendritic cells, peripheral blood mononuclear cells, etc. means. Therefore, the subject from which the antigen-presenting cell used in the present invention is derived is the same molecule (for example, HLA-DRB1 * 08: 02 molecule, HLA-DRB1) as the MHC class II molecule to which the added WT1 peptide can bind.
- HLA-DRB1 * 13 02 molecule
- HLA-DRB1 * 14 03 molecule
- HLA-DRB1 * 14 05 molecule
- HLA-DQB1 * 03 02 molecule
- HLA-DQB1 * 04 01 molecule It must have more MHC class II molecules).
- the addition of the WT1 peptide to the antigen-presenting cell can be performed directly by adding the WT1 peptide, or by adding a polynucleotide encoding the WT1 peptide or a polynucleotide encoding the WT1 peptide, Alternatively, it may be performed indirectly by adding cells containing the expression vector.
- the addition of the WT1 peptide to the antigen-presenting cell is performed by bringing the WT1 peptide into contact with the antigen-presenting cell, or by adding a polynucleotide encoding the WT1 peptide or an expression vector containing the polynucleotide to the antigen-presenting cell. This can be done by introducing.
- the polynucleotide encoding the WT1 peptide, the expression vector containing the polynucleotide encoding the WT1 peptide, and the cell containing the expression vector can be obtained by techniques well known to those skilled in the art. Specifically, the polynucleotide used in the present invention can be determined based on the amino acid sequence of the WT1 peptide (for example, the amino acid sequence shown in SEQ ID NO: 2). The polynucleotide can be produced by, for example, a DNA or RNA synthesis method, a PCR method, or the like.
- the type of expression vector containing the above-mentioned polynucleotide, the sequence contained in addition to the above-mentioned polynucleotide sequence, etc. can be appropriately selected according to the type, purpose, etc. of the host into which the expression vector is introduced, plasmid, phage vector, virus vector Etc.
- a cell containing the expression vector can be produced, for example, by transforming a host cell. Examples of host cells include E. coli, yeast, insect cells, animal cells and the like.
- a method for introduction into a host cell a conventional method such as a calcium phosphate method, a DEAE-dextran method, an electroporation method, or a lipid for gene introduction can be used.
- TCR / CD3 complex on T cell surface recognizes antigen peptide via MHC class II molecule on antigen presenting cell surface, and T cell surface integrin is integrin ligand on antigen presenting cell surface. It is activated by being stimulated with.
- the activation of helper T cells in this specification includes not only the activation of helper T cells but also the induction and proliferation of helper T cells.
- the helper T cell activated in the present invention may be an undifferentiated T cell (for example, naive T cell).
- Activated helper T cells have a function of activating the immune system by promoting induction, proliferation, and activation of B cells and cytotoxic T cells.
- helper T cells activated in vitro using the method of the present invention can be used for the treatment or prevention of cancer or the like, or as an adjuvant for these.
- the activation of helper T cells can be evaluated by measuring production and secretion amount of cytokines such as interferon (for example, interferon ⁇ ) and interleukin.
- the present invention provides a composition for activating helper T cells or cytotoxic T cells by adding WT1 peptide to antigen-presenting cells.
- the activation of cytotoxic T cells may be performed through the activation of helper T cells.
- the active ingredient contained in the composition of the present invention include a WT1 peptide, a polynucleotide encoding the WT1 peptide, a vector containing the polynucleotide, and a cell containing the vector. Any molecule may be used as long as it can be presented on the surface of the presenting cell. These factors can be obtained by methods well known to those skilled in the art as described above.
- the WT1 peptide used in the present invention contains HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA -It has the ability to bind to either DQB1 * 03: 02 molecule or HLA-DQB1 * 04: 01 molecule.
- the WT1 peptide used in the present invention may have the ability to bind to at least two or more of the MHC class II molecules.
- the WT1 peptide used in the present invention may have the ability to bind to any MHC class II molecule among HLA-DR molecules, HLA-DQ, and HLA-DP molecules.
- composition of the present invention is HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule And, when administered to a subject having any one or more MHC class II molecules of HLA-DQB1 * 04: 01 molecules, helper T cells and / or cytotoxic T cells in the subject are active The immune system is activated.
- the WT1 gene is also used for various cancers and tumors such as leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma and other hematopoietic tumors, stomach cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin Since it is highly expressed in solid cancers such as cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer, the composition of the present invention can also be used as an adjuvant for the treatment or prevention of cancer. Alternatively, helper T cells, cytotoxic T cells and the like activated using the composition of the present invention can be used, for example, as an adjuvant for the treatment of the cancer.
- the composition of the present invention contains, for example, a carrier, an excipient, or an additive in addition to the WT1 peptide, the polynucleotide encoding the WT1 peptide, a vector containing the polynucleotide, or a cell containing the vector. May be.
- the WT1 peptide or the like contained in the composition of the present invention comprises a known MHC class I-restricted WT1 peptide because it activates helper T cells and / or cytotoxic T cells specifically for the WT1 peptide. Or you may apply with these.
- the application method of the composition of the present invention can be appropriately selected depending on conditions such as the desired degree of activation of helper T cells and / or cytotoxic T cells, the state of antigen-presenting cells, and the like.
- Examples of the application method include intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, nasal administration, oral administration, and the like, or addition to an antigen-presenting cell culture medium.
- the amount of the WT1 peptide, etc. contained in the composition of the present invention, the form of the composition, the number of applications, etc. are the degree of activation of the desired helper T cells and / or cytotoxic T cells, the state of antigen-presenting cells, etc. It can select suitably according to conditions.
- the present invention provides a method for treating or preventing cancer in a subject, comprising the step of activating helper T cells or cytotoxic T cells by adding a WT1 peptide to antigen-presenting cells,
- the WT1 peptide is HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule, and
- a method is provided that has the ability to bind to any MHC class II molecule selected from HLA-DQB1 * 04: 01 molecules.
- the method of the present invention is a method for activating helper T cells and / or cytotoxic T cells to activate a subject's immune system to treat or prevent cancer in the subject.
- the step of activating cytotoxic T cells may be performed through a step of activating helper T cells.
- the addition of the WT1 peptide to the antigen-presenting cell can be performed directly by adding the WT1 peptide, by adding a polynucleotide encoding the WT1 peptide or a polynucleotide encoding the WT1 peptide, or the expression vector May be carried out indirectly by the addition of cells containing.
- the polynucleotide encoding the WT1 peptide, the expression vector containing the polynucleotide encoding the WT1 peptide, and the cell containing the expression vector can be obtained by methods well known to those skilled in the art as described above.
- the target to which the method of the present invention can be applied is HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * Any MHC class II molecule positive target selected from 03:02 molecules and HLA-DQB1 * 04: 01 molecules.
- the cancer to which the present invention can be applied may be any, for example, hematopoietic tumors such as leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, stomach cancer, colon cancer, lung cancer, breast cancer, germ cell Examples include solid cancers such as cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, and ovarian cancer.
- the method of the present invention may be used in combination with a method for treating or preventing cancer using a MHC class I molecule-restricted WT1 peptide or a pharmaceutical composition therefor.
- the present invention provides the use of a WT1 peptide, a polynucleotide encoding the WT1 peptide, a vector containing the polynucleotide, or a cell containing the vector for producing the composition. Furthermore, the present invention provides a WT1 peptide, a polynucleotide encoding the WT1 peptide, a vector containing the polynucleotide, or a cell containing the vector, which is used for activating helper T cells or cytotoxic T cells. .
- the present invention provides the WT1 peptide, a polynucleotide encoding the WT1 peptide for activating helper T cells and / or cytotoxic T cells by adding a WT1 peptide to an antigen-presenting cell,
- a vector comprising a polynucleotide, a kit comprising a cell comprising the vector, wherein the WT1 peptide comprises HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA -DRB1 * 14: 05 molecules, HLA-DQB1 * 03: 02 molecules, and HLA-DQB1 * 04: 01 molecules that are capable of binding to any MHC class II molecule.
- the kit is used in the method for activating the helper T cell or cytotoxic T cell.
- the kit of the present invention may contain, for example, a means for obtaining antigen-presenting cells, a means for evaluating the activity of helper T cells and / or cytotoxic T cells, and the like.
- an instruction manual is attached to the kit. Helper T cells or cytotoxic T cells can be efficiently activated using the kit of the present invention.
