JP2006280324A - HLA class II restricted WT1 antigen peptide - Google Patents

Abstract

Disclosed is a polypeptide that is an HLA class II-restricted WT1 antigen peptide.
To achieve the above object, the polypeptide is a WT1-derived polypeptide, which is an HLA class II-restricted WT1 antigen peptide consisting of a specific amino acid sequence. WT1-specific CD4 ⁺ T cells activated by the polypeptide recognize polypeptides bound to HLA class II molecules on leukemic cells and are directly cytotoxic to the leukemic cells in an HLA class II-restricted manner. Indicates. The polypeptide is useful in the field of immunotherapy against malignant tumors including leukemia, such as cellular immunotherapy, vaccine therapy, gene therapy, and the like.
[Selection figure] None

Description

  The present invention relates to HLA class II restricted WT1 antigen peptides.

  The WT1 gene is a zinc finger type transcription factor isolated as a causative gene in chromosome 11p13 of a Wilms tumor patient (see Patent Document 1), and its gene product, WT1 protein, It has a structure consisting of a suppression domain, an activation domain, and a zinc finger.

  It is known that WT1 protein is hardly expressed in normal cells and is specifically expressed in malignant tumors. Among them, WT1 protein is expressed particularly in the case of most leukemias. Based on these characteristics, the WT1 gene and WT1 protein are used to detect cancers including leukemia and solid cancer, and to detect and diagnose cancer (see Patent Documents 2 and 3), as well as cancer therapeutic effects and prognosis (See Patent Document 4), it is also used as a target gene / target protein.

Furthermore, in recent years, immunotherapy using WT1 as a target antigen for malignant tumors has attracted attention, and a plurality of groups have identified WT1-derived antigenic peptides (epitopes) recognized by CD8 ⁺ cytotoxic T cells. (For example, see Patent Document 5). WT1-specific HLA class I-restricted CD8 ⁺ cytotoxic T cells induced in vivo and externally by WT1 antigenic peptides can efficiently destroy leukemia cells and solid tumor cells. Based on such findings, for example, immunovaccine therapy of cancer using HLA class I-restricted WT1 antigen peptide as a vaccine, or WT1-specific CD8 ⁺ cytotoxicity produced in vitro using the WT1-specific antigen peptide Attempts have been made such as cellular immunotherapy to return T cells to the patient's body.

On the other hand, CD4 ⁺ T cells recognize antigenic peptides presented on major histocompatibility complex (MHC) class II molecules. CD4 ⁺ T cells specific for tumor-associated antigens such as WT1 are known to play a central role in regulating host immune responses to malignant tumors and infectious diseases. Despite its importance, there has been little identification of HLA class II restricted tumor associated antigen epitopes compared to HLA class I restricted tumor associated antigen epitopes. This is also true for the WT1 protein.
US Pat. No. 5,350,840 WO97 / 39354 pamphlet JP 2000-308500 A JP 2002-48793 A Special Table 2002-52599 gazette

  Accordingly, an object of the present invention is to provide a polypeptide that is an HLA class II-restricted WT1 antigen peptide.

In order to achieve the object, the polypeptide of the present invention is the polypeptide according to any one of (1) to (3) below.
(1) A polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 30.
(2) A polynucleotide comprising an amino acid sequence in which one to several amino acid residues of the polypeptide of (1) are deleted, substituted or added, and the polypeptide of (1) And a polypeptide having an amino acid sequence homology of 80% or more.
(3) A polypeptide having an amino acid sequence in which one to several amino acid residues of the polypeptide of (1) are deleted, substituted or added, and binds to an HLA class II molecule. HLA class II restricted WT1 antigen peptide that can be specifically recognized by CD4 ⁺ T cells.

In view of the importance of CD4 ⁺ T cells in immunotherapy, the present inventor has conducted extensive research and has developed an HLA class II-restricted antigen peptide (HLA class II-restricted) specific for WT1 protein that can be recognized by CD4 ⁺ T cells. WT1 antigen peptide) was identified, and it was found that this WT1 antigen peptide can promote proliferation and activation of WT1-specific CD4 ⁺ T cells. As a result of further research, the WT1-specific CD4 ⁺ T cells can produce T _H 1 cytokines. Furthermore, the WT1-specific CD4 ⁺ T cells themselves are HLA class II-restricted against leukemia cells and the like. The inventors have found that the present invention exhibits sex cytotoxicity and have reached the present invention.

