JP2013116891A - Pharmaceutical composition for treating meningioma - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for treating meningioma that is useful for the treatment of meningioma.SOLUTION: The pharmaceutical composition for treating meningioma comprises as an effective ingredient a substance exhibiting cytotoxicity to a WT1-expressing cell or a substance having activity of inducing cytotoxicity to a WT1-expressing cell. The substance exhibiting cytotoxicity to a WT1-expressing cell is, for example, a substance selected from the group consisting of "a cell exhibiting WT1-expressing cell-specific cytotoxic activity" and "an antibody specifically recognizing a WT1-expressing cell". The substance having activity of inducing cytotoxicity to a WT1-expressing cell is, for example, a substance selected from the group consisting of "WT1", "a WT1-derived peptide", "a cell presenting a WT1-derived peptide", "a receptor specifically recognizing a WT1-expressing cell" and "a nucleic acid coding a receptor specifically recognizing a WT1-expressing cell".

Description

  The present invention is a meningioma containing as an active ingredient a substance that exhibits cytotoxic activity against WT1-expressing cells, or a substance that induces cytotoxic activity against WT1-expressing cells, which is useful in the treatment of meningiomas The present invention relates to a therapeutic pharmaceutical composition.

  Brain tumors are thought to have been caused by individual mutations in the nerve cells and glial cells that make up the brain, and show a very diverse morphological image depending on the cells that originated. It has become. Internationally, the WHO classification is widely used. Tissue classification by WHO classification and its frequency of occurrence were: glioma 28%, meningiomas 26%, pituitary adenoma 17%, schwannoma 11%, germ cell tumor 2.1%, lymphoma hematopoietic tumor (frequency) Unknown). The most common glioma is astrocytoma, which accounts for about 80% of gliomas.

  The WT1 gene is derived from a gene isolated as one of the causative genes of Wilms tumor (Non-patent Document 1), and is a growth factor gene (PDGFα chain, CSF-1, IGF-II), growth factor receptor gene (IGF) -IR, EGFR) and other genes (RAR-α, C-myb, C-myc, bcl-2, ornithine decarboxylase, N-myc) are repressed. On the other hand, the WT1 gene is known to promote transcription of RbAp46, Dax-1, and bcl-2 genes. The WT1 gene is highly expressed in solid cancers such as leukemia, stomach cancer, colon cancer, lung cancer, breast cancer, germ cell cancer, liver cancer, skin cancer, bladder cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer and the like. (Patent Document 1). These diseases are involved in tumor development and play an important role in the onset and progression of solid cancers and hematopoietic tumors.

  In patients with hematopoietic tumors and the like, it is known that a humoral immune response to WT1-derived proteins and a cellular immune response by CD8 positive T cells are naturally induced. A plurality of cytotoxic T lymphocyte (CTL) epitope peptides that are recognized by CD8 positive T cells from WT1 have been identified. For example, human leukocyte type antigen HLA-A0201-restricted 126-134 peptide, HLA-A2402-restricted 235-243 peptide, and the like have been reported. CTLs that recognize these peptides have been reported to damage HLA-restricted tumor cells that express the WT1 antigen. The expression of WT1 gene in normal cells excluding normal hematopoietic stem cells is extremely low, but normal hematopoietic stem cells are said to have the same expression as tumor cells. However, in human in vitro colony formation inhibition experiments using WT1 peptide-specific CTLs and in vivo experiments using WT1 peptide vaccine in mice, no damage to normal hematopoietic stem cells has been observed (Non-patent Document 2). .

  Currently, many tumor antigens expressed on the surface of various tumor cells have been found, and the expression intensity in each tumor cell and normal tissue has been reported. Treatments based on cellular immunity or humoral immunity targeting these tumor antigens are being studied, but there are a wide variety of tumors and tumor antigens, and selection and selection of appropriate tumor antigens for the treatment of each tumor Trial and error continues to find therapeutic strategies based on tumor antigens.

US Pat. No. 7,696,180

Molecular Cell Biology, Vol. 11, p. 1707 (1991) Japanese Journal of Clinical Oncology (Jpn. J. Clin. Oncol.), 40, 377-387 (2010)

  An object of the present invention is to provide a pharmaceutical composition for treating meningiomas useful in the treatment of meningiomas.

  As a result of diligent efforts to solve the above-mentioned problems, the present inventors use, as an active ingredient, a substance that exhibits cytotoxicity against WT1-expressing cells or a substance that has an activity of inducing cytotoxicity against WT1-expressing cells. The present inventors have found a pharmaceutical composition for treating meningiomas and have completed the present invention.

That is, if the present invention is outlined,
[1] A pharmaceutical composition for treating meningiomas comprising, as an active ingredient, a substance that exhibits cytotoxicity against WT1-expressing cells, or a substance that has an activity of inducing cytotoxicity against WT1-expressing cells,
[2] A pharmaceutical composition for the treatment of meningioma according to [1], which contains, as an active ingredient, cells having WT1-expressing cell-specific cytotoxicity,
[3] A pharmaceutical composition for treating meningiomas according to [2], comprising cells expressing a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that specifically recognizes WT1-expressing cells,
[4] A pharmaceutical composition for treating meningiomas according to [1], comprising an active ingredient selected from WT1, a WT1-derived peptide, and a cell presenting a WT1-derived peptide,
[5] The pharmaceutical composition for meningioma treatment according to [4], wherein the cell presenting the WT1-derived peptide is an antigen-presenting cell,
[6] A pharmaceutical composition for treating meningiomas according to [1], which contains an active ingredient selected from a receptor that specifically recognizes WT1-expressing cells or a nucleic acid encoding the receptor,
[7] A pharmaceutical composition for treating meningiomas according to [6], which contains a nucleic acid encoding TCR or CAR that specifically recognizes WT1-expressing cells,
[8] The pharmaceutical composition according to [7], wherein a viral vector particle contains a nucleic acid encoding TCR or CAR that specifically recognizes a WT1-expressing cell;
[9] The pharmaceutical composition for treating meningiomas according to [1], comprising as an active ingredient an antibody that specifically recognizes WT1-expressing cells or a polypeptide containing an antigen recognition site of the antibody.

  The present invention provides a pharmaceutical composition for treating meningiomas useful in the treatment of meningiomas. The pharmaceutical composition for treating meningiomas of the present invention exhibits high cytotoxicity or high activity inducing cytotoxicity against meningiomas.

It is a figure which shows the expression of the WT1 gene in meningioma. It is a figure which shows the structure of the WT1-TCR expression vector used in the Example. It is a figure which shows the intracellular cytokine amount of the cell which expresses WT1-TCR. It is a figure which shows the cytotoxic activity of the cell which expresses WT1-TCR. It is a figure which shows the tumor size of a malignant skull base meningioma mouse model. It is a figure which shows the number of CD8 positive cells which infiltrated the normal tissue and the tumor. It is a figure which shows the survival rate of a malignant skull base meningioma mouse model.

  As used herein, “meningioma” is a generic term for tumors that originate from the meninges (buffy, arachnoid, dura mater) wrapping the brain, and benign meningiomas, dysmorphic meningiomas, and anaplastic meningiomas including. When the tumor becomes huge, the pressure in the entire cranial lumen increases, causing headaches, nausea and the like. Several established meningioma cell lines have been reported, including IOMM-Lee [Neurosurgery, 27 (3), 389-395 (1990)], HKBMM [Humumell (Hum Cell), No. 17 (4), 211-217 (2004)].

  In this specification, “WT1” is a protein encoded by a gene isolated as one of the causative genes of Wilms tumor as described above. The amino acid sequence is described in NCBI RefSeq: NP_000369.3, and the base sequence is described in NCBI RefSeq: NM_000378.4. The amino acid sequence of WT1 is shown in SEQ ID NO: 14. The expression of WT1 in cells can be quantified by the method described in Patent Document 1, for example.