- the present invention provides an antigen-presenting cell in which a complex of an antigen peptide containing a WT1 peptide and an MHC class II molecule is presented.
- the MHC class II molecule is HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * Any of 03:02 molecules and HLA-DQB1 * 04: 01 molecules may be used, and at least two or more of the above MHC class II molecules may be used.
- the antigen-presenting cells of the present invention may be prepared using techniques known among those skilled in the art.
- a cell having antigen-presenting ability is isolated from a cancer patient, and the isolated cell is pulsed with the WT1 peptide (for example, a peptide having the amino acid sequence shown in SEQ ID NO: 2) or a polynucleotide encoding the WT1 peptide, Alternatively, it may be prepared by introducing an expression vector containing the polynucleotide into a cell and presenting a complex of an antigen peptide containing a WT1 peptide and an MHC class II molecule on the cell surface (Cancer Immunol.Immunother.
- a cell having an antigen-presenting ability is not limited as long as it expresses an MHC class II molecule capable of presenting a WT1 peptide on the cell surface. Spheres or dendritic cells are preferred.
- the presence of the antigen-presenting cell of the present invention is confirmed by an increase in cytotoxic T cell activity that is confirmed by an increase in the amount of interferon ⁇ .
- the antigen-presenting cell of the present invention is effectively used in cell therapy (for example, dendritic cell therapy) as an active ingredient of a pharmaceutical composition.
- the present invention provides a helper T cell that recognizes a complex of an antigen peptide containing a WT1 peptide and an MHC class II molecule.
- the MHC class II molecule is HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * Any of 03:02 molecules and HLA-DQB1 * 04: 01 molecules may be used, and at least two or more of the above MHC class II molecules may be used.
- helper T cell of the present invention for example, an antigen peptide containing a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03
- Helper T cells that recognize a complex with any MHC class II molecule selected from the molecule, HLA-DRB1 * 14: 05, HLA-DQB1 * 03: 02, and HLA-DQB1 * 04: 01 Can be mentioned.
- the helper T cell of the present invention can be easily prepared and obtained by those skilled in the art using a method known in the art (Iwata, M. et al., Eur. J. Immunol, 26, 2081 (1996). )) (This document is incorporated herein by reference).
- the present invention provides a cytotoxic T cell activated by a helper T cell that recognizes a complex of an antigen peptide containing a WT1 peptide and an MHC class II molecule.
- the cytotoxic T cell of the present invention include an antigen peptide containing a peptide consisting of the amino acid sequence shown in SEQ ID NO: 2, HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14 : 3 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule, and a helper that recognizes a complex with any MHC class II molecule selected from HLA-DQB1 * 04: 01 molecule Mention may be made of cytotoxic T cells activated by T cells.
- the cytotoxic T cell of the present invention can be easily prepared by a person skilled in the art by a known method.
- a patient's peripheral blood lymphocytes are isolated and stimulated in vitro with a peptide (eg, a peptide having the amino acid sequence set forth in SEQ ID NO: 2), a polynucleotide encoding the peptide, or an expression vector containing the peptide.
- a peptide eg, a peptide having the amino acid sequence set forth in SEQ ID NO: 2
- a polynucleotide encoding the peptide or an expression vector containing the peptide.
- the cytotoxic T cells prepared as described above can be used as an active ingredient of a pharmaceutical composition for treating or preventing cancer or the like.
- the present invention provides an HLA tetramer having an antigen peptide containing the WT1 peptide and an MHC class II molecule.
- the MHC class II molecules are HLA-DRB1 * 08: 02 molecules, HLA-DRB1 * 13: 02 molecules, HLA-DRB1 * 14: 03 molecules, HLA-DRB1 * 14: 05 molecules, HLA-DQB1 * 03: 02 Or any of the HLA-DQB1 * 04: 01 molecules, or at least two or more of the MHC class II molecules.
- the HLA tetramer means a tetramer obtained by biotinylating a complex (HLA monomer) in which an HLA protein is associated with a peptide and binding it to avidin.
- HLA monomer a complex in which an HLA protein is associated with a peptide and binding it to avidin.
- HLA tetramers those containing various antigen peptides are commercially available, and the HLA tetramers of the present invention can be easily prepared (Science 279: 2103-2106 (1998), Science 274: 94-96 (1996). )) (This document is incorporated herein by reference).
- the tetramer of the present invention is preferably fluorescently labeled so that the helper T cells and cytotoxic T cells of the present invention bound by known detection means such as flow cytometry and fluorescence microscope can be easily selected or detected.
- the HLA tetramer in the present invention is not limited to a tetramer, and a multimer such as a pentamer or a dendrimer can be used as necessary.
- a multimer refers to a multimer obtained by combining two or more complexes (HLA monomers) each having an HLA protein associated with a peptide using a known technique.
- the present invention activates a helper T cell or cytotoxic T cell comprising any of the above composition, antigen-presenting cell, helper T cell, cytotoxic T cell or tetramer as an active ingredient
- a pharmaceutical composition is provided.
- the pharmaceutical composition of the present invention may contain any one or more of the above compositions, antigen-presenting cells, helper T cells, cytotoxic T cells or tetramers as an active ingredient.
- the pharmaceutical composition of the present invention can be used for treating or preventing cancer.
- the pharmaceutical composition of the present invention comprises various cancers and tumors expressing WT1, for example, hematopoietic tumors such as leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, stomach cancer, colon cancer, lung cancer, breast cancer, germ cells It can be applied to solid cancers such as cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer.
- WT1 hematopoietic tumors
- hematopoietic tumors such as leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, stomach cancer, colon cancer, lung cancer, breast cancer, germ cells
- solid cancers such as cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer.
- the pharmaceutical composition of the present invention comprises HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * It can be used to administer to subjects with any MHC class II molecule selected from 03:02 molecules and HLA-DQB1 * 04: 01 molecules.
- the pharmaceutical composition of the present invention may be used in combination with other methods for treating or preventing cancer or pharmaceutical compositions therefor.
- the pharmaceutical composition of the present invention may contain a helper T cell or cytotoxic T cell activator, proliferator, inducer, or the like, or contains a known MHC class I-restricted WT1 peptide. May be.
- the pharmaceutical composition of the present invention may contain, for example, a carrier, an excipient and the like in addition to the active ingredient.
- the administration method of the pharmaceutical composition of the present invention can be appropriately selected according to conditions such as the type of disease, the state of the subject, and the target site. Examples of the method include, but are not limited to, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration, nasal administration, and oral administration.
- the amount of the active ingredient contained in the pharmaceutical composition of the present invention, the dosage form of the pharmaceutical composition, the number of administrations, and the like can be appropriately selected according to conditions such as the type of disease, the condition of the subject, and the target site.
- the present invention treats cancer, comprising the step of administering to a subject an effective amount of any of the above compositions, antigen-presenting cells, helper T cells, cytotoxic T cells or tetramers.
- a method for prevention wherein the subject is HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA A method is provided that has any MHC class II molecule selected from -DQB1 * 03: 02 molecules and HLA-DQB1 * 04: 01 molecules.
- Cancers that can be treated or prevented by the method of the present invention include various cancers and tumors that express WT1, for example, hematopoietic tumors such as leukemia, myelodysplastic syndrome, multiple myeloma, malignant lymphoma, stomach cancer, colon cancer, lung cancer, Solid cancers such as breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer and ovarian cancer.
- the method of the present invention may be used in combination with other methods for treating or preventing cancer, for example, methods for treating or preventing cancer using a known MHC class I molecule-restricted WT1 peptide.
- the present invention provides the use of any of the above compositions, antigen presenting cells, helper T cells, cytotoxic T cells or tetramers for producing the above pharmaceutical compositions. Furthermore, the present invention provides the composition, antigen-presenting cell, helper T cell, cytotoxic T cell or tetramer, which is used for activating helper T cells or cytotoxic T cells. Furthermore, the present invention provides the composition, antigen-presenting cell, helper T cell, cytotoxic T cell or tetramer used for the treatment or prevention of cancer.
- the present invention relates to an antibody that specifically binds to the WT1 peptide or a polynucleotide encoding the WT1 peptide (hereinafter also referred to as anti-WT antibody).