Since the polypeptide of the present invention can promote the proliferation and activation of WT1-specific CD4 ⁺ T cells, the polypeptide of the present invention can be used for vaccine therapy against malignant tumors including leukemia, for example.

For example, WT1-specific CD4 ⁺ T cells can be produced by co-culturing the polypeptide of the present invention with lymphocytes and antigen-presenting cells removed from a patient. The WT1-specific CD4 ⁺ T cells are directly cytotoxic to leukemia cells. This cytotoxic function against leukemia cells by WT1-specific CD4 ⁺ T cells is a function that the present inventors have found for the first time. Therefore, returning the WT1-specific CD4 ⁺ T cells to the body of the patient enables more effective cellular immunotherapy for malignant tumors including leukemia.

Further, the antigen-presenting cell itself is activated by the recognition of the polypeptide of the present invention bound to the HLA class II molecule on the surface of the antigen-presenting cell such as a dendritic cell by the WT1-specific CD4 ⁺ T cell. Thus, WT1-specific CD8 ⁺ cytotoxic T cells can be produced more efficiently. Thus, for example, WT1-specific CD8 ⁺ cytotoxic T cells can be produced by co-culturing the polypeptide of the present invention with lymphocytes and antigen-presenting cells removed from a patient. By returning the WT1-specific CD8 ⁺ T cells to the body of the patient, cellular immunotherapy for malignant tumors including leukemia becomes possible.

Furthermore, a dendritic cell that presents an antigen of the polypeptide of the present invention can be produced by co-culturing the polypeptide of the present invention with, for example, a dendritic cell removed from a patient. According to this dendritic cell, it is possible to activate WT1-specific CD4 ⁺ T cells and activate WT1-specific CD8 ⁺ cytotoxic T cells. By returning the dendritic cells to the body of the patient, more effective cellular immunotherapy for malignant tumors including leukemia becomes possible.

  The HLA genotype of the HLA class II molecule on which the polypeptide of the present invention is presented is preferably HLA-DP5.

  The polynucleotide of the present invention is a polynucleotide encoding the polynucleotide of the present invention.

  The vector of the present invention is a vector containing the polynucleotide of the present invention.

  The first pharmaceutical composition of the present invention is a pharmaceutical composition used for immunotherapy, comprising a polypeptide of the present invention, a polynucleotide of the present invention, a vector of the present invention, or a derivative thereof. is there.

The CD4 ⁺ T cell production method of the present invention is a production method comprising the steps of collecting lymphocytes from a patient and incubating the lymphocytes, antigen-presenting cells, and the polypeptide of the present invention.

The CD8 ⁺ T cell production method of the present invention is a production method comprising the steps of collecting lymphocytes from a patient, and incubating the lymphocytes, antigen-presenting cells, and the polypeptide of the present invention.

  The method for producing dendritic cells of the present invention is a method comprising the steps of collecting dendritic cells from a patient and incubating the dendritic cells with the polypeptide of the present invention. As another aspect, the method for producing a dendritic cell of the present invention is a production method including a step of collecting a dendritic cell from a patient and a step of introducing the vector of the present invention into the dendritic cell.

The second pharmaceutical composition of the present invention is a pharmaceutical composition used for immunotherapy, wherein the CD4 ⁺ T cells produced by the method for producing CD4 ⁺ T cells of the present invention and the CD8 ⁺ T cells of the present invention are produced. A pharmaceutical composition comprising at least one of CD8 ⁺ T cells produced by the method and dendritic cells produced by the method for producing dendritic cells of the present invention.

The CD4 ⁺ T cell line of the present invention is a CD4 ⁺ T cell line that proliferates specifically in response to the polypeptide of the present invention.

The polypeptide of the present invention is an antigenic peptide of a WT1 protein that binds on an HLA class II molecule, preferably an HLA-DP5 type HLA class II molecule and can be specifically recognized by CD4 ⁺ T cells.

  In the present invention, “antigenic peptide” refers to a polypeptide constituting an antigenic determinant (epitope). The antigen peptide does not need to be a minimum unit of an antigenic determinant, and examples thereof include a polypeptide composed of 8 to 26, 9 to 24, 10 to 22, or 11 to 20 amino acids. It is done.