  In the present specification, the “T cell” is also referred to as a T lymphocyte and means a cell derived from the thymus among lymphocytes involved in an immune response. T cells include helper T cells, suppressor T cells, regulatory T cells, cytotoxic T lymphocytes (CTL), naive T cells, memory T cells, αβ T cells expressing TCRs of α and β chains, γ Γδ T cells that express TCRs of the chain and δ chain are included. Examples of “cell populations containing T cells” include blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, peripheral blood mononuclear cells (PBMC), blood cell lineage collected, isolated, purified and derived from these Examples of the cell population include cells, hematopoietic stem cells, and cord blood mononuclear cells. In addition, various cell populations derived from blood cells containing T cells can be used in the present invention. These cells may be activated in vivo (in vivo) or ex vivo (ex vivo) by cytokines such as anti-CD3 antibodies and IL-2. Any of these cells collected from a living body or those obtained by culturing in vitro can be used as they are or after being cryopreserved.

  As used herein, “TCR” is a receptor present on the cell surface of a T cell that recognizes an antigen-MHC molecule (major histocompatibility complex) presented on the cell surface. As the TCR, a heterodimer composed of an α chain and a β chain and a heterodimer composed of a γ chain and a δ chain are known, and any of them can be suitably used in the present invention.

  As used herein, “chimeric antigen receptor (CAR)” refers to a fusion protein comprising an extracellular domain that binds to an antigen, a transmembrane domain derived from a polypeptide different from the extracellular domain, and at least one intracellular domain. Say. Sometimes called “chimeric receptor”, “T-body”, “chimeric immune receptor (CIR)”. An “extracellular domain that binds to an antigen” refers to any oligo or polypeptide that can bind to an antigen, and an “intracellular domain” refers to a signal that results in the activation or inhibition of a biological process in the cell. By any oligo or polypeptide known to function as a transducing domain.

  The pharmaceutical composition for meningioma treatment of the present invention contains a substance that exhibits cytotoxicity against WT1-expressing cells or a substance having an activity of inducing cytotoxicity against WT1-expressing cells as an active ingredient. The substance that exhibits cytotoxicity against WT1-expressing cells is selected from the group consisting of, for example, “cells having WT1-expressing cell-specific cytotoxic activity” and “antibodies that specifically recognize WT1-expressing cells” It is. Substances having an activity of inducing cytotoxicity against WT1-expressing cells include, for example, “WT1”, “WT1-derived peptide”, “cell presenting WT1-derived peptide”, “receptor specifically recognizing WT1-expressing cell” And “a nucleic acid encoding a receptor that specifically recognizes WT1-expressing cells”. Each substance is described in detail below.

(1) Nucleic acid encoding a receptor that specifically recognizes WT1 In one embodiment of the present invention, the pharmaceutical composition for treating meningiomas of the present invention comprises a nucleic acid encoding a receptor that specifically recognizes WT1-expressing cells. Is contained as an active ingredient. Examples of the receptor include TCR and CAR. Therefore, in a further aspect of the present invention, the pharmaceutical composition for meningioma treatment of the present invention contains, as an active ingredient, a nucleic acid encoding TCR or CAR that specifically recognizes WT1-expressing cells.
In one embodiment of the present invention, the pharmaceutical composition for meningioma treatment of the present invention contains, as an active ingredient, a nucleic acid encoding TCR that specifically recognizes WT1-expressing cells. Here, “TCR specifically recognizing WT1-expressing cells” means a TCR having the ability to bind to a complex of a WT1-derived peptide and MHC. The nucleic acid encoding the TCR can be prepared by selecting a T cell that specifically recognizes a WT1-expressing cell from a cell population containing a T cell present in a living body. For example, cell populations containing T cells (eg, PBMC) are collected from mammals and cultured in the presence of WT1 or WT1-derived peptides while stimulating these cell populations. From this cell population, specificity to WT1-expressing cells and specific recognition of WT1-expressing cells from CTLs, helper T cells, and naive T cells isolated using cell surface antigens such as CD8 and CD4 as indicators Nucleic acids encoding TCR can be prepared. The collection of a cell population containing T cells is preferably performed from a living body having a disease such as a WT1-positive tumor. For example, HLA-A2402-restricted CTL clone TAK-1 [Blood, 95, 286-293 (2000)] is a VCR20 / J33 / Cα TCRα chain gene and Vβ5.1 / J2. It has a 1 / Cβ2 TCRβ chain gene. The specificity of WT1-expressing cells of T cells can be measured by using, for example, a chromium release assay or a proliferation assay to compare solubility and proliferation with negative controls. In addition, the production of cytokines can be measured to evaluate the WT1-expressing cell specificity of T cells.

  Using a DNA extracted from a TCR-expressing cell that specifically recognizes a WT1-expressing cell as a template, the TCR gene can be amplified and cloned based on the nucleic acid sequence of the TCR constant region. Further, RNA is extracted from cells by a conventional method to synthesize cDNA, and 5'-rapid amplification using this as a template and antisense primers complementary to nucleic acids encoding the constant regions of TCR α chain and β chain, respectively. It can be prepared by performing the application of cDNA end (RACE). 5'-RACE may be performed by a known method, for example, using a commercially available kit such as SMART PCR cDNA Synthesis Kit (manufactured by Clontech). The DNA amplified by the above method is incorporated into a plasmid vector, and E. coli is transformed. A plasmid is prepared from the transformant, and the base sequence of the inserted DNA is determined. By comparing the obtained base sequence with the sequence of a known TCR α chain or β chain gene, DNA unrelated to the TCR gene amplified by 5′-RACE can be excluded.

  When a TCR-encoding nucleic acid that specifically recognizes a WT1-expressing cell is introduced into a T cell, the expression of the endogenous TCR α chain and TCR β chain originally expressed by the T cell is suppressed by siRNA, thereby expressing WT1 expression. A method for obtaining a T cell that expresses a TCR recognizing a cell at a high ratio is known (International Publication No. 2008/153029 pamphlet). When the nucleic acid is applied to the method, the base sequence is a sequence different from the base sequence corresponding to the RNA on which siRNA that suppresses the expression of the endogenous TCR α chain and TCR β chain acts (codon conversion type sequence). Thus, T cells expressing a high ratio of TCR that specifically recognizes WT1-expressing cells can be obtained. The base sequence can be prepared by introducing a silent mutation into a nucleic acid encoding TCR obtained from nature or chemically synthesizing an artificially designed nucleic acid.

  In another embodiment of the present invention, the pharmaceutical composition for meningioma treatment of the present invention contains, as an active ingredient, a nucleic acid encoding CAR that specifically recognizes WT1-expressing cells. The nucleic acid is a nucleic acid encoding a fusion protein comprising an extracellular domain that binds to WT1, a transmembrane domain derived from a polypeptide different from the extracellular domain, and at least one intracellular domain. CAR is reviewed in Current Opinion in Immunology, Vol. 21, pp. 215-223 (2009).

  An extracellular domain that binds to WT1 is a domain that includes an oligo or polypeptide that can bind to WT1. This domain confers specificity to CAR by binding to and interacting with WT1. Examples of extracellular domains that can be used for CAR include those derived from anti-WT1 antibodies (H and L chains) and TCR variable regions (TCRα, TCRβ, TCRγ, TCRδ). While it may be effective to use the whole of these proteins, for example, antibody Fab fragments or antibody variable regions [H chain V region (VH) and L chain V region (VL)] can be used. . In particular, a single chain antibody (scFv) can be preferably used.