- the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody. Specific examples include an antibody that specifically binds to a peptide having the amino acid sequence shown in SEQ ID NO: 2. Methods for producing these antibodies are already well known, and the antibodies of the present invention can also be produced according to these conventional methods (Current protocols Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley and Sons.
- a peptide having the amino acid sequence shown in SEQ ID NO: 2 can be used as an immunogen to immunize non-human animals such as rabbits, and obtained from the sera of these animals by conventional methods.
- the peptide used in the present invention (a peptide having the amino acid sequence shown in SEQ ID NO: 2) is immunized to a non-human animal such as a mouse, and the obtained spleen cells and myeloma cells are treated with cells.
- the anti-WT antibody of the present invention can also be produced by enhancing the immunological reaction using various adjuvants depending on the host. Examples of such an adjuvant include mineral gels (for example, Freund's adjuvant, aluminum hydroxide, etc.), surface active substances, human adjuvants and the like.
- the anti-WT antibody of the present invention can be used for affinity chromatography, immunological diagnosis and the like.
- the immunological diagnosis can be appropriately selected from immunoblotting, radioimmunoassay (RIA), enzyme immunoassay (ELISA), fluorescence or luminescence assay.
- the present invention relates to HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03
- a method for determining the presence or amount of a WT1 peptide in any MHC class II molecule positive subject selected from: 02 molecules and HLA-DQB1 * 04: 01 molecules comprising: (A) reacting a sample obtained from the subject with the anti-WT antibody; (B) examining the presence or amount of the anti-WT antibody contained in the sample; A method comprising the steps is provided.
- Samples used in the step (a) include HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecules and those obtained from subjects with any MHC class II molecule selected from HLA-DQB1 * 04: 01 molecules can be used.
- Examples of the sample used in the step (a) include body fluids such as blood and lymphocytes, and tissues. Obtaining a sample, reaction with an antibody, and the like can be appropriately performed by those skilled in the art using known techniques. Since the step (b) in the present invention includes, for example, determining the localization, site, amount, etc.
- the anti-WT antibody can be used for cancer diagnosis, prognosis diagnosis and the like.
- the anti-WT antibody may be labeled.
- a known label such as a fluorescent label or a radioactive label can be used. By labeling, the presence or amount of the WT1 peptide can be determined easily and rapidly.
- the present invention relates to a kit for determining the presence or amount of a WT1 peptide comprising the anti-WT antibody as an essential component.
- the kit of the present invention may contain, for example, anti-WT antibody acquisition means, evaluation means, and the like.
- an instruction manual is attached to the kit.
- the present invention in yet another embodiment, comprises HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * Determine the presence or amount of WT1-specific helper T cells or WT1-specific cytotoxic T cells in any MHC class II molecule positive subject selected from 03:02 molecules and HLA-DQB1 * 04: 01 molecules
- a method (A) stimulating a sample obtained from said subject with WT1 peptide; (B) examining the presence or amount of cytokines, helper T cells or cytotoxic T cells, Providing a method wherein the increase in the presence or amount of cytokines, helper T cells or cytotoxic T cells is indicative of the presence or amount of WT1-specific helper T cells or WT1-specific cytotoxic T cells .
- the sample of the present invention may be any sample as long as it contains antigen-presenting cells.
- the sample used in the present invention may be derived from a healthy person or a cancer patient. By using these cells derived from a healthy person, for example, it is possible to diagnose whether or not the patient is suffering from or has a predisposition to cancer. By using these cells derived from cancer patients, for example, it is possible to predict whether WT1 immunotherapy has an effect in cancer patients.
- the obtained sample may be cultured before and after stimulation with the WT1 peptide, and the culture conditions can be appropriately determined by those skilled in the art. Stimulation of these cells using the WT1 peptide can be performed using a known technique such as electroporation, and may be performed either in vitro or in vivo. Cytokine production, helper T cells, and cytotoxic T cell responses exist, or cytokine production, helper T cells, or cytotoxic T cell responses can be examined by known methods.
- the present invention relates to a kit for determining the presence or amount of a WT1 peptide comprising the WT1 peptide as an essential component.
- the kit of the present invention may contain, for example, a sample obtaining means, an evaluation means such as a cytokine, and the like.
- an instruction manual is attached to the kit.
- the present invention also provides the following: A composition comprising a WT1 peptide for activating a helper T cell by adding a WT1 peptide to an antigen-presenting cell, the helper T cell comprising an HLA-DRB1 * 08: 02 molecule, an HLA-DRB1 * Any MHC class selected from 13:02 molecules, HLA-DRB1 * 14: 03 molecules, HLA-DRB1 * 14: 05 molecules, HLA-DQB1 * 03: 02 molecules, and HLA-DQB1 * 04: 01 molecules
- a pharmaceutical composition for treating or preventing cancer comprising as an active ingredient any one of a WT1 peptide, a polynucleotide encoding the WT1 peptide, an expression vector containing the polynucleotide, or a cell containing the expression
- the present invention also provides the following: (1) A composition comprising a WT1 peptide, a polynucleotide encoding the WT1 peptide, an expression vector containing the polynucleotide, or a cell containing the expression vector for activating helper T cells, Cells are HLA-DRB1 * 08: 02 molecules, HLA-DRB1 * 13: 02 molecules, HLA-DRB1 * 14: 03 molecules, HLA-DRB1 * 14: 05 molecules, HLA-DQB1 * 03: 02 molecules, and HLA A composition that recognizes a complex of any MHC class II molecule selected from DQB1 * 04: 01 molecules and the WT1 peptide.
- a WT1 peptide, a polynucleotide encoding the WT1 peptide, an expression vector containing the polynucleotide, or a composition containing a cell containing the expression vector for activating cytotoxic T cells, -DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule, and HLA-DQB1 * 04 A composition administered to a subject having any MHC class II molecule selected from: 01 molecules.
- a composition comprising a WT1 peptide, a polynucleotide encoding the WT1 peptide, an expression vector containing the polynucleotide, or a cell containing the expression vector for treating or preventing cancer, comprising HLA-DRB1 * 08:02, HLA-DRB1 * 13: 02, HLA-DRB1 * 14: 03, HLA-DRB1 * 14: 05, HLA-DQB1 * 03: 02, and HLA-DQB1 * 04: 01
- the composition according to any one of (1) to (3), comprising a WT1 peptide.
- the WT1 peptide is a peptide comprising the amino acid sequence: Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His (SEQ ID NO: 2), a mutant or a modified form thereof.
- An antigen-presenting cell in which a complex of an antigen peptide containing a WT1 peptide and an MHC class II molecule is presented, wherein the MHC class II molecule is an HLA-DRB1 * 08: 02 molecule, an HLA-DRB1 * Any MHC class selected from 13:02 molecules, HLA-DRB1 * 14: 03 molecules, HLA-DRB1 * 14: 05 molecules, HLA-DQB1 * 03: 02 molecules, and HLA-DQB1 * 04: 01 molecules An antigen-presenting cell that is an II molecule.
- the WT1 peptide is a peptide comprising the amino acid sequence: Lys Arg Tyr Phe Lys Leu His His Leu Gln Met His Ser Arg Lys His (SEQ ID NO: 2), a mutant or a modification thereof, Antigen presenting cell. (9) The antigen-presenting cell according to (8) above, wherein the WT1 peptide is a peptide comprising the amino acid sequence: Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His (SEQ ID NO: 2).
- a helper T cell that recognizes a complex of an antigen peptide containing a WT1 peptide and an MHC class II molecule, wherein the MHC class II molecule is an HLA-DRB1 * 08: 02 molecule, an HLA-DRB1 * 13: Any MHC class II molecule selected from 02 molecules, HLA-DRB1 * 14: 03 molecules, HLA-DRB1 * 14: 05 molecules, HLA-DQB1 * 03: 02 molecules, and HLA-DQB1 * 04: 01 molecules Helper T cells.
- the antigen-presenting cell according to any one of (7) to (9), the helper T cell according to any of (10) to (12), or the cytotoxic T cell according to (13) A pharmaceutical composition for treating or preventing cancer, comprising any of cells as an active ingredient.