In the present invention, “binding to an HLA class II molecule and being specifically recognized by CD4 ⁺ T cells” means, for example, that a polypeptide is an HLA class II molecule on the surface of an antigen-presenting cell such as a dendritic cell. Above, when presented with an appropriate co-stimulatory factor, can specifically activate CD4 ⁺ T cells, or when a polypeptide binds to an HLA class II molecule on the cell surface of macrophages or B cells In addition, it means that the macrophages and B cells can be activated by specific CD4 ⁺ T cells.

  The binding between the polypeptide of the present invention and the HLA class II molecule is not particularly limited, and the polypeptide expressed inside the cell or the polypeptide taken into the cell by the eating and drinking action is HLA class II in the cell. It may be a case where it binds to a molecule, or it may be a case where a polypeptide existing outside the cell directly binds to a class II molecule already present on the cell surface.

In the present invention, an HLA class II molecule is a molecule corresponding to a human MHC class II molecule, and an HLA-DP type is an HLA class II molecule, and an alloantigen type of an HLA class II molecule is a DP type. That is, it means a gene product of HLA-DP gene, and HLA-DP5 type means that the genotype of the allele of HLA-DP gene is HLA-DP5 (HLA-DPB1 ^* 0501). . In addition, it is said that the HLA gene frequency of HLA-DP5 in Japanese accounts for about 50%.

  The polypeptide of the present invention includes a polypeptide consisting of the amino acid sequences set forth in SEQ ID NOs: 1-30. The amino acid sequence of SEQ ID NO: 1 corresponds to the 336th to 355th amino acid sequence of the WT1 protein (accession number AAO61088). The amino acid sequences of SEQ ID NOs: 2 to 10 respectively correspond to sequences that are deleted one amino acid at a time from the C-terminal side of the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 11 corresponds to the 337th to 355th amino acid sequences of the WT1 protein, and the amino acid sequences of SEQ ID NOS: 12 to 20 are each missing one amino acid from the C-terminal side of the amino acid sequence of SEQ ID NO: 11. It corresponds to the sequence that was lost. The amino acid sequence of SEQ ID NO: 21 corresponds to the 338th to 355th amino acid sequences of the WT1 protein, and the amino acid sequences of SEQ ID NOS: 22 to 30 are missing one amino acid from the C-terminal side of the amino acid sequence of SEQ ID NO: 21, respectively. It corresponds to the sequence that was lost.

  The polypeptide of the present invention includes a polypeptide that is homologous to the polypeptide consisting of the amino acid sequences set forth in SEQ ID NOs: 1-30. Here, that the amino acid sequences are “homologous” means that they are sufficiently similar to maintain their functions. For example, even if there are differences in amino acid sequences due to substitution, deletion or addition of amino acid residues, if these do not affect the function of the polypeptide of the present invention, they can be said to be homologous. In addition, the number of residues of different amino acids is, for example, 1-6, 1-5, 1-4, 1-3, 1-2, or 1. In addition, when two or more amino acid sequences show, for example, 80%, 84%, 88%, 92%, 96% identity or similarity, they can be said to be homologous. The identity and similarity refer to a relationship determined by comparing these sequences between two or more base sequences or amino acid sequences. Whereas the identity indicates the ratio of the same character string when aligned, the similarity includes not only the same character but also a similarly substituted character. Identity and similarity can be easily determined by various computer programs known in the art.

  The method for producing the polypeptide of the present invention is not particularly limited. For example, the polypeptide of the present invention may be produced by a conventionally known chemical synthesizer, and an expression vector containing the polynucleotide of the present invention is appropriately used for Escherichia coli, yeast, insect cells, etc. May be introduced into a suitable host and biologically produced.

  The polynucleotide of the present invention is a polynucleotide encoding the polypeptide of the present invention. The polynucleotide of the present invention includes a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 31 or a complementary sequence thereof. The polynucleotide of the present invention consisting of the base sequence of SEQ ID NO: 31 is a polynucleotide encoding the polypeptide of the present invention consisting of the amino acid sequence of SEQ ID NO: 1. Moreover, the polynucleotide of this invention contains the polynucleotide which codes polypeptide of this invention which consists of an amino acid sequence of sequence number 2-30. The base sequences of these polynucleotides can be easily determined by referring to the base sequence of SEQ ID NO: 31. That is, three nucleotides corresponding to the deleted amino acid may be deleted from the base sequence of SEQ ID NO: 31.