  The intracellular domain of CAR is a molecule capable of transmitting a signal into a cell when an extracellular domain existing in the same molecule binds (interacts) with WT1. The intracellular domain of CAR is exemplified by an intracellular domain containing a primary cytoplasmic signaling sequence derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In addition, examples include intracellular domains containing secondary cytoplasmic signal transduction sequences derived from CD2, CD4, CD5, CD8α, CD8β, CD28, CD137, CD134, ICOS, GITR and CD154.

  The transmembrane domain of CAR is, for example, α, β chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 of T cell receptor, The transmembrane domain of CD134, CD137, ICOS, CD154, GITR can be used. Alternatively, an artificially designed arrangement may be used.

  A TCR or CAR that specifically recognizes a WT1-expressing cell transmits a signal into the cell via its intracellular domain to stimulate the cell. Stimulated cells produce cytokines and the like, exhibit cytotoxicity against WT1-expressing cells, or induce cytotoxicity of other immune cells. As the nucleic acid encoding TCR or CAR, a nucleic acid obtained by amplifying a nucleic acid obtained from a living body and a nucleic acid obtained by linking these nucleic acids may be used, or a nucleic acid having the same sequence may be chemically synthesized. May be used. Furthermore, a nucleic acid having a base sequence different from that of the nucleic acid encoding the same amino acid sequence as that of the nucleic acid can be prepared and used. When designing a nucleic acid having a base sequence different from that of the nucleic acid, it is possible to select a codon to be used based on the frequency of codon usage in the cell and other purposes.

A nucleic acid encoding TCR or CAR that specifically recognizes WT1-expressing cells can be linked to another nucleic acid so that it is expressed under the control of a suitable promoter. As the promoter, any of those that constitutively promote expression and those that are induced by drugs or the like (for example, tetracycline or doxorubicin) can be used. For example, phosphoglycerate kinase (PGK) promoter, Xist promoter, β-actin promoter, RNA polymerase II promoter and other mammalian promoters, SV40 early promoter, cytomegalovirus promoter, herpes simplex virus thymidine kinase promoter, various retroviruses Virus-derived promoters such as the LTR promoter can be used. In addition, in order to achieve efficient transcription of the nucleic acid, a promoter or other regulatory element cooperating with the transcription start site, for example, a nucleic acid containing an enhancer sequence or terminator sequence may be linked. Furthermore, a gene that can serve as a marker for confirming nucleic acid expression (for example, a drug resistance gene, a gene encoding a reporter enzyme, or a gene encoding a fluorescent protein) may be incorporated. It should be noted that the nucleic acid encoding the TCRα chain polypeptide and the nucleic acid encoding the TCRβ chain polypeptide may be transcribed and translated by different promoters, using an internal ribosome entry site (IRES) or a self-digestible 2A peptide. It may be transcribed and translated by one promoter.
Moreover, the nucleic acid encoding TCR or CAR that specifically recognizes WT1 may be contained in the virus vector particle. Examples of virus vectors include retrovirus vectors (including oncoretrovirus vectors, lentivirus vectors, pseudotype vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, or Sendai virus. Viral vectors such as vectors, Epstein-Barr virus (EBV) vectors, herpes simplex virus (HSV) vectors can be used. As the above-mentioned viral vector, those lacking replication ability are preferable so that they cannot self-replicate in infected cells. By inserting a nucleic acid encoding TCR or CAR that specifically recognizes WT1 into an appropriate viral vector by a conventional method, the nucleic acid can be contained in a viral vector particle.

The present invention provides a pharmaceutical composition for treating meningioma comprising a nucleic acid encoding TCR or CAR that specifically recognizes WT1-expressing cells as an active ingredient together with a pharmaceutically acceptable excipient. Suitable excipients are well known to those skilled in the art and include, for example, phosphate buffered saline (eg, 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4). , Hydrochlorides, hydrobromides, phosphates, sulfates and other aqueous solutions containing mineral salts, physiological saline, solutions such as glycol or ethanol, and acetates, propionates, malonates, benzoic acids Including salts of organic acids such as salts. Adjuvants such as wetting or emulsifying agents and pH buffering agents can also be used. Pharmaceutically acceptable excipients are described in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). The composition can be in a known form suitable for parenteral administration, eg, injection or infusion. Furthermore, formulation adjuvants such as suspending agents, preservatives, stabilizers and / or dispersants, preservatives can be used to extend the shelf life during storage. The composition may be in dry form for reconstitution with a suitable sterile liquid prior to use. For microparticle-mediated administration, DNA can be coated on particles such as microscopic gold particles. When the active ingredient is a nucleic acid, the dosage is, for example, in the range of 0.001 mg to 10 mg of the nucleic acid per kg of body weight at a time. For example, when administered to a human patient, it is administered in the range of 0.001 to 50 mg for a patient weighing 60 kg. When the active ingredient is a virus vector particle, the dose is administered to a subject weighing 60 kg at a time, for example, in the range of about 1 × 10 ³ pfu to 1 × 10 ¹⁵ pfu as a virus titer. .

(2) Cells having WT1-specific cytotoxic activity In another embodiment of the present invention, the pharmaceutical composition for treating meningiomas of the present invention contains cells having WT1-expressing cell-specific cytotoxic activity as active ingredients. The cell is, for example, a cell that expresses TCR or CAR that specifically recognizes a WT1-expressing cell. Such cells can be obtained by collecting cells that specifically recognize WT1 from the living body as described in (1) above, or encode TCR or CAR that specifically recognizes WT1 described in (1) above. It can be prepared by a method such as introducing a nucleic acid into a cell, stimulating a T cell or a precursor cell thereof with an antigen-presenting cell presenting a WT1-derived peptide. When these cells bind to WT1-expressing cells via TCR or CAR, signals are transmitted into the cells and activated. Activation of cells expressing TCR or CAR varies depending on the type of host cell and intracellular domain, and can be confirmed using, for example, cytokine release, improvement of cell proliferation rate, changes in cell surface molecules, etc. as indicators. . For example, the release of cytotoxic cytokines (tumor necrosis factor, lymphotoxin, etc.) from activated cells results in the destruction of meningioma cells that express WT1. In addition, other immune cells such as B cells, dendritic cells, NK cells, macrophages and the like are stimulated by cytokine release and changes in cell surface molecules. Accordingly, the compositions of the present invention are useful in adoptive immunotherapy.

  The introduction of a nucleic acid encoding TCR or CAR to prepare the cells is performed ex vivo in humans. As a cell into which a nucleic acid is introduced, a mammal, for example, a cell derived from a human or a cell derived from a non-human mammal such as monkey, mouse, rat, pig, cow or dog can be used. As the cell type, for example, cells collected, isolated, purified, or derived from blood (peripheral blood, umbilical cord blood, etc.), body fluid such as bone marrow, tissue or organ can be used. PBMC, immune cells [dendritic cells, B cells, hematopoietic stem cells, macrophages, monocytes or NK cells, hematopoietic cells (neutrophils, basophils, monocytes)], hematopoietic stem cells, cord blood mononuclear cells, fibers Blast cells, preadipocytes, hepatocytes, blood cells, skin keratinocytes, mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, various cancer cell lines, or neural stem cells can be used. In the present invention, it is particularly preferable to use T cells, T cell progenitor cells (hematopoietic stem cells, lymphocyte progenitor cells, etc.) or cell populations containing these. T cells include CD8 positive T cells or CD4 positive T cells, regulatory T cells, cytotoxic T cells, and tumor infiltrating lymphocytes. Cell populations containing T cells and T cell progenitor cells include PBMC. The cells may be collected from a living body, expanded, or established as a cell line. When it is desired to transplant a cell into which a nucleic acid is introduced or a cell differentiated from the cell, it is preferable to introduce the nucleic acid into a cell collected from the living body itself or the same kind of living body.