- the present invention also provides the following: (1) A method for activating helper T cells, comprising a step of activating helper T cells by adding WT1 peptide to antigen-presenting cells, wherein the helper T cells are HLA-DRB1 * 08: 02 molecules , HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule, and HLA-DQB1 * 04: 01 molecule
- a method for recognizing a complex of any MHC class II molecule and the WT1 peptide comprising a step of activating helper T cells by adding WT1 peptide to antigen-presenting cells, wherein the helper T cells are HLA-DRB1 * 08: 02 molecules , HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQ
- Cytotoxic T cell activation method comprising HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 A WT1 peptide, a polynucleotide encoding the WT1 peptide, a polynucleotide encoding the WT1 peptide, a subject having any MHC class II molecule selected from a molecule, HLA-DQB1 * 03: 02 molecule, and HLA-DQB1 * 04: 01 molecule, the polynucleotide Administering an expression vector comprising: or a cell comprising said expression vector.
- HLA-DRB1 * 08 02 molecule
- HLA-DRB1 * 13 02 molecule
- HLA-DRB1 * 14 03 molecule
- HLA-DRB1 * 14 05
- a WT1 peptide a polynucleotide encoding the WT1 peptide
- a polynucleotide encoding the WT1 peptide a subject having any MHC class II molecule selected from a molecule, HLA-DQB1 * 03: 02 molecule, and HLA-DQB1 * 04: 01 molecule
- the polynucleotide Administering an expression vector comprising: or a cell comprising said expression vector.
- the WT1 peptide is a peptide comprising the amino acid sequence: Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His (SEQ ID NO: 2), a mutant or a modified form thereof.
- the present invention also provides the following: (1) Use of a WT1 peptide, a polynucleotide encoding a WT1 peptide, an expression vector containing the polynucleotide, or a cell containing the expression vector for the manufacture of a medicament for activating helper T cells,
- the helper T cells are HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule , And use that recognizes a complex of any one of the MHC class II molecules selected from HLA-DQB1 * 04: 01 molecules and the WT1 peptide.
- a WT1 peptide, a polynucleotide encoding the WT1 peptide, an expression vector containing the polynucleotide, or a cell containing the expression vector for the manufacture of a medicament for activating cytotoxic T cells
- the drug is HLA-DRB1 * 08: 02 molecule, HLA-DRB1 * 13: 02 molecule, HLA-DRB1 * 14: 03 molecule, HLA-DRB1 * 14: 05 molecule, HLA-DQB1 * 03: 02 molecule , And use to be administered to a subject having any MHC class II molecule selected from HLA-DQB1 * 04: 01 molecules.
- HLA-DRB1 * 08 02 molecule
- HLA-DRB1 * 13 02 molecule
- HLA-DRB1 * 14 03 molecule
- HLA-DRB1 * 14 05 molecule
- HLA-DQB1 * 03 02 molecule
- HLA -Use HLA-Use that is to be administered to a subject having any MHC class II molecule selected from DQB1 * 04: 01 molecules.
- Example 1 Establishment of WT1 peptide (SEQ ID NO: 2) specific Th1 clone cells
- WT1-332 specific Th1 clone cells
- Th1 clone cells specific Th1 clone cells
- Test materials The main test materials are as follows. Test substance Storage conditions: -30 °C freezer * WT1-332: Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His (SEQ ID NO: 2)
- test substance Dissolved to 20 mg / mL with 10 mM acetic acid, sterilized by filtration, and stored frozen ( ⁇ 30 ° C. setting).
- PBMC Peripheral mononuclear cells for feeder cells
- Cell name PBMC 2
- Origin Peripheral blood of healthy adult blood donors Anonymized to be connectable under the control of a personal information manager, and written informed consent was taken, and blood was collected and prepared in this study.
- Specimen condition One of the feeders is different from the cloning sample.
- Collection conditions Heparin sodium blood collection 40 mL
- B-LCL cell line for feeder 5 Cell line name: B-LCL cell line 6) Origin: Human peripheral blood 7) Source: RIKEN Cell Bank or IHWG Cell Bank
- PBMC peripheral blood of healthy volunteers by specific gravity centrifugation, and a part thereof was used for induction of WT1-332-specific T cells. The remaining cells were stored frozen in a cell banker (Juji Field Co., Ltd.) and used as antigen-presenting cells or feeder cells during restimulation.
- PBMCs are seeded in a 24-well plate at 1.5 x 10 6 cells / well x 10 wells, and a final concentration of 20 ⁇ g / mL WT1-332 and a final concentration of 10 ng / mL IL -7 was added to start the culture (Day 0, total medium volume: 2 mL / well). Restimulation was performed one week later. For re-stimulation, first, WT1-332 with a final concentration of 20 ⁇ g / mL was added to PBMC for antigen-presenting cells adjusted to a cell concentration of 3.0 ⁇ 10 6 cells / mL or less, and cultured for 2 hours.
- MMC mitomycin C
- adherent cells were removed, seeded at 1.15-1.43 ⁇ 10 6 cells / well, together with the same number of WT1 / MMC-treated antigen-presenting cells, and a final concentration of 10 ng / mL Of IL-7 was added to resume the culture (Day 7).
- Two days later, half of the culture medium was exchanged with a medium containing 40 U / mL IL-2, and culture was continued for another week while another half of the culture medium was exchanged with a medium containing 20 U / mL IL-2.
- Intracellular cytokine staining The cultured cells were collected on Day 14, seeded in 2 wells of 2.0 ⁇ 10 5 in 96-well round-bottom plates, 20 ⁇ g / mL WT1-332 was added to one, and solvent was added to the other. Further, Brefeldin A at a final concentration of 1 ⁇ was added and cultured for 2 hours. Collect cells, add PE-labeled anti-human CD4 antibody and FITC-labeled anti-human CD8 antibody, react at 4 ° C for 15 minutes, wash with Staining Buffer, add Cytofix Cytoperm Fix / Perm, and at 4 ° C for 20 minutes Processed. Washed with Perm./Wash Buffer, added PerCP-labeled anti-human IFN- ⁇ antibody, reacted at 4 ° C. for 30 minutes, washed with Perm./Wash Buffer, and analyzed by FACS.
- ICS Intracellular cytokine staining
- CD4 + cells were isolated from cultured cells using MACS Microbeads.
- the required number of sets was prepared according to the number of samples to be cloned, and PHA was added at a final concentration of 200 ng / mL to obtain a PHA-containing feeder cell mixture.
- PHA was added at a final concentration of 200 ng / mL to obtain a PHA-containing feeder cell mixture.
- select a specimen with a high CD4 + IFN- ⁇ + cell ratio from the ICS results and use the prepared PHA-containing feeder cell mixture to obtain a CD4 + cell concentration of 10 cells / mL.
- amplification stimulation by PHA stimulation was applied in the same manner as above, but at the time of seeding, the culture dish was placed in a 24-well plate, the total amount of PBMC was 1.0 ⁇ 10 6 cells / well, and B-LCL The total cell volume was changed to 1.0 ⁇ 10 5 cells / well, the final PHA concentration was 50 ng / mL, and the number of Th1 clone cells to be amplified was changed to 2.0 ⁇ 10 5 cells / well or less.
- IL-2 was added 3 days after the start of the culture, and the IL-2 content of the medium to be added was 200 U / mL.
- Th1 clonal cells were seeded in 96-well round-bottom plates in 1 or 3 wells. To this was added 10 ⁇ M acetic acid or 20 ⁇ g / mL of each peptide, and culture was started (total culture volume: 200 ⁇ L / well). After about 24 hours, the culture supernatant was collected.
- IFN- ⁇ concentration in each culture supernatant was measured after 4-fold dilution of each culture supernatant with Assay Diluent (Becton Dickinson). IFN- ⁇ concentration was measured using the BD OptEIA ELISA set (human IFN- ⁇ , Becton Dickinson) according to the package insert. The antibody dilution concentration was 500 times and the calibration curve range was 18.75-1200 pg. In addition, the color reaction time was changed to 5 minutes, and when the measurement range was exceeded, an extrapolated value was treated, and when the absorbance was less than 0, it was treated as 0.
- Th1 clonal cells confirmed to have antigen reactivity were cryopreserved in a cell banker to obtain a master cell bank.
- the IFN- ⁇ concentration in the culture supernatant of each group was measured by ELISA.
- Th1 clone cells In the specific Th1 clone cells (hereinafter referred to as Th1 clone cells) established by adding WT1-332 in Example 1, WT1-332 is a specific HLA. We investigated whether T cells could be activated in a restricted manner, and confirmed the restricted alleles.