Furthermore, the polynucleotide of the present invention includes a polynucleotide comprising a base sequence homologous to the base sequence encoding the amino acid sequence of SEQ ID NOs: 1 to 30 or a complementary sequence thereof. The phrase “homologous” in the nucleotide sequence of a polynucleotide means that the polypeptide encoded by the polynucleotide is sufficiently similar to maintain the function as the polypeptide of the present invention. For example, even if there are differences in the base sequence due to point mutations, deletions or additions, they can be said to be homologous if they do not affect the function of the gene. The number of different bases is, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1. Also, in two or more sequences, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% identity These are said to be homologous. In addition, when one of the two polynucleotides hybridizes with a polynucleotide comprising the other complementary sequence under stringent conditions, they can be said to be homologous. The stringent conditions are not particularly limited. For example, in a solution containing 6 × SSC, 0.5% SDS, 5 × Denhardt, 0.01% denatured salmon sperm nucleic acid, a temperature of [Tm−25 ° C.] And conditions for keeping the temperature overnight. The Tm is obtained by the following formula, for example.
Tm = 81.5-16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) − (600 / N)
In the above formula, N is the chain length of the oligonucleotide probe, and% G + C is the content of guanine and cytosine residues in the oligonucleotide probe primer.

  The method for producing the polynucleotide of the present invention is not particularly limited, and may be produced by chemical synthesis, for example, or produced by various gene amplification methods using primers designed based on the known WT1 gene, Alternatively, it can be cloned into an appropriate vector.

  The vector of the present invention is a vector containing the polynucleotide of the present invention. The vector of the present invention is preferably an expression vector, and the polynucleotide of the present invention is preferably linked so that it can be expressed in a host cell into which the vector of the present invention is introduced. The type of the vector of the present invention is not particularly limited, and may be, for example, a viral vector or a plasmid vector.

  The first pharmaceutical composition of the present invention may be the polypeptide of the present invention, a derivative thereof, or the vector of the present invention itself, and further comprises a pharmaceutically acceptable adjuvant, carrier or excipient. But you can. In addition, the derivative of the polypeptide of the present invention is not particularly limited, and includes those modified by a conventionally known modification method, for example, salts, sugar chain modified products, lipidated products, and the like.

The first pharmaceutical composition containing the polypeptide of the present invention can be used for vaccine therapy of malignant tumors including administering to a patient an effective amount as a vaccine. In the body of a patient to which the vaccine is administered, the polypeptide of the present invention is taken up by antigen-presenting cells such as dendritic cells and bound to HLA class II molecules and presented on the antigen-presenting cells. Then, WT1-specific CD4 ⁺ T cells are stimulated for proliferation and activated. On the other hand, when the polypeptide of the present invention bound to the HLA class II molecule on the surface of the antigen-presenting cell is recognized by the WT1-specific CD4 ⁺ T cell, the antigen-presenting cell itself is also activated. Specific CD8 ⁺ cytotoxic T cells are stimulated to be activated and activated. The activated WT1-specific CD8 ⁺ cytotoxic T cells destroy malignant tumor cells that express WT1 in the patient. This makes it possible to treat malignant tumors.

Furthermore, the present inventors have found for the first time that the WT1-specific CD4 ⁺ T cells can exhibit HLA class II-restricted cytotoxicity against malignant tumor cells. That is, according to the vaccine using the first pharmaceutical composition containing the polypeptide of the present invention, the synergy between the cytotoxicity of WT1-specific CD8 ⁺ T cells and the cytotoxicity of WT1-specific CD4 ⁺ T cells. The effect enables more effective treatment of malignant tumors.

When the first pharmaceutical composition containing the polypeptide of the present invention is used as a vaccine, it is preferable to use a WT1-specific HLA class I-restricted antigen peptide (HLA class I-restricted WT1 antigen peptide) in combination. The peptide can bind to HLA class I molecules on the surface of antigen-presenting cells including dendritic cells and the like to proliferate and stimulate WT1-specific CD8 ⁺ cytotoxic T cells. The HLA class I-restricted WT1 antigen peptide is not particularly limited and, for example, a conventionally known peptide can be used. For example, CMTWNQMNL (Blood 95; 286-293, 2000, SEQ ID NO: 32) and RMFPNAPYL (Blood 95; 2198) -2203, 2000, SEQ ID NO: 33).