A nucleic acid encoding TCR or CAR that specifically recognizes a WT1-expressing cell can be inserted into the vector and the vector introduced into the cell. For example, retrovirus vectors (including oncoretrovirus vectors, lentivirus vectors, pseudotype vectors), adenovirus vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors or Sendai virus vectors, Epstein-Bar Viral vectors such as viral (EBV) vectors and herpes simplex virus (HSV) vectors can be used. As the above-mentioned viral vector, those lacking replication ability are preferable so that they cannot self-replicate in infected cells.
When a nucleic acid encoding a TCR that specifically recognizes a WT1-expressing cell is introduced into a T cell, as described in (1) above, the expression of an endogenous TCR α chain and a TCR β chain that the T cell originally expresses is expressed. By suppressing with siRNA, T cells that express TCR that recognizes WT1-expressing cells at a high ratio can be obtained.

  Also described in liposomes and WO 96/10038 pamphlet, WO 97/18185 pamphlet, WO 97/25329 pamphlet, WO 97/30170 pamphlet and WO 97/31934 pamphlet. Non-viral vectors can also be used in the present invention in combination with condensing agents such as cationic lipids. Furthermore, the nucleic acid of the present invention can be introduced into cells by calcium phosphate transfection, DEAE-dextran, electroporation, or particle bombardment.

  For example, when a retroviral vector is used, an appropriate packaging cell can be selected based on the LTR sequence and the packaging signal sequence possessed by the vector, and retroviral particles can be prepared and used. it can. For example, PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP + E-86 and GP + envAm-12 (US Pat. No. 5,278,056), Psi-Crip [American Academy of Sciences, Vol. 85, No. 6460-6464] Page (1988)] is exemplified. Retroviral particles can also be produced using 293 cells or 293T cells with high transfection efficiency. Retrovirus vectors produced based on many types of retroviruses and packaging cells that can be used for packaging the vectors are widely available from various companies.

Cells having cytotoxic activity specific for WT1-expressing cells may be cultured and / or stimulated using an appropriate medium and / or stimulating molecule prior to administration to a subject. Stimulatory molecules include cytokines, appropriate proteins, and other components. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, IFN-γ and the like, and a medium containing IL-2 is preferably used. Although there is no limitation in particular as a density | concentration in the culture medium of IL-2, For example, it is 0.01-1 * 10 < ⁵ > U / mL suitably, More preferably, it is 1-1 * 10 < ⁴ > U / mL. Examples of suitable proteins include CD3 ligand, CD28 ligand, and anti-IL-4 antibody. In addition, a lymphocyte stimulating factor such as lectin can also be added. Further, serum or plasma may be added to the medium. The amount added to these media is not particularly limited, but is 0 to 20% by volume, and the amount of serum or plasma used can be changed depending on the culture stage. For example, the serum or plasma concentration can be decreased in stages and used. The origin of serum or plasma may be either self (meaning that the origin is the same as the cell being cultured) or non-self (meaning that the origin is different from the cell being cultured), but preferably Self-derived ones are used from the viewpoint of safety.

The cell culture equipment used for cell culture is not particularly limited, and for example, a petri dish, a flask, a bag, a large culture tank, a bioreactor, or the like can be used. As the bag, a CO ₂ gas permeable bag for cell culture can be used. Moreover, when manufacturing a large cell population industrially, a large culture tank can be used. Moreover, although culture | cultivation can be implemented by any of an open system and a closed system, it is preferable to culture | cultivate by a closed system from a viewpoint of the safety | security of the cell population obtained suitably.

In the present invention, a pharmaceutical composition containing, as an active ingredient, a cell having cytotoxic activity specific to WT1-expressing cells is used parenterally administered to a subject. Parenteral administration methods include methods such as intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. In order to enhance the antitumor action, it can also be administered to meningiomas or nearby tissues, such as subcutaneously. The dose is appropriately selected according to the state, weight, age, etc. of the subject, but usually, the number of cells is about 10 ⁷ to 10 ⁹ cells per time for a subject weighing 60 kg, preferably About 5 × 10 ^{7 to} 5 × 10 ⁸ cells are administered. The pharmaceutical composition of the present invention can be administered once or multiple times. The pharmaceutical composition of the present invention can be in a known form suitable for parenteral administration, for example, injection or infusion. The pharmaceutical composition of the present invention may optionally contain a pharmacologically acceptable excipient. Examples of the pharmacologically acceptable excipient include those described in (1) above. The pharmaceutical composition of the present invention may also contain physiological saline, phosphate buffered saline (PBS), a medium and the like in order to stably maintain the cells. Although it does not specifically limit as a culture medium, Media, such as RPMI, AIM-V, and X-VIVO10, are generally mentioned.

(3) Receptor specifically recognizing WT1 In another embodiment of the present invention, the pharmaceutical composition for treating meningiomas of the present invention contains a receptor specifically recognizing WT1-expressing cells as an active ingredient. Examples of the receptor include TCR and CAR. The TCR or CAR can be produced using the nucleic acid (1) or the cell (2). Therefore, in a further aspect of the present invention, the pharmaceutical composition for meningioma treatment of the present invention contains TCR or CAR specifically recognizing WT1-expressing cells as an active ingredient.

The administration route of the pharmaceutical composition containing the receptor as an active ingredient may be oral administration or parenteral administration, but parenteral administration such as intramuscular administration, subcutaneous administration, intravenous administration and intraarterial administration is preferable. In order to enhance the antitumor action, it can also be administered to meningioma or nearby subcutaneous. The dose may be any amount that is effective for treatment, and is appropriately selected according to the size and symptoms of the tumor. Usually, the effective amount of polypeptide per day for the subject is 0. The dose is 0001 μg to 1000 μg, preferably 0.001 μg to 1000 μg, and can be administered once or divided into several times. Preferably, it is divided into several times and administered every few days to several months.
A pharmaceutical composition containing the above receptor as an active ingredient is appropriately mixed with pharmacologically acceptable excipients, adjuvants such as wetting agents or emulsifiers, pH buffering agents, suspending agents, preservatives, stabilizing agents. And / or may contain formulation aids such as dispersants. Examples of the pharmacologically acceptable excipient include those described in (1) above. The pharmaceutical composition of the present invention may be, for example, an oral dosage form such as a tablet, powder or liquid, or a parenteral dosage form such as an injection or infusion. Moreover, the dry form for reconstitution with a suitable aseptic liquid before use may be sufficient.

(4) WT1 and WT1-derived peptide In another aspect of the present invention, the pharmaceutical composition for meningioma treatment of the present invention contains WT1 or a WT1-derived peptide as an active ingredient. In the present invention, natural WT1 expressed by an organism not genetically modified or recombinant WT1 prepared by genetic recombination technology can be used. Furthermore, a variant of WT1 can be used. A variant is a polypeptide that differs from a native protein in substitutions, deletions, additions and / or insertions of one or more, preferably no more than 10, amino acids so that the immunogenicity of the protein is retained. For example, WT1 variants are substituted with 1-3 amino acid residues in the immunogenic portion, resulting in improved response to antigen-specific antisera and / or T cell lines compared to native proteins. ing. In particular, it is preferable that the binding property to MHC (major histocompatibility complex) class I molecule or MHC class II molecule is improved. WT1 or WT1-derived peptides are taken up by antigen-presenting cells, and then individual WT1-derived peptides generated by intracellular degradation bind to MHC molecules to form complexes, and these complexes are formed on the surface of antigen-presenting cells. Presented in high density. CTL specific for this complex proliferate and induce cytotoxicity against WT1-expressing cells.