- Outline of the test B-LCL cells with appropriate HLA class II alleles subjected to WT1-332 pulse are used as antigen-presenting cells.
- the amount of IFN- ⁇ produced is measured.
- HLA restriction of Th1 clonal cells was evaluated. Specifically, Th1 clone cells were cultured under PHA stimulation using B-LCL cells for feeders and allogeneic PBMC prepared from peripheral blood as feeder cells.
- B-LCL cells treated with WT1-332 or solvent were used as antigen-presenting cells, co-cultured with the collected Th1 clone cells, and the amount of IFN- ⁇ produced was measured to stimulate WT1-332-specific antigen stimulation. The presence or absence was evaluated.
- HLA class II restriction of each Th1 clone cell was evaluated by using various B-LCL cells having different types as antigen-presenting cells.
- B-LCL cells for feeders Culture was started at 1 ⁇ 10 5 cells / mL from cryopreserved cells on Day 0, collected on Day 3, and used as feeder B-LCL cells upon PHA stimulation.
- PBMC Preparation of PBMC Prepared by peripheral gravity blood from healthy volunteers by Day 2 and after measuring the number of cells, cell pellets were collected, resuspended in a cell banker (Mitsubishi Chemical Rulece Co., Ltd.), and then placed in a cryotube. The aliquot was dispensed and stored frozen in a freezer set at ⁇ 80 ° C. At the time of PHA stimulation, cryopreserved PBMC was thawed as needed and used as feeder PBMC.
- Th1 clone cells were thawed and seeded On Day 2, start culture from cryopreserved cells in 20 U / mL IL-2 containing medium at 2 x 10 6 cells / mL or less, collect on Day 3, and recover Th1 during PHA stimulation Clone cells were used.
- Antigen-presenting B-LCL cells for restriction evaluation were thawed and seeded On day 11, the culture was started from cryopreserved cells at about 1 ⁇ 10 5 cells / mL, and the cells were collected on day 15 and used for the restriction evaluation test.
- Th1 clonal cells were collected, and seeded in 96-well round bottom plates at 1.0-2.5 ⁇ 10 4 cells / 100 ⁇ L / well, 3 wells according to the number of collected cells.
- Distribute 4-7 mL of antigen-presenting B-LCL cells adjusted to a cell concentration of 1 ⁇ 10 6 cells / mL into two conical tubes, with acetic acid at a final concentration of 10 ⁇ M on one side and a final concentration of 20 ⁇ g on the other side.
- IFN- ⁇ concentration in each culture supernatant was measured after each culture supernatant was diluted 100-fold with Assay Diluent (Becton Dickinson). IFN- ⁇ concentration was measured using the BD OptEIA ELISA set (human IFN- ⁇ , Becton Dickinson) according to the package insert.
- the calibration curve range was 5-640 pg / mL and the color reaction time was The time was changed to 10 minutes, and when the measurement range was exceeded, it was treated as an extrapolated value, and when the absorbance was less than 0, it was treated as 0.
- HLA-DRB1 * 08 02 molecule
- HLA-DRB1 * 13 02 molecule
- HLA-DRB1 * 14 03 molecule
- HLA-DRB1 * 14 05 molecule
- HLA-DQB1 * 03 02 molecule
- a composition and a pharmaceutical composition for treating and / or preventing cancer by activating helper T cells and / or cytotoxic T cells are provided, the field of pharmaceuticals such as WT1 gene is increased.
- the present invention can be used in the development and manufacturing fields of various hematopoietic tumors and solid cancers that are expressed.
- SEQ ID NO: 2 Synthetic peptide
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Abstract
Description
(1)WT1ペプチドを抗原提示細胞に添加して、ヘルパーT細胞を活性化させる工程を含む、ヘルパーT細胞の活性化方法であって、該WT1ペプチドが、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子に結合する能力を有するものである方法、
(2)WT1ペプチドが、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子のうちの少なくとも2つのMHCクラスII分子に結合する能力を有するものである、(1)記載の方法、
(3)WT1ペプチドが、さらに、HLA-DRB1*01:01分子、HLA-DRB1*04:01分子、HLA-DRB1*04:03分子、HLA-DRB1*04:05分子、HLA-DRB1*04:06分子、HLA-DRB1*08:03分子、HLA-DRB1*09:01分子、HLA-DRB1*11:01分子、HLA-DRB1*15:01分子、HLA-DRB1*15:02分子、HLA-DRB3*02:02分子、HLA-DRB4*01:01分子、HLA-DPB1*02:01分子、HLA-DPB1*03:01分子、HLA-DPB1*05:01分子、およびHLA-DPB1*09:01分子から選択されるいずれかのHLAクラスII分子に結合する能力を有するものである、(1)または(2)記載の方法、
(4)WT1ペプチドの抗原提示細胞への添加が、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリペプチドを含む発現ベクター、または該発現ベクターを含む細胞の添加により行われる、(1)から(3)のいずれか1つ記載の方法、
(5)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、(1)~(4)のいずれか1つ記載の方法、
(6)WT1ペプチドを抗原提示細胞に添加して、ヘルパーT細胞を活性化させるための、WT1ペプチドを含む組成物であって、該WT1ペプチドが、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子に結合する能力を有するものである組成物、
(7)WT1ペプチドが、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子のうちの少なくとも2つのMHCクラスII分子に結合する能力を有するものである、(6)記載の組成物、
(8)WT1ペプチドが、さらに、HLA-DRB1*01:01分子、HLA-DRB1*04:01分子、HLA-DRB1*04:03分子、HLA-DRB1*04:05分子、HLA-DRB1*04:06分子、HLA-DRB1*08:03分子、HLA-DRB1*09:01分子、HLA-DRB1*11:01分子、HLA-DRB1*15:01分子、HLA-DRB1*15:02分子、HLA-DRB3*02:02分子、HLA-DRB4*01:01分子、HLA-DPB1*02:01分子、HLA-DPB1*03:01分子、HLA-DPB1*05:01分子、およびHLA-DPB1*09:01分子から選択されるいずれかのHLAクラスII分子に結合する能力を有するものである、(6)または(7)記載の組成物、