  A conventionally well-known method can be used for the preparation method of the 1st pharmaceutical composition containing polypeptide of this invention used as a vaccine. The vaccine may be prepared from the polypeptide of the present invention and a pharmaceutically acceptable excipient, for example, as a solution or suspension used as an injection. The vaccine may be prepared in a solid form. Examples of the excipient include water, salt water, dextrose, glycerol, ethanol and the like, and combinations thereof. If desired, the vaccine may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccine. The adjuvant is not particularly limited, and conventionally known adjuvants can be used. Among them, those that promote the uptake of the polypeptide of the present invention by antigen-presenting cells such as dendritic cells in the body of the patient are preferable. The polypeptide of the present invention may be formulated into a vaccine as a neutral or pharmaceutically acceptable salt.

  The vaccine is usually administered parenterally by, for example, but not limited to, subcutaneous injection, intradermal, dermal or intramuscular injection. Formulations suitable for other administration methods include, for example, suppositories, oral, buccal, sublingual, intraperitoneal, intrathecal, anal and intracranial formulations. The vaccine is preferably administered in a therapeutically effective amount for the patient and dosage form.

  In the present invention, the malignant tumor to be treated is a malignant tumor that expresses the WT1 protein, and examples thereof include various leukemias and various solid tumors. Examples of the leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), childhood ALL, and the like, and examples of the solid tumor include lung, breast , Thyroid and gastrointestinal cancers, and melanomas, but are not limited to these.

  The first pharmaceutical composition comprising the polynucleotide of the present invention and the vector induces a WT1-specific immune response by inoculating the patient's body, preferably in a muscle cell, and expressing the polypeptide of the present invention. It can be used for DNA (RNA) vaccine therapy. The preparation method as a vaccine is not specifically limited, It can prepare similarly to the vaccine containing the polypeptide of the above-mentioned this invention.

The method for producing CD4 ⁺ T cells of the present invention comprises collecting lymphocytes from a patient, and incubating the lymphocytes, antigen-presenting cells, and the polypeptide of the present invention, so that WT1-specific CD4 ⁺ T cells can be produced. Inducing proliferation and activation, and recovering the proliferated and activated CD4 ⁺ T cells. The antigen-presenting cells can be autologous, allogeneic, and allogeneic antigen-presenting cells that match the patient for HLA class II. The antigen-presenting cell is preferably one that presents the polypeptide of the present invention to CD4 ⁺ T cells and expresses HLA class II molecules and costimulatory molecules that can be activated by proliferation stimulation, for example, dendritic cells. .

The WT1-specific CD4 ⁺ T cells produced by the CD4 ⁺ T cell production method of the present invention have a characteristic function of exhibiting HLA class II-restricted cytotoxicity against malignant tumor cells including leukemia cells. . Furthermore, since the WT1-specific CD4 ⁺ T cells activate antigen-presenting cells that present the polypeptide of the present invention, the WT1-specific CD8 ⁺ cytotoxic T cells are stimulated to be activated and activated. Become. The activated WT1-specific CD8 ⁺ cytotoxic T cells destroy malignant tumor cells that express WT1 in the patient.

A second pharmaceutical composition of the present invention, as one aspect, is a pharmaceutical composition comprising the WT1-specific CD4 ⁺ T cells, and an effective amount of this pharmaceutical composition is administered to a patient to treat a malignant tumor. Cellular immunotherapy to treat is possible.

Method for producing CD8 ⁺ T cells of the present invention, lymphocytes taken from the patient, by incubating a polypeptide of the lymphocytes and antigen presenting cells and the present invention, firstly, the WT1-specific CD4 ⁺ T cells Activating the antigen-presenting cells, thereby activating and proliferating WT1-specific CD8 ⁺ cytotoxic T cells, and recovering the proliferated and activated CD8 ⁺ T cells. It is a manufacturing method including. In order to produce more efficiently, it is preferable to use the aforementioned HLA class I-restricted WT1 antigen peptide in combination. As the antigen-presenting cells, self- and allogeneic antigen-presenting cells can be used. As the antigen-presenting cell, the polypeptide of the present invention can be presented to CD4 ⁺ T cells, express HLA class II molecules and co-stimulatory molecules that can be activated by proliferation stimulation, and can activate CD8 ⁺ T cells. Those that express HLA class I molecules and costimulatory molecules are preferred, for example, dendritic cells.