A WT1-derived peptide is a peptide comprising an immunogenic portion of WT1 and of any length comprising a WT1-derived sequence of at least 9 amino acids. For example, it is 9 to 400 amino acids, preferably 9 to 200 amino acids, more preferably 9 to 50 amino acids, and particularly preferably 9 to 25 amino acids. A WT1-derived peptide is a peptide that is recognized and specifically bound by the TCR and binds to an MHC class I molecule or an MHC class II molecule. The binding of these molecules to WT1-derived peptides can be detected using any assay known in the art, for example, the ability of the peptides to bind to MHC class I is ¹²⁵ I labeled β2 microglobulin. The ability to promote the incorporation of (β2m) into the MHC class I / β2m / peptide heterotrimeric complex can be monitored or assessed by peptide competition assays. For example, amino acid numbers 6-22, amino acid numbers 117-139, amino acid numbers 244-262, amino acid numbers 244-252, amino acid numbers 235-243, amino acid numbers 136-144 of human WT1 (SEQ ID NO: 14) as WT1-derived peptides, Peptides having amino acid numbers 225 to 233, amino acid numbers 10 to 18, amino acid numbers 126 to 134, amino acid numbers 37 to 45, and amino acid numbers 37 to 46 can be used.

  WT1 or a WT1-derived peptide can be linked with a signal sequence or a linker sequence for enhancing binding to a solid support at the N-terminus thereof. For example, FLAG, 6 × His consisting of 6 His (histidine) residues, 10 × His, influenza agglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag HSV-tag, E-tag, SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment, GST (glutathione-S-transferase), immunoglobulin constant region (Fc), β -Known peptides such as galactosidase and MBP (maltose binding protein) can be bound. The protein or peptide may be prepared by treating the fusion protein thus prepared with an appropriate protease and recovering the target peptide.

  WT1 or WT1-derived peptides can be prepared from the nucleic acids that encode them. For example, it can be prepared by a suitable host cell transformed with any expression vector containing a nucleic acid encoding recombinant WT1 or a WT1-derived peptide. Suitable host cells can be prokaryotic cells, yeast cells and higher eukaryotic cells. For example, E. coli, yeast cells or mammalian cells (eg COS or CHO) can be used. Naturally expressed WT1 and WT1 or WT1-derived peptides produced by host cells are concentrated using a filter and purified using an appropriate purification matrix (eg, an affinity matrix or ion exchange resin). May be.

  WT1 or a WT1-derived peptide can be obtained by synthesis based on its amino acid sequence. Peptide synthesis can be performed according to the methods used for chemical synthesis of ordinary peptides. Commonly used synthetic methods include, for example, Peptide Synthesis, Interscience Publishing, (1966), The Proteins, Volume 2, Academic Press Inc. Publication (1976), peptide synthesis, Maruzen Co., Ltd. (1975), peptide synthesis basics and experiments, Maruzen Co., Ltd. (1985), International Publication No. WO99 / 67288, etc.

  The pharmaceutical composition for meningioma treatment containing WT1 or a WT1-derived peptide as an active ingredient can contain a pharmacologically acceptable carrier or excipient so that cellular immunity is effectively established. Furthermore, a delivery system for transporting proteins and peptides can be used. For example, non-specific immune response enhancers such as adjuvants or liposomes can be utilized to enhance the level of T cell responses to WT1 or WT1-derived peptides. As the adjuvant, those described in the literature [Clinical Microbiological Review (Clin. Microbiol. Rev.), Vol. 7, pp. 277-289 (1994)] can be used. Examples include body-derived components, cytokines, plant-derived components, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin and pluronic polyol, polyanions, peptides, and oil emulsions (emulsion formulations). In addition, the pharmaceutical composition of the present invention may be a liposome preparation, a particulate preparation bound to beads having a diameter of several μm, a preparation bound to lipid, and the like.

  As a method for administering a pharmaceutical composition for treating meningiomas containing WT1 or a WT1-derived peptide as an active ingredient, oral administration, intradermal administration, subcutaneous administration, intravenous injection, etc. can be used. Systemic administration, meningioma or the vicinity It may be administered locally to any tissue, for example subcutaneously. The pharmaceutical composition of the present invention may be, for example, an oral dosage form such as a tablet, powder or liquid, or a parenteral dosage form such as an injection or infusion. Moreover, the dry form for reconstitution with a suitable aseptic liquid before use may be sufficient. The dose of protein or peptide can be appropriately adjusted depending on the age, weight, administration method, etc. of the subject, but is usually 0.001 mg to 1000 mg, preferably 0.01 mg to 100 mg, more preferably for a subject with a body weight of 60 kg. It is 0.1 mg to 10 mg, and can be administered once or divided into several times. Preferably, it is divided into several times and administered every few days to several months. A person skilled in the art can appropriately select an appropriate dose. The compositions of the invention can also be administered as part of a sustained release formulation (a formulation such as a capsule or sponge that provides a slow release of the active ingredient after administration). Such formulations can be prepared using well known techniques and can be administered, for example, orally, rectally, subcutaneously, or by implantation at the desired target site.

(5) Cells presenting WT1-derived peptides In another aspect of the present invention, the pharmaceutical composition for treating meningiomas of the present invention contains cells presenting WT1-derived peptides (antigen-presenting cells) as active ingredients. The antigen-presenting cell may be an antigen-presenting cell on which a complex of the peptide described in (4) and an MHC molecule (HLA antigen) is presented. For such antigen-presenting cells, cells having antigen-presenting ability are isolated from the living body, and WT1 or WT1-derived peptide is pulsed outside the body, or a vector expressing WT1 or WT1-derived peptide is introduced into the cell. And can be prepared by presenting it on the cell surface. The “cell having antigen-presenting ability” is not particularly limited as long as it is a cell (macrophage, B-cell, etc.) expressing an MHC molecule capable of presenting a WT1-derived peptide on the cell surface. Dendritic cells that are considered high are particularly preferred. When using dendritic cells, for example, lymphocytes are separated from the peripheral blood of a living body by Ficoll method, then non-adherent cells are removed, and adherent cells are cultured in the presence of GM-CSF and IL-4 to form dendritic cells. Antigen-presenting cells can be prepared by inducing cells and culturing and pulsing the dendritic cells with peptides. The antigen-presenting cell is useful for inducing a cell expressing TCR that specifically recognizes WT1 described in (2) above from a cell population containing T cells.

In the present invention, a pharmaceutical composition containing cells presenting a WT1-derived peptide as an active ingredient is used parenterally administered to a subject. The pharmaceutical composition of the present invention can be in a known form suitable for parenteral administration, for example, injection or infusion. The pharmaceutical composition of the present invention may optionally contain a pharmacologically acceptable excipient. Examples of the pharmacologically acceptable excipient include those described in (1) above. The pharmaceutical composition of the present invention preferably contains physiological saline, phosphate buffered saline (PBS), a medium and the like in order to stably maintain antigen-presenting cells. Although it does not specifically limit as a culture medium, Media, such as RPMI, AIM-V, and X-VIVO10, are generally mentioned. Parenteral administration methods include methods such as intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. In order to enhance the antitumor action, it can also be administered to meningiomas or nearby tissues, such as subcutaneously. Furthermore, it can administer with an immunostimulant, an immunomodulator, etc. The dose is appropriately selected according to the condition, body weight, age, etc. of the subject. Usually, the number of cells is about 10 ⁷ to 10 ⁹ cells per subject, preferably 5 for a subject weighing 60 kg. About 10 ^{7 to} 5 × 10 ⁸ cells are administered. By administering antigen-presenting cells to a living body, WT1-specific CTL is efficiently activated in the living body, and cellular immunity is induced against meningioma cells.