(9)WT1ペプチドの抗原提示細胞への添加が、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞の添加により行われる、(6)~(8)のいずれか1つ記載の組成物、
(10)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、(6)~(9)のいずれか1つ記載の組成物、
(11)WT1ペプチドを含む抗原ペプチドとMHCクラスII分子との複合体が提示されている抗原提示細胞であって、前記MHCクラスII分子が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子である、抗原提示細胞、
(12)WT1ペプチドを含む抗原ペプチドとMHCクラスII分子との複合体を認識するヘルパーT細胞であって、前記MHCクラスII分子が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子である、ヘルパーT細胞、
(13)(11)記載のヘルパーT細胞により活性化される、細胞傷害性T細胞、
(14)(6)~(10)のいずれか1つ記載の組成物、(11)記載の抗原提示細胞、(12)記載のヘルパーT細胞、または(13)記載の細胞傷害性T細胞のいずれかを有効成分として含む、癌を治療または予防するための医薬組成物、
(15)WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞のいずれかを有効成分として含む、細胞傷害性T細胞を活性化させるための医薬組成物であって、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に投与するための医薬組成物、
(16)WT1ペプチドに特異的に結合する抗体であって、該WT1ペプチドが、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子に結合する能力を有するものである抗体、
(17)HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子陽性対象におけるWT1特異的ヘルパーT細胞の存在または量を決定する方法であって、
(a)前記対象から取得した試料をWT1ペプチドを用いて刺激し、
(b)サイトカインまたはヘルパーT細胞の存在または量を調べる、
工程を含み、サイトカインまたはヘルパーT細胞の存在または量の増大がWT1特異的ヘルパーT細胞の存在または量を示すものである方法、
を提供する。
(a)前記対象から取得した試料を上記抗WT抗体と反応させ、次いで、
(b)前記試料に含まれる上記抗WT抗体の存在または量を調べる、
工程を含む方法を提供する。前記工程(a)において用いられる試料として、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象から取得されたものを用いることができる。前記工程(a)に用いられる試料は、例えば、血液、リンパ球などの体液、組織などを挙げることができる。試料の取得や抗体との反応などは、当業者であれば公知の手法を用いて適宜行うことができる。本発明における工程(b)は、例えば、上記抗WT抗体の局在、部位、量等を決定することを含むので、癌の診断、予後診断などに用いることができる。上記抗WT抗体は標識されていてもよい。標識としては、蛍光標識、放射性標識などの公知のものを使用することができる。標識することによりWT1ペプチドの存在または量の決定を簡便かつ迅速に行うことが可能となる。
(a)前記対象から取得した試料をWT1ペプチドを用いて刺激し、
(b)サイトカイン、ヘルパーT細胞または細胞傷害性T細胞の存在または量を調べる、
工程を含み、サイトカイン、ヘルパーT細胞または細胞傷害性T細胞の存在または量の増大がWT1特異的ヘルパーT細胞またはWT1特異的細胞傷害性T細胞の存在または量を示すものである方法を提供する。本発明の試料は、抗原提示細胞を含むものであればいかなるものであってもよく、例えば、末梢血単核球、浸潤性リンパ球、腫瘍細胞、腹水中の細胞、胸水中の細胞、脳脊髄液中の細胞、骨髄細胞、またはリンパ節細胞などを挙げることができる。本発明に用いられる試料は、健常人由来であっても、あるいは癌患者由来であってもよい。健常人由来のこれらの細胞を用いることにより、例えば、癌に罹患しているかどうか、あるいはその素因を有するかどうかを診断することなどが可能になる。癌患者由来のこれらの細胞を用いることにより、例えば、癌患者においてWT1免疫療法が効果を有するかどうかを予測することなどが可能になる。本発明の方法において、取得した試料は、WT1ペプチドによる刺激の前後に培養されていてもよく、前記培養条件は当業者が適宜決定することができる。WT1ペプチドを用いたこれらの細胞の刺激は、エレクトロポレーションなどの公知の手法を用いて行うことができ、インビトロまたはインビボのいずれにおいて行われてもよい。サイトカイン産生、ヘルパーT細胞、細胞傷害性T細胞の反応が存在するか、あるいはサイトカイン産生量、ヘルパーT細胞、または細胞傷害性T細胞の反応量は既知の方法により調べることができる。
WT1ペプチドを抗原提示細胞に添加して、ヘルパーT細胞を活性化させるための、WT1ペプチドを含む組成物であって、該ヘルパーT細胞が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子と該WT1ペプチドとの複合体を認識するものである、組成物、および前記組成物を有効成分として含む、癌を治療または予防するための医薬組成物;
WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞のいずれかを有効成分として含む、癌を治療または予防するための医薬組成物であって、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に投与するための医薬組成物;および
WT1ペプチドを抗原提示細胞に添加して、ヘルパーT細胞を活性化させる工程を含む、ヘルパーT細胞の活性化方法であって、該ヘルパーT細胞が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子と該WT1ペプチドとの複合体を認識するものである、方法。
(1)ヘルパーT細胞を活性化させるための、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞を含む組成物であって、該ヘルパーT細胞が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子と該WT1ペプチドとの複合体を認識するものである、組成物。
(2)細胞傷害性T細胞を活性化させるための、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞を含む組成物であって、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に投与される、組成物。
(3)癌を治療または予防するための、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞を含む組成物であって、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に投与される、組成物。
(4)WT1ペプチドを含む、上記(1)~(3)のいずれかに記載の組成物。
(5)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、上記(1)~(4)のいずれかに記載の組成物。
(6)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチドである、上記(5)記載の組成物。
(7)WT1ペプチドを含む抗原ペプチドとMHCクラスII分子との複合体が提示されている抗原提示細胞であって、前記MHCクラスII分子が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子である、抗原提示細胞。
(8)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、上記(7)記載の抗原提示細胞。
(9)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチドである、上記(8)記載の抗原提示細胞。
(10)WT1ペプチドを含む抗原ペプチドとMHCクラスII分子との複合体を認識するヘルパーT細胞であって、前記MHCクラスII分子が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子である、ヘルパーT細胞。
(11)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、上記(10)記載のヘルパーT細胞。
(12)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチドである、上記(11)記載のヘルパーT細胞。
(13)上記(12)記載のヘルパーT細胞により活性化される、細胞傷害性T細胞。
(14)上記(7)~(9)のいずれかに記載の抗原提示細胞、上記(10)~(12)のいずれかに記載のヘルパーT細胞、または上記(13)記載の細胞傷害性T細胞のいずれかを有効成分として含む、癌を治療または予防するための医薬組成物。
(1)WT1ペプチドを抗原提示細胞に添加して、ヘルパーT細胞を活性化させる工程を含む、ヘルパーT細胞の活性化方法であって、該ヘルパーT細胞が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子と該WT1ペプチドとの複合体を認識するものである、方法。
(2)WT1ペプチドの抗原提示細胞への添加が、WT1ペプチドと抗原提示細胞とを接触させることにより、またはWT1ペプチドをコードするポリヌクレオチドもしくは該ポリヌクレオチドを含む発現ベクターを抗原提示細胞に導入することにより行われる、上記(1)記載の方法。
(3)細胞傷害性T細胞の活性化方法であって、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞を投与することを含む、方法。
(4)癌を治療または予防するための方法であって、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞を投与することを含む、方法。
(5)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、上記(1)~(4)のいずれかに記載の方法。
(6)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチドである、上記(5)記載の方法。
(1)ヘルパーT細胞を活性化させるための医薬の製造ための、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞の使用であって、該ヘルパーT細胞が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子と該WT1ペプチドとの複合体を認識するものである、使用。