In another aspect, the second pharmaceutical composition of the present invention is a pharmaceutical composition comprising WT1-specific CD8 ⁺ cytotoxic T cells produced by the method for producing CD8 ⁺ T cells of the present invention. By administering an effective amount of this pharmaceutical composition to a patient, cellular immunotherapy for treating malignant tumors becomes possible. The second pharmaceutical composition of the present invention may further contain both the WT1-specific CD4 ⁺ T cells and the WT1-specific CD8 ⁺ cytotoxic T cells. Thereby, more effective treatment becomes possible by the synergistic effect of both.

The 1st manufacturing method of the dendritic cell of this invention is a manufacturing method including the process of extract | collecting a dendritic cell from a patient, and the process of incubating the said dendritic cell and the polypeptide of this invention. Examples of the step of collecting the dendritic cells include two methods. The first method is to collect dendritic cells themselves from the patient's own blood using a blood component separation device, and the second method is to collect immature dendritic cells from the patient, This is a method of differentiating into dendritic cells with GM-CSF, IL-4, TNF or the like which is one kind of cytokine. By incubating the collected dendritic cells with the polypeptide of the present invention, dendritic cells with enhanced immunity-inducing ability of WT1-specific CD4 ⁺ T cells can be produced. Furthermore, it is preferable to use the aforementioned HLA class I-restricted WT1 antigen peptide in the incubation because dendritic cells with enhanced immunity-inducing ability against WT1-specific CD8 ⁺ T cells can be produced.

The 2nd manufacturing method of the dendritic cell of this invention is a manufacturing method including the process of extract | collecting a dendritic cell from a patient, and the process of introduce | transducing the vector of this invention into the said dendritic cell. Examples of the step of collecting the dendritic cells include the two methods described above. By introducing the vector of the present invention into the collected dendritic cells, it is possible to produce dendritic cells in which the peptide of the present invention is expressed in the cells and the immunity-inducing ability of WT1-specific CD4 ⁺ T cells is enhanced. Furthermore, when an expression vector containing a polynucleotide encoding the above-mentioned HLA class I-restricted WT1 antigen peptide is also introduced, dendritic cells with enhanced immunity-inducing ability against WT1-specific CD8 ⁺ T cells can be produced. Therefore, it is preferable. The gene introduction method is not particularly limited, and examples thereof include a method using a viral vector, a lipofection method, an electroporation method, a microinjection method, a cell fusion method, a DEAE dextran method, and a calcium phosphate method.

The dendritic cells produced by the method for producing dendritic cells of the present invention can be used, for example, as antigen-presenting cells in the method for producing WT1-specific CD4 ⁺ or CD8 ⁺ T cells of the present invention, in addition to the pharmaceutical composition described below. Can also be used.

The 2nd pharmaceutical composition of this invention is a pharmaceutical composition containing the dendritic cell manufactured by the manufacturing method of the dendritic cell of this invention as another aspect. The second pharmaceutical composition of the present invention contains dendritic cells with enhanced immunity-inducing ability specifically for WT1, and can be used for dendritic cell therapy of malignant tumors. The second pharmaceutical composition of the present invention may further contain the above-mentioned WT1-specific CD4 ⁺ T cells and / or WT1-specific CD8 ⁺ cytotoxic T cells in addition to the dendritic cells. .

The CD4 ⁺ T cell line of the present invention is a WT1-specific CD4 ⁺ T cell line that proliferates in response to the polypeptide of the present invention. The method for producing the CD4 ⁺ T cell line of the present invention is similar to the method for producing ordinary T cell clones, in which IL-2 is added to lymphocytes, antigen-presenting cells, and the polypeptide of the present invention. It can be produced by culturing for a period. By isolating a T cell receptor gene from the CD4 ⁺ T cell line of the present invention and using the gene for gene transfer, a WT1-specific CD4 ⁺ T cell (helper T cell) can be easily prepared.

  The present invention will be further described below using examples.