(6) Antibody specifically recognizing WT1 In another aspect of the present invention, the pharmaceutical composition for treating meningiomas of the present invention comprises an antibody specifically recognizing WT1 or a polymorph containing an antigen recognition site of the antibody. A peptide is contained as an active ingredient. These antibodies and polypeptides may be derived from polyclonal antibodies or monoclonal antibodies. These antibodies and polypeptides specifically bind to WT1 present on the surface of meningioma cells and induce cellular immunity against meningioma cells.

  Antibodies that specifically recognize WT1 can be prepared by techniques known to those skilled in the art. For example, the WT1 or WT1-derived peptide described in (4) above is used as an antigen to inject mammals such as mice, rats, rabbits, sheep or goats to induce immunity. In particular, a relatively short peptide may be bound to a carrier protein [for example, bovine serum albumin or KLH (keyhole limpet hemocyanin)]. Immunization is performed by administering 20 to 200 μg of an antigen-adjuvant mixture at a time subcutaneously, intramuscularly or intraperitoneally in an animal. Polyclonal antibodies specific for WT1 are purified from immunized mammals periodically, and from antisera, for example, by affinity chromatography using a polypeptide coupled to a suitable solid support.

  A monoclonal antibody that specifically recognizes WT1 is produced using a hybridoma produced by a so-called cell fusion method. The hybridoma is established by fusing a population of anti-WT1 antibody-producing cells and myeloma cells to form a hybridoma, cloning the hybridoma, and selecting a clone that produces an antibody that recognizes WT1. Preparation of a monoclonal antibody from a cloned hybridoma can be achieved by using a known method, for example, a method for purifying a hybridoma in the peritoneal cavity of a laboratory animal and purifying a monoclonal antibody from ascites. Furthermore, a technique for humanizing the thus obtained animal-derived monoclonal antibody is also known.

Fragments such as F (ab ′) ₂ , Fab ′, Fab and the like obtained by allowing a proteolytic enzyme such as pepsin or papain to act on the antibody as a polypeptide containing the antigen recognition site of the antibody and removing the Fc part of the antibody Can be used. Furthermore, based on the obtained monoclonal antibody, a recombinant antibody produced by genetic engineering, a chimeric antibody in which the constant region is substituted with the constant region of another antibody, a bispecific antibody (bivalent antibody), scFv , Fab ₃ , Diabody, Tribody, Tetrabody, Minibody, Bis-scFv, (scFv) ₂ -Fc, intact-IgG can also be used. These polypeptides are described in detail in Nature Biotechnology, Vol. 23, No. 9, pages 1126-1136 (2005).
Furthermore, the antigen recognition site of the antibody can be used as the extracellular domain of CAR described in (1) above.

  A pharmaceutical composition containing an antibody that specifically recognizes WT1 or a polypeptide containing an antigen recognition site of the antibody, if necessary, an appropriate pharmaceutically acceptable carrier that is inert to the polypeptide, It can be formulated by mixing with a medium or the like. Examples of the carrier or medium include sterilized water and physiological saline, stabilizers, excipients, antioxidants (such as ascorbic acid), buffers (such as phosphoric acid, citric acid, and other organic acids), preservatives, Surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), binders and the like can be mentioned. In addition, other low molecular weight polypeptides, serum albumin, proteins such as gelatin and immunoglobulin, amino acids such as glycine, glutamine, asparagine, arginine and lysine, sugars and carbohydrates such as polysaccharides and monosaccharides, and sugars such as mannitol and sorbitol Alcohol may be included. In the case of an aqueous solution for injection, for example, isotonic solutions containing physiological saline, glucose and other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and appropriate dissolution You may use together with adjuvants, such as alcohol (ethanol etc.), polyalcohol (propylene glycol, PEG, etc.), nonionic surfactant (polysorbate 80, HCO-50), etc.

  If necessary, the antibody of the present invention or a polypeptide containing the antigen recognition site thereof may be enclosed in microcapsules (microcapsules such as hydroxymethylcellulose, gelatin, poly [methylmethacrylic acid], etc.), and a colloid drug delivery system ( Liposomes, albumin microspheres, microemulsions, nanoparticles, nanocapsules, etc.). Furthermore, a method of using a drug as a sustained-release drug is also known and can be applied to the pharmaceutical composition of the present invention.

  The dosage of the pharmaceutical composition containing an antibody specifically recognizing WT1 or a polypeptide containing the antigen recognition site of the antibody depends on the type of dosage form, administration method, age and weight of the subject, patient symptom, disease In consideration of the type and the degree of progression, etc., the final decision will be made as appropriate according to the judgment of the doctor. In general, for a subject weighing 60 kg, 0.1 to 2000 mg per day is 1 to several times. Can be administered separately. More preferred is 1-1000 mg / day, even more preferred is 50-500 mg / day, and most preferred is 100-300 mg / day. These doses vary depending on the body weight and age of the subject, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. The administration period is also preferably determined as appropriate according to the course of healing of the subject.

  It is also conceivable to perform gene therapy by incorporating a nucleic acid encoding an antibody that specifically recognizes WT1 or a polypeptide containing an antigen recognition site of the antibody into a gene therapy vector. As a method for administering these nucleic acids, in addition to direct administration with a naked plasmid, it can be packaged in liposomes, or introduced into cells using the viral vector described in (2) above, or colloidal gold particles, etc. The bead carrier can be coated and administered. The nucleic acid administration method, dosage, excipient that can be administered simultaneously, and the like are the same as those of the nucleic acid encoding a receptor that specifically recognizes WT1 in (1) above.

The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to the following examples.
Among the operations described in this specification, the basic operations are described in 2001, issued by Cold Spring Harbor Laboratory. Edited by T. Maniatis et al., Molecular Cloning: The method described in A Laboratory Manual 3rd edition (Molecular Cloning: A Laboratory Manual 3rd ed.).

Example 1 WT1 expression in meningiomas 29 samples of meningioma cells collected from meningioma patients with informed consent, and malignant meningioma cell lines HKBMM cells, Ktelmgl cells, IOMM-Lee cells Prepared. RNA was extracted from each cell using a kit (Ambion). Subsequently, cDNA was prepared from each extracted RNA using a Transscriptor First Strand cDNA Synthesis kit (Roche). Similarly, cDNA was also prepared from Human Total RNA Master Panel II (Clontech) as a normal brain RNA sample. The prepared cDNA was stored in a freezer at -20 ° C.

  The expression of the WT1 gene was quantified using a LightCycler (registered trademark) 480 Probe Master (Roche). 20 μL of a reaction solution containing each cDNA, a primer (SEQ ID NO: 15 and 16) having a final concentration of 0.5 μM, and a 0.2 μM probe (SEQ ID NO: 17) was prepared, and PCR was performed using a LightCycler (registered trademark) 480 system. As an internal control, Universal ProbeLibrary Human GAPD Gene Assay (manufactured by Roche) was used, and relative WT1 expression levels were compared by a comparative Ct method (ΔΔCt method). The relative value of the WT1 expression level in each sample when the expression level of WT1 in the normal brain is 1 is shown in FIG. In FIG. 1, the vertical axis represents the relative value of WT1 to normal brain expression, and the horizontal axis represents the sample number and meningioma cell line name. As shown in FIG. 1, in meningioma cells and meningioma-derived cell lines collected from patients, the expression level of the WT1 gene is increased several to several tens of times as compared with the expression in normal brain. I understood that.