(2)細胞傷害性T細胞を活性化させるための医薬の製造のための、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞の使用であって、該医薬がHLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に投与されるものである、使用。
(3)癌を治療または予防するための医薬の製造のための、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞の使用であって、該医薬がHLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に投与されるものである、使用。
(4)WT1ペプチドを含む医薬の製造のための、上記(1)~(3)のいずれかに記載の使用。
(5)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、上記(1)~(4)のいずれかに記載の使用。
(6)WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチドである、上記(5)記載の使用。
WT1ペプチド(配列番号2)(以降WT1-332と表記)特異的Th1クローン細胞(以下、Th1クローン細胞)の樹立を目的に、以下の検討を行った。
主な試験材料は以下の通りである。
被験物質
*WT1-332:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)
10 mM酢酸で20 mg/mLに溶解し、濾過滅菌して冷凍保存(-30℃設定)した。
1)細胞名:PBMC
2)由来:健康成人血液提供者末梢血
個人情報管理者の管理下で連結可能匿名化、及び文書によるインフォームドコンセントを行い、本試験にて採血、調製した。
3)検体条件:フィーダーの内、いずれかがクローニング実施検体と異なること。
4)採取条件:加ヘパリンナトリウム採血40 mL
5)細胞株名:B-LCL細胞株
6)由来:ヒト末梢血
7)入手先:理研セルバンクもしくはIHWGセルバンク
ヒトAB型血清及び牛胎仔血清(FBS)は、非働化処理後、0.2 μmフィルターで濾過して用いた。培地は、PBMC分離用に20 U/mLヘパリン HBSSを、B-LCL細胞用に10% FBS/ RPMI-1640(100 units/mL penicillin, 100 μg/mL streptomycin)培地を、その他の培養には10% ヒトAB型血清/AIM-V(インビトロジェン)培地を用いた。細胞の培養は、各培地中、37℃-5%CO2インキュベーターで行った。
PBMCの調製
健常人ボランティア末梢血から比重遠心法によりPBMCを調製し、その一部をWT1-332特異的T細胞の誘導に用いた。残りの細胞はセルバンカー(十慈フィールド株式会社)中に凍結保存し、再刺激時の抗原提示細胞もしくはフィーダー細胞として使用した。
調製したPBMCを1.5×106個/ウェル×10 ウェルで24ウェルプレートへ播種し、最終濃度20 μg/mL のWT1-332及び最終濃度10 ng/mL のIL-7を添加して培養を開始した(Day0,総培地量:2 mL/ウェル)。
1週間後に再刺激を行った。再刺激にはまず、細胞濃度を3.0×106個/mL以下に調製した抗原提示細胞用PBMCに最終濃度20 μg/mLのWT1-332を添加して2時間培養し、さらにマイトマイシンC(MMC)溶液(最終濃度=50 μg/mL)を添加して45分間培養し、AIM-Vで洗浄して抗原提示細胞とした。次に、培養していたPBMCを回収後、接着細胞を除去し、1.15-1.43×106個/ウェルで播種し、同数のWT1/MMC処理済みの抗原提示細胞と共に、最終濃度10 ng/mL のIL-7を添加して培養を再開した(Day7)。2日後に40 U/mL IL-2含有培地で培養液の半量交換を行い、さらに隔日で20 U/mL IL-2含有培地で培養液の半量交換を行いながら1週間培養を続けた。
Day14に培養細胞を回収し、96ウェル丸底プレートに2.0×105個ずつ2ウェルに播種し、一方に20 μg/mL WT1-332を、他方に溶媒を添加して4時間培養した後、さらに最終濃度1×のBrefeldin Aを添加して2時間培養した。細胞を回収し、PE標識抗ヒトCD4抗体、FITC標識抗ヒトCD8抗体を添加し、4℃、15分間反応させ、Staining Bufferで洗浄し、Cytofix Cytoperm Fix/Permを添加し、4℃、20分間処理した。Perm./Wash Buffer で洗浄し、PerCP標識抗ヒトIFN-γ抗体を添加して4℃、30分間反応させ、Perm./Wash Bufferで洗浄後、FACSにより解析した。
凍結保存細胞から約3×105個/mLで培養開始し、サブコンフルエント時に継代操作を行い、PHA刺激時のフィーダー用B-LCL細胞とした。
メーカーの推奨プロトコールに準じて、培養細胞からMACS Microbeadsを用いてポジティブセレクションを行い、CD4+細胞を単離した。
各フィーダー用B-LCL細胞及びPBMC(細胞濃度3×106/mL以下)を、マイトマイシンC溶液(最終濃度=50 μg/mL)でCO2インキュベーター中45分間処理し、AIM-Vで洗浄後、PBMC2種とB-LCL細胞2種の計4種の細胞を混和(各PBMCの最終濃度2.5×105個/mL、各B-LCL細胞の最終濃度2.5×104個/mL)した。これをクローニングする検体数に応じて、必要セット数調製し、最終濃度200 ng/mLでPHAを添加して、PHA含有フィーダー細胞混和液とした。
次に、分画したCD4+細胞の内、ICSの結果からCD4+IFN-γ+細胞比率が高い検体を選択し、準備したPHA含有フィーダー細胞混和液で、CD4+細胞濃度が10個/mLになるよう調整し、96 ウェル プレートに100 μL/ウェル(PBMC総量:5.0×104cells/ウェル、B-LCL細胞:5.0×103個/ウェル、PHA:200 ng/mL、CD4+細胞:1個/ウェル)で播種し、培養を開始した。5日後に培養液と等量の80 U/mL IL-2含有培地を添加し、その後隔日で80 U/mL IL-2含有培地で培養液の半量交換を行った。この間に、明らかな細胞増幅が観察されたウェルは、48ウェルプレートにスケールアップし培養を続けた。
クローニング開始から10日から14日間隔で、上記と同様にPHA刺激による増幅刺激を加えたが播種時に、培養皿を24ウェルプレートに、PBMC総量を1.0×106 個/ウェルに、B-LCL細胞総量を1.0×105個/ウェルに、PHA最終濃度を50ng/mLに、増幅させるTh1クローン細胞数は2.0×105個/ウェル以下に変更した。また、IL-2添加のタイミングを培養開始から3日後としさらに添加する培地のIL-2含有量を200 U/mLとした。
クローニングし、増幅させたTh1クローン細胞の抗原反応性を確認するため、回収したTh1クローン細胞を96ウェル丸底プレートに1ウェルもしくは3ウェルで播種した。これに10 μM酢酸もしくは、20 μg/mLの各ペプチドを添加して、培養を開始し(総培養液量:200 μL/ウェル)、約24時間後に培養上清を回収した。
各培養上清中のIFN-γ濃度は、各培養上清をAssay Diluent(Becton Dickinson社)で4倍希釈後に測定した。IFN-γ濃度の測定には、BD OptEIA ELISA set(human IFN-γ、Becton Dickinson社)を用い添付文書に従い測定したが、抗体の希釈濃度を500倍に、検量線の範囲を18.75-1200 pg/mLに、また、発色反応時間を5分に変更し、測定域を超えた場合は外挿値、吸光度が0を下回った場合は0として扱った。
抗原反応性が確認されたTh1クローン細胞をセルバンカー中で凍結保存し、マスターセルバンクとした。
WT1-332刺激下で14日間培養を行ったCD4+細胞中に、WT1-332特異的Th1細胞が有意に誘導されているか否かを評価するため、下式に従いWT1-332特異的Th1細胞比率(%)と、WT1-332非特異的Th1比率(%)を算出した。
WT1-332特異的Th1細胞比率(%)
=WT1-332刺激時のCD4+細胞内IFN-γ+細胞数 / 総生細胞数×
分画後CD4+細胞比率 / 分画前CD4+細胞比率 ×100
WT1-332非特異的Th1細胞比率(%)
=AcOH添加時のCD4+細胞内IFN-γ+細胞数 / 総生細胞数×100×
分画後CD4+細胞比率 / 分画前CD4+細胞比率 ×100
各実験でWT1-332特異的Th1比率からWT1-332非特異的Th1比率を除した値が高かった6~7検体についてクローニングを実施した。
各群の培養上清中IFN-γ濃度をELISAにより測定し、溶媒添加時に対してWT1-332添加時のIFN-γ産生量が500 pg/mL以上多く、1.2倍以上高い場合に抗原反応性が維持されているものと判断し、その後の操作を行った。
種々のTh1クローン細胞の樹立を行うことができた。得られたクローンの一部は、タイピングの結果、新規の拘束アレルを有していることが確認された。結果を表4に示す。これらのクローンを用いて、実施例2においてWT1-332が特定のHLA拘束性にTh1細胞を活性化できるか検討行った。
*HLAタイピングを実施していないHLA座はN.T.と表記した。
実施例1においてWT1-332添加により樹立された特異的Th1クローン細胞(以下、Th1クローン細胞)において、WT1-332が特定のHLA拘束性にT細胞を活性化できるかの検討を行い、またその拘束アレルを確認した。
培地類の調製
ヒトAB型血清及びFBSは非働化処理後、0.2 μmフィルターで濾過して用いた。培地は、PBMC分離用に20 U/mLヘパリン HBSSを、B-LCL細胞用に10% FBS及び1% P/S(100 units/mL ペニシリン, 100 μg/mL ストレプトマイシン)含有RPMI-1640を、その他の培養には10% ヒトAB型血清含有AIM-V培地を用いた。細胞の培養は、各培地中、37℃-5%CO2インキュベーターで行った。
適当なHLAクラスIIアレルを持ったB-LCL細胞にWT1-332パルスを行ったものを抗原提示細胞とし、Th1クローン細胞と共培養後、産生されるIFN-γ量を測定することにより、Th1クローン細胞のHLA拘束性を評価した。
具体的には、フィーダー用B-LCL細胞及び末梢血より調製した他家PBMCをフィーダー細胞として用い、PHA刺激下でTh1クローン細胞を培養した。次に、WT1-332或いは溶媒で処理したB-LCL細胞を抗原提示細胞として、回収したTh1クローン細胞と共培養し、産生されたIFN-γ量を測定してWT1-332特異的抗原刺激の有無を評価した。それぞれのTh1クローン細胞のHLAクラスII型に基づき、一部異なる型を有する種々のB-LCL細胞を抗原提示細胞として用いることにより、各Th1クローン細胞のHLAクラスII拘束性を評価した。
Day 0に凍結保存細胞から1×105個/mLで培養開始し、Day 3に回収してPHA刺激時のフィーダー用B-LCL細胞とした。
Day 2までに健常人ボランティア末梢血から比重遠心法により調製し、細胞数を測定後、細胞ペレットを回収し、セルバンカー(三菱化学メディエンス株式会社)に再懸濁後、クライオチューブに分注して-80℃設定の冷凍庫中で凍結保存した。PHA刺激時に凍結保存PBMCを随時融解し、フィーダー用PBMCとして供した。