Based on the identification of HLA class II-restricted WT1 antigen peptide and the establishment of WT1-specific CD4 ⁺ T cell line WT1 protein amino acid sequence, 43 types of peptides consisting of 20 amino acids overlapping 10 amino acids are synthesized comprehensively. CD4 ⁺ T cells specifically reacting with any of these polypeptides were isolated by a conventionally known technique. That is, peripheral blood mononuclear cells (PBMC) derived from healthy individuals were stimulated three times with the synthetic polypeptide at a concentration of 10 μg / mL, and proliferative response to added WT1 peptide and cytotoxicity to growing cells. I investigated. Cells that showed a positive response to the WT1 peptide were continuously added to the wells once every 1-2 weeks in a culture medium containing interleukin-2 (IL-2) with mitomycin C-treated autologous PBMC and WT1 peptide. Cultured.

As a result, a CD4 ⁺ T cell line NIK-1 was established which grew specifically in response to a specific WT1 peptide (WT1 _336-355 KLSHLQMHSRKHTGEKPYQC: SEQ ID NO: 1).

NIK-1 produced a large amount of T _H 1 cytokines such as interferon γ, interleukin (IL) -2, and IL-12 when stimulated with the WT1 peptide in the presence of autologous PBMC. Moreover, NIK-1 showed strong cytotoxicity against self B-LCL (B cell-derived EB virus transformed strain) presenting the WT1 peptide. Thus, the cytotoxicity of NIK-1 against self and various allogenic (allogeneic) LCL cells was measured with and without the addition of the WT1 peptide. The cytotoxicity was measured using a conventionally known chromium 51-releasing cytotoxicity assay. The ratio of NIK-1 to target cells was 10: 1, 5: 1 and 2.5: 1. These results are shown in Table 1 below.

As shown in Table 1 above, the NIK-1 strain is an autologous LCL and allogenic (allogeneic) cell that expresses HLA-DP5, and exhibits cytotoxicity only when the WT1 peptide is added. -DP5 ^- against allogeneic cells, or without WT1 peptide, it did not show cytotoxicity. Therefore, it was suggested that the cytotoxicity of NIK-1 shows HLA-DP5 restriction.

  Therefore, in order to confirm the HLA-DP5 restriction of the WT1 peptide and NIK-1 strain, NIK was similarly used as a target cell using a cell in which HLA-DP5 or HLA-DP9 was introduced into an L cell of a cultured cell. -1 cytotoxicity was examined. Before adding the effector NIK-1 cells to the target cells, an anti-HLA-DR monoclonal antibody (L243), an anti-HLA-DQ monoclonal antibody (HU-11) or an anti-HLA-DP monoclonal antibody ( B7 / 21) was incubated with target cells for 30 minutes at the optimal concentration. The results are shown in Table 2 below.

As shown in Table 2, NIK-1 showed cytotoxic to HLA-DP5 ⁺ L cells presenting WT1 peptide, the HLA-DP9 ⁺ L cells did not show cytotoxicity. Furthermore, the cytotoxicity of NIK-1 was inhibited by adding anti-HLA-DP monoclonal antibody. From these results, it was confirmed that the NIK-1 strain is a WT1-specific HLA-DP5-restricted CD4 ⁺ T cell line.

Next, a deletion polypeptide of the WT1 peptide (SEQ ID NO: 1) is prepared, and NIK-1 can be recognized by NIK-1 by measuring the cytotoxicity of NIK-1 when placed on autologous LCL cells. We have identified shorter antigenic epitopes. An example is shown in Table 3 below. As a result, the minimum amino acid sequence showing 50% or more cytotoxicity was WT1 _337-347 LSHLQMHSRKH (SEQ ID NO: 19).

HLA-DP5-restricted cytotoxic target cells for leukemia cells of NIK-1 cell line are newly derived from various leukemia cell lines, lymphoma cell lines, and patients with acute myeloid leukemia (AML) or acute lymphoma leukemia (ALL) NIK-1 cytotoxicity was measured using isolated leukemia cells. In most leukemia cell lines, HLA-DP5 was negative, and in most types of leukemia cells, expression of HLA class II molecules was positive. The results are shown in Table 4 below.

  As shown in Table 4 above, NIK-1 showed strong cytotoxicity to HLA-DP5-positive leukemia cell lines expressing WT1, but HLA-DP5-negative leukemia cell lines and HLA not expressing WT1. -DP5 positive and negative lymphoma cell lines showed no cytotoxicity. NIK-1 also efficiently destroyed newly isolated HLA-DP5-positive leukemia cells, but did not destroy HLA-DP5-negative leukemia cells. Therefore, it was suggested that the WT1-specific cytotoxicity of NIK-1 against leukemia cells is HLA-DP5-restricted.