Example 2 Production of WT1-TCRα and β Genes An J et al., International Journal of Hematology (Int. J Hematol.), Vol. 93, pp. 176-185 (2011), 235-243 of tumor antigen WT1 Nucleic acid fragments containing the HCR-A2402-restricted TCR α-chain gene (Vα20 / J33 / Cα) and TCRβ-chain gene (Vβ5.1 / J2.1 / Cβ2), which recognize the peptide of HLA-A2402, were prepared. Next, the base sequences encoding the amino acid sequences of the C region shown in SEQ ID NOs: 3 and 6 of the TCR α chain gene thus obtained are changed to the base sequences of SEQ ID NOs: 2 and 5, respectively. Converted. Thus, a codon-converted WT1-TCR α-chain gene encoding the same amino acid sequence as the wild-type TCR gene but having a different base sequence encoding the C region was prepared. Similarly, codon conversion in which the base sequences encoding the amino acid sequences of the C region shown in SEQ ID NOs: 9 and 12 of the TCR β chain gene (SEQ ID NOs: 7 and 10 respectively) were converted to the base sequences of SEQ ID NOs: 8 and 11, respectively. A β-chain gene of the type WT1-TCR was prepared. The details of the above base sequence conversion are shown in Table 1. In addition, an artificial gene shown in SEQ ID NO: 13 was synthesized. This artificial gene transcribes four types of single-stranded RNA that forms a stem-loop structure. These single-stranded RNAs generate siRNAs that suppress the expression of the wild-type TCR α chain and wild-type TCR β chain genes targeting the nucleotide sequences of SEQ ID NOs: 1, 4, 7, and 10 in the cell, respectively. The amino acid sequence encoded by the wild type and the codon-converted base sequence is the same, but since a mutation has been introduced into the base sequence of the codon-converted TCR, a double strand transcribed from the DNA described in SEQ ID NO: 13 The expression of codon-converted TCR is not suppressed by siRNA.

Example 3 Preparation of WT1-TCR Expression Retroviral Vector Using pMSCVneo (Clontech) as a template, PCR was performed using a primer added with a restriction enzyme XhoI recognition sequence and a primer added with a restriction enzyme EcoRI recognition sequence. The MSCV 3 ′ LTR site was amplified and cut with XhoI and EcoRI. The cleaved DNA fragment was cloned into the XhoI-EcoRI site of the pMT vector [pMT vector described in Gene Ther, Vol. 11, pages 94-99 (2004)] to prepare pMS. Next, pDON-5 vector (manufactured by Takara Bio Inc.) was cleaved with restriction enzymes MluI and NotI, and the untranslated region of human EF1α was excised and cloned into the MluI-NotI site of the pMS vector to obtain pMS3.

A nucleic acid fragment containing the codon-converted WT1-TCR α-chain gene prepared in Example 2 and a nucleic acid fragment containing an artificial gene that generates siRNA were cloned into the ApaI-NotI site of pMS3, and pMS3-WT1-A- si was obtained.
PCR was performed using Mouse Genomic DNA (manufactured by Takara Bio Inc.) as a template and primers with restriction enzyme MluI, NotI and BglII recognition sequences and primers with restriction enzyme XhoI, NotI and BamHI recognition sequences. Went. A nucleic acid fragment was prepared by cleaving the amplified DNA fragment containing the obtained PGK promoter sequence with NotI and BamHI.
Finally, the nucleic acid fragment containing the codon-converted WT1-TCR β-chain gene prepared in Example 2 and the nucleic acid fragment containing the PGK promoter sequence were cloned into the NotI-XhoI site of pMS3-WT1-A-si. The plasmid vector pMS3-WT1-siTCR was obtained. The structure between LTRs of pMS3-WT1-siTCR is shown in FIG.

Example 4 Preparation of WT1-TCR-expressing retrovirus solution and infection of human PBMC E. coli JM109 was transformed with pMS3-WT1-siTCR prepared in Example 3 to obtain a transformant. The plasmid DNA retained by this transformant was purified using QIAGEN Plasmid Midi Kit (manufactured by Qiagen) and subjected to the following operation as transfection DNA.
The prepared pMS3-WT1-siTCR was transfected into 293T cells. For this operation, Retrovirus Packaging Kit Eco (manufactured by Takara Bio Inc.) was used according to the product protocol. A supernatant containing ecotropic virus was obtained from the obtained transduced cells, and filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore). Using this supernatant, ecotropic virus was infected to PG13 cells (ATCC CRL-10686) by a method using polybrene. The obtained cells were cultured, and the culture supernatant was collected. The supernatant was filtered through a 0.45 μm filter, and the WT1-TCR expression retrovirus solution obtained was designated as MS3-WT1-siTCR.
Peripheral blood mononuclear cells (PBMC) isolated from human peripheral blood from which informed consent was obtained, MS3-WT1-siTCR was applied twice by a standard method using RetroNectin (registered trademark, manufactured by Takara Bio Inc.). WT1-TCR-introduced cells were prepared by infection and the cells were collected.

Example 5 Intracellular Cytokine Measurement The WT1-TCR-introduced cells prepared in Example 4 were measured for T cell phenotype and intracellular IFN-γ synthesis by the simultaneous flow cytometry evaluation method. First, WT1-TCR-introduced cells prepared in Example 4 were suspended in RPMI-1640 culture solution (manufactured by SIGMA) at a concentration of 1 × 10 ⁶ cells / mL. This suspension is a HLA-A0207 meningioma cell line seeded in a 24-well cell culture plate (manufactured by FALCON) at 0.5 mL / well and suspended therein at a concentration of 1 × 10 ⁶ cells / mL. Ktelmgl cells or IOMM-Lee cells, which are HLA-A2402 meningioma cell line, were added at 0.5 mL / well, respectively. As a control, PBMC (WT1-TCR non-introduced cells) not infected with virus was used instead of WT1-TCR-introduced cells, and the same operation was performed. These plates were cultured at 37 ° C. in a 5% CO ₂ incubator (culture 0 hour). After 1 hour of culture, BD GolgiStop (manufactured by BD PharMingen) was added to each well, and the culture was further continued. After 8 hours of culture, the cells were collected and washed twice with FACS buffer (phosphate buffered saline containing 1% FCS and 0.1% NaN ₃ ). Fluorescein isothiocyanate (FITC) -conjugated anti-CD8 antibody (BD PharMingen) and allophycocyanin (APC) -conjugated anti-IFN-γ antibody (BD PharMingen) were added to the washed cells and stained on ice for 30 minutes. Subsequently, intracellular cytokine staining was performed using a Cytofix / Cytoperm kit (BD PharMingen). These were analyzed with a flow cytometer (BD FACScanto II, manufactured by BD Biosciences). The result is shown in FIG. In FIG. 3, (a) shows Ktelmgl cells and WT1-TCR non-introduced cells (NGMC), (b) shows Ktelmgl cells and WT1-TCR-introduced cells (GMC), and (c) shows IOMM-Lee cells and WT1-TCR non-introduced cells. Introduced cells, (d) are the results of co-culture of IOMM-Lee cells and WT1-TCR-introduced cells. The vertical axis represents the amount of IFN-γ, and the horizontal axis represents the amount of CD8. As shown in FIG. 3- (d), intracellular expression of IFN-γ was observed in CD8 positive T cells by co-culture of IOMM-Lee cells and WT1-TCR-introduced cells. On the other hand, intracellular expression of IFN-γ was not observed in the co-culture of the combinations of FIGS. 3- (a) to (c). This showed that WT1-TCR-introduced cells specifically produced IFN-γ for HLA-A2402-positive meningioma cells.