Day 2に、凍結保存細胞から20 U/mL IL-2含有培地中で2×106cells/mL以下で培養開始し、Day 3に回収してPHA刺激時のTh1クローン細胞とした。
Day 3に、各フィーダー用B-LCL細胞及びPBMCを、マイトマイシンC溶液(最終濃度=50 μg/mL)でCO2インキュベーター中45分間処理し、AIM-Vで洗浄後、PBMC2種とB-LCL細胞2種の計4種の細胞を混和(各PBMCの最終濃度1.0×106個/mL、各B-LCL細胞の最終濃度1.0×105個/mL)した。これを、播種するTh1クローン細胞に対して必要セット数調製し、最終濃度100 ng/mLでPHAを添加して、PHA含有フィーダー細胞混和液とした。次に、培養していたTh1クローン細胞を24 ウェルプレートに0.5mL/個(細胞濃度4.0×105個/mL)で播種し、さらにPHA含有フィーダー細胞混和液を0.5 mL/ウェルで播種し、培養を行った。Day 6に培養液と等量の200 U/mL IL-2含有培地を添加し、Day 8、Day 10、Day 12、Day14には40 U/mL IL-2含有培地で培養液の半量交換を行い、Day 15に拘束性評価試験に供した。尚、培養途中で細胞がコンフルエントに達した場合は、20 U/mL IL-2含有培地で継代培養を行った。
Day 11に、凍結保存細胞から約1×105個/mLで培養開始し、Day 15に細胞を回収し拘束性評価試験に用いた。
Day 15にTh1クローン細胞を回収し、回収細胞数に応じて96ウェル丸底プレートに1.0-2.5×104cells/100 μL/ウェル、3ウェルで播種した。細胞濃度を1×106個/mLに調製した抗原提示用B-LCL細胞を4-7mLずつコニカルチューブ2本に分注し、一方に最終濃度10 μMで酢酸を、他方に最終濃度20 μg/mLでWT1-332を添加して2時間培養した後、AIM-Vで洗浄し、Th1クローン細胞を播種した96ウェルプレートへ2.5×104cells/100 μL/ウェルで添加し、16時間以上培養した。尚、WT1-332反応性を確認する為に、同プレートに、B-LCL細胞の代わりにWT1-332或いは溶媒を添加したウェルを用意した。
各培養上清中のIFN-γ濃度は、各培養上清をAssay Diluent(Becton Dickinson)で100倍希釈後に測定した。IFN-γ濃度の測定には、BD OptEIA ELISA set(human IFN-γ, Becton Dickinson)を用い添付文書に従い測定したが、検量線の範囲を5-640 pg/mLに、また、発色反応時間を10分に変更し、測定域を超えた場合は外挿値、吸光度が0を下回った場合は0として扱った。
各培養上清中のIFN-γ量
(i)Th1クローン細胞CloneR82-1 (DRB1*08:02/14:03, DPB1*02:01, DQB1*03:01/03:02) のHLA拘束性評価試験
CloneR82-1のIFN-γ産生は、WT1-332パルスDRB1*08:02(+) B-LCL (HEV#0052およびHEV#0324)の刺激によって検出された(図1)。したがって、CloneR82-1はWT1-332特異的HLA-DRB1*08:02-拘束性Th1クローンであることが判明した。
CloneR132-1のIFN-γ産生は、WT1-332パルスDRB1*13:02(+) B-LCL (HEV#0046)の刺激によってのみ検出された(図2)。したがって、CloneR132-1は、WT1-332特異的HLA-DRB1*13:02-拘束性Th1クローンであることが判明した。
CloneR143-1のIFN-γ産生は、WT1-332パルスDRB1*14:03(+) B-LCL (HEV#0052)の刺激によってのみ検出された(図3)。したがって、CloneR143-1はWT1-332特異的HLA-DRB1*14:03拘束性Th1クローンであることが確認された。
Clone R145-2のIFN-γ産生は、WT1-332パルスDRB1*14:05(+) B-LCL (ISH5)の刺激によってのみ検出された(図4)。したがって、Clone R145-2はWT1-332特異的HLA-DRB1*14:05拘束性Th1クローンであることが判明した。
CloneQ32-1のIFN-γ産生は、WT1-332パルスDQB1*03:02(+) B-LCL (HEV#0052およびHEV#0238)の刺激によって検出された(図5)。したがって、CloneQ32-1はWT1-332特異的HLA-DQB1*03:02拘束性Th1クローンであることが確認された。
CloneQ41-1のIFN-γ産生は、WT1-332をパルスした種々の抗原提示細胞(HEV#0174、HEV#0013、HEV#0035、およびHEV#0050)の刺激によって検出されたが、WT1-332をパルスしたHEV#0201およびHEV#0073では検出されなかった(図6)。DQB1*04:01は、HEV#0174、HEV#0013、HEV#0035、および0050においてのみ発現している。したがって、CloneQ41-1はWT1-332特異的HLA-DQB1*04:01拘束性Th1クローンであることが確認された。
Claims (14)
- ヘルパーT細胞を活性化させるための、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞を含む組成物であって、該ヘルパーT細胞が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子と該WT1ペプチドとの複合体を認識するものである、組成物。
- 細胞傷害性T細胞を活性化させるための、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞を含む組成物であって、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に投与される、組成物。
- 癌を治療または予防するための、WT1ペプチド、WT1ペプチドをコードするポリヌクレオチド、該ポリヌクレオチドを含む発現ベクター、または該発現ベクターを含む細胞を含む組成物であって、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子を有する対象に投与される、組成物。
- WT1ペプチドを含む、請求項1~3のいずれかに記載の医薬組成物。
- WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、請求項1~4のいずれかに記載の組成物。
- WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチドである、請求項5記載の組成物。
- WT1ペプチドを含む抗原ペプチドとMHCクラスII分子との複合体が提示されている抗原提示細胞であって、前記MHCクラスII分子が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子である、抗原提示細胞。
- WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、請求項7記載の抗原提示細胞。
- WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチドである、請求項8記載の抗原提示細胞。
- WT1ペプチドを含む抗原ペプチドとMHCクラスII分子との複合体を認識するヘルパーT細胞であって、前記MHCクラスII分子が、HLA-DRB1*08:02分子、HLA-DRB1*13:02分子、HLA-DRB1*14:03分子、HLA-DRB1*14:05分子、HLA-DQB1*03:02分子、およびHLA-DQB1*04:01分子から選択されるいずれかのMHCクラスII分子である、ヘルパーT細胞。
- WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチド、その変異体または修飾体である、請求項10記載のヘルパーT細胞。
- WT1ペプチドが、アミノ酸配列:Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His(配列番号2)を含むペプチドである、請求項11記載のヘルパーT細胞。
- 請求項12記載のヘルパーT細胞により活性化される、細胞傷害性T細胞。
- 請求項7~9のいずれかに記載の抗原提示細胞、請求項10~12のいずれかに記載のヘルパーT細胞、または請求項13記載の細胞傷害性T細胞のいずれかを有効成分として含む、癌を治療または予防するための医薬組成物。
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CA2892660A1 (en) | 2014-06-26 |
CA2892660C (en) | 2022-02-15 |
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US9833493B2 (en) | 2017-12-05 |
CN104995203B (zh) | 2019-06-28 |
UA118962C2 (uk) | 2019-04-10 |
EP2933261A1 (en) | 2015-10-21 |
JP6530192B2 (ja) | 2019-06-12 |
AR094026A1 (es) | 2015-07-08 |
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EP2933261A4 (en) | 2016-07-06 |
KR20150096697A (ko) | 2015-08-25 |
SG10201705028WA (en) | 2017-07-28 |
KR102158225B1 (ko) | 2020-09-21 |
CN104995203A (zh) | 2015-10-21 |
AU2013365067B2 (en) | 2017-10-05 |
ES2805337T3 (es) | 2021-02-11 |
EP2933261B1 (en) | 2020-06-10 |
MY175935A (en) | 2020-07-15 |
PH12015501361B1 (en) | 2015-09-02 |
SG11201504530VA (en) | 2015-07-30 |
CN110195040A (zh) | 2019-09-03 |
HK1216755A1 (zh) | 2016-12-02 |
IL239056A0 (en) | 2015-07-30 |
TW201429487A (zh) | 2014-08-01 |
PH12015501361A1 (en) | 2015-09-02 |
BR112015013697A2 (pt) | 2017-11-14 |
TWI646970B (zh) | 2019-01-11 |
US20150328278A1 (en) | 2015-11-19 |
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