  Therefore, in order to confirm NIK-1 HLA-DP5 restriction, before adding NIK-1 as an effector to target cells, anti-HLA-DR monoclonal antibody (L243), anti-HLA-DQ monoclonal antibody (HU- 11) or anti-HLA-DP monoclonal antibody (B7 / 21) was incubated with target cells at an optimal concentration for 30 minutes to measure NIK-1 cytotoxicity. The results are shown in Table 5 below. The target cells in the table are the same as the target cells in Table 4 above.

  As shown in Table 5 above, the cytotoxicity of NIK-1 against leukemia cell lines and leukemia cells was inhibited by anti-HLA-DP monoclonal antibodies. This indicates that the WT1-specific cytotoxicity of NIK-1 against leukemia cells is HLA-DP5-restricted.

It was confirmed that the cytotoxic pathway of NIK-1, which is a WT1-specific CD4 ⁺ T cell line, against leukemia cells is perforin. Conkanamycin A (CMA) is a vacuolar H ⁺ -ATPase inhibitor that inhibits perforin-type cytotoxicity by increasing the pH of soluble granules and accelerating the degradation of perforin, and therefore CMA is an effective tool for measuring perforin-mediated cytotoxicity. Therefore, before incubating the effector cell NIK-1 with the target cell, it was pretreated with 100 nmol CMA (manufactured by Wako Pure Chemical Industries, Ltd.) for 2 hours, incubated with the target cell in the presence of CMA, and NIK- The cytotoxicity of 1 was measured. The results are shown in Table 6 below. The target cells in the table are the same as the target cells in Table 4 above.

As shown in Table 6 above, when NIK-1 was treated with CMA, a decrease in cytotoxicity against leukemia cells was observed. Therefore, it was suggested that the main pathway of WT1-specific CD4 ⁺ T cell cytotoxicity against leukemia cells is perforin-dependent granule exocytosis.

  As described above, the present invention is useful, for example, in the medical field of malignant tumors including leukemia, particularly in the field of immunotherapy including cellular immunotherapy, vaccine therapy, gene therapy, and the like.

Claims (11)

The polypeptide according to any one of (1) to (3) below.
(1) A polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 30.
(2) A polynucleotide comprising an amino acid sequence in which one to several amino acid residues of the polypeptide of (1) are deleted, substituted or added, and the polypeptide of (1) And a polypeptide having an amino acid sequence homology of 80% or more.
(3) A polypeptide comprising an amino acid sequence in which one to several amino acid residues in the amino acid sequence of the polypeptide (1) are deleted, substituted or added, and binds to an HLA class II molecule. HLA class II restricted WT1 antigen peptide that can be specifically recognized by CD4 ⁺ T cells.   The polypeptide according to claim 1, wherein the HLA genotype of the HLA class II molecule is HLA-DP5.   A polynucleotide encoding the polypeptide according to claim 1 or 2.   A vector comprising the polynucleotide according to claim 3.   A pharmaceutical composition used for immunotherapy, comprising the polypeptide according to claim 1 or 2, the polynucleotide according to claim 3, the vector according to claim 4, or a derivative thereof. A method for producing CD4 ⁺ T cells, the step of collecting lymphocytes from a patient, and the step of incubating the lymphocytes, antigen-presenting cells, and the polypeptide or derivative thereof according to claim 1 or 2. Manufacturing method. A method for producing CD8 ⁺ T cells, the step of collecting lymphocytes from a patient, and the step of incubating the lymphocytes, antigen-presenting cells, and the polypeptide or derivative thereof according to claim 1 or 2. Manufacturing method.   A method for producing dendritic cells, comprising the steps of collecting dendritic cells from a patient, and incubating the dendritic cells with the polypeptide or derivative thereof according to claim 1 or 2. .   A method for producing dendritic cells, comprising the steps of collecting dendritic cells from a patient and introducing the vector according to claim 4 into the dendritic cells. A pharmaceutical composition for use in immunotherapy, comprising CD4 ⁺ T cells produced by the production method according to claim 6, CD8 ⁺ T cells produced by the production method according to claim 7, and claim 8 Or a pharmaceutical composition comprising at least one cell selected from the group consisting of dendritic cells produced by the production method according to 9. A CD4 ⁺ T cell lines, which proliferate in response specifically to a polypeptide according to claim 1 or 2 CD4 ⁺ T cell line.