Example 6 Cytotoxic activity of WT1-TCR-introduced cells against meningioma cells Meningioma cell line Ktelmgl cells or IOMM-Lee cells were washed 3 times with RPMI 1640 medium without fetal calf serum (FCS), and 1 × 10 ^6. The cells were each suspended in 1 mL of RPMI 1640 medium containing 10% FCS. To these cell suspensions, a 2.5 mM Calcein-AM (manufactured by Dojindo Laboratories) solution suspended in 10 μL of DMSO was added and labeled at 37 ° C. for 1 hour. The obtained meningioma cell line was used as a target cell for measurement of cytotoxic activity.

  Target of Ktelmgl cell or IOMM-Lee cell comprising WT1-TCR-introduced cell or WT1-TCR non-introduced cell (effector cell) prepared in Example 4 and the labeled target cell and 20 times its amount of unlabeled cell The cells were suspended in RPMI 1640 medium containing 10% FCS in a ratio of 50: 1, 25: 1, 12: 1, 6: 1, 3: 1, 1: 1, and 100 μL of the suspension was added to a 96-well V-bottom plate. Put in well. After reacting at 37 ° C. for 5 hours, the supernatant was collected by centrifugation, and the fluorescence intensity of the supernatant was measured with a fluorescence plate reader (ex: 485, em: 538 nm). From the measured fluorescence intensity, the specific cytotoxic activity was calculated according to the following formula:

Cytotoxic activity (%) = [sample (fluorescence intensity) −spontaneous emission (fluorescence intensity)] / [complete emission (fluorescence intensity) −spontaneous emission (fluorescence intensity)] × 100

  In the above formula, spontaneous release is the fluorescence intensity in a well to which no effector cells are added, and indicates the amount of spontaneous release from target cells. Moreover, complete release refers to the fluorescence intensity when Triton X-100 is added to target cells and destroyed.

  The result is shown in FIG. The horizontal axis represents the effector cell number / target cell number ratio (E / T ratio), and the vertical axis represents the cytotoxic activity (%). WT1-TCR-introduced cells show cytotoxic activity against IOMM-Lee cells, an HLA-A2402-positive meningioma cell line, and cytotoxic activities against Ktelmgl cells, an HLA-A0207 meningioma cell line There wasn't. Non-introduced cells did not show cytotoxic activity against IOMM-Lee cells and Ktelmgl cells. From the above results, it was revealed that TCR that specifically recognizes WT1 imparts PBMC with specific cytotoxic activity against meningioma cells according to its HLA restriction.

Example 7 Therapeutic effect of WT1-TCR-introduced cells on meningioma model mice To prepare IOMM-Lee cells expressing green fluorescent protein (GFP), pRetroQ-AcGFP-C1 Vector (manufactured by Clontech) was used. The GFP gene was introduced into IOMM-Lee cells to prepare IOMM-Lee-GFP cells.
NOD. A malignant skull base meningioma mouse model was prepared by transplanting IOMM-Lee-GFP cells to the skull base of Cg-Prkdcscid Il2rggtSug / Jic (NOG) mouse (manufactured by Central Institute for Experimental Animals). After reaching 2-5 mm (about 5 days after transplantation), treatment was started using the WT1-TCR-introduced cells and non-introduced cells prepared in Example 4. Each cell 5 × 10 ⁷ cells / mouse was administered into the tail vein of 10 mice.

On the 12th day after transplantation of IOMM-Lee-GFP cells, the mouse head was fixed with 10% neutral buffered formalin (manufactured by Wako Pure Chemical Industries, Ltd.) for 48 hours. Tumor size was calculated from the fluorescence region. The calculated tumor size is shown in FIG. As shown in FIG. 5, administration of WT1-TCR-introduced cells was shown to suppress tumor growth in a mouse model of malignant skull base meningioma.
In addition, the mouse head fixed with formalin was decalcified with Decalifying Solution B (manufactured by Wako Pure Chemical Industries) for 96 hours and embedded in paraffin to prepare a 5 μm continuous sliced specimen. An anti-human CD8 antibody (manufactured by MBL), Vector M. O. M.M. Immunohistochemical staining was performed using an Immunodetection Kit (manufactured by Vector Laboratories). CD8 positive cells infiltrating normal tissues (oral mucosa and submucosa soft tissue) and CD8 positive cells infiltrating the tumor were measured using a microscope. The result is shown in FIG. As shown in FIG. 6, there was no difference between CD8 positive cells infiltrating normal tissues in the WT1-TCR introduced cell administration group and the non-introduced cell administration group. In contrast, there were more CD8 positive cells infiltrating tumor cells in the WT1-TCR-introduced cell administration group than in the non-introduced cell administration group.
Furthermore, the survival rate of each mouse was followed up to the 28th day after IOMM-Lee-GFP cell transplantation. The result is shown in FIG. The horizontal axis represents the number of days after transplantation, and the vertical axis represents the survival rate. As shown in FIG. 7, administration of WT1-TCR-introduced cells was shown to prolong survival in a malignant skull base meningioma mouse model.

  The present invention provides a pharmaceutical composition for treating meningiomas useful in the treatment of meningiomas.

SEQ ID NO: 1: A part of wild type TCR alpha chain coding sequence
SEQ ID NO: 2: A part of codon modified TCR alpha chain coding sequence
SEQ ID NO: 3: A part of TCR alpha chain amino acid sequence
SEQ ID NO: 4: A part of wild type TCR alpha chain coding sequence
SEQ ID NO: 5: A part of codon modified TCR alpha chain coding sequence
SEQ ID NO: 6: A part of TCR alpha chain amino acid sequence
SEQ ID NO: 7: A part of wild type TCR beta chain coding sequence
SEQ ID NO: 8: A part of codon modified TCR beta chain coding sequence
SEQ ID NO: 9: A part of TCR beta chain amino acid sequence
SEQ ID NO: 10: A part of wild type TCR beta chain coding sequence
SEQ ID NO: 11: A part of codon modified TCR beta chain coding sequence
SEQ ID NO: 12: A part of TCR beta chain amino acid sequence
SEQ ID NO: 13: siRNA generating sequence for wild type TCR genes
SEQ ID NO: 15: WT1 forward primer
SEQ ID NO: 16: WT1 reverse primer
SEQ ID NO: 17: WT1 probe

Claims (9)

  A pharmaceutical composition for treating meningiomas comprising, as an active ingredient, a substance that exhibits cytotoxicity against WT1-expressing cells, or a substance that has an activity of inducing cytotoxicity against WT1-expressing cells.   The pharmaceutical composition for treating meningiomas according to claim 1, comprising cells having WT1-expressing cell-specific cytotoxicity as active ingredients.   The pharmaceutical composition for the treatment of meningioma according to claim 2, comprising cells expressing a T cell receptor (TCR) or a chimeric antigen receptor (CAR) that specifically recognizes WT1-expressing cells.   The pharmaceutical composition for meningioma treatment according to claim 1, comprising an active ingredient selected from WT1, WT1-derived peptide, and cells presenting WT1-derived peptide.   The pharmaceutical composition for meningioma treatment according to claim 4, wherein the cell presenting the WT1-derived peptide is an antigen-presenting cell.   The pharmaceutical composition for treating meningioma according to claim 1, comprising an active ingredient selected from a receptor that specifically recognizes WT1-expressing cells or a nucleic acid encoding the receptor.   The pharmaceutical composition for treating meningiomas according to claim 6, comprising a nucleic acid encoding TCR or CAR that specifically recognizes WT1-expressing cells.   The pharmaceutical composition according to claim 7, wherein the viral vector particle contains a nucleic acid encoding TCR or CAR that specifically recognizes WT1-expressing cells.   The pharmaceutical composition for treating meningioma according to claim 1, comprising an antibody that specifically recognizes WT1-expressing cells or a polypeptide containing an antigen recognition site of the antibody as an active ingredient.

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