Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/48—Reproductive organs
  • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
  • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

• the present invention relates to a glypican-3-specific T cell receptor, a method for producing the T cell receptor, and a medicament containing the T cell receptor.
• HCC hepatocellular carcinoma
• Immunotherapy is considered to be one of the potential treatments for HCC.
• Glypican-3 GPC3
• GPC3 Glypican-3
• CAR human chimeric antigen receptor
• Patent Document 1 a treatment method using a GPC3-specific antibody and a human chimeric antigen receptor (CAR) targeting GPC3 has been reported.
• Patent Document 2 a T cell receptor (TCR) specific to the HLA-A02-restricted GPC3 367-375 peptide has also been reported (Patent Document 2).
• Glypican-3 was isolated as a developmentally regulated transcript from rat small intestine (Non-Patent Document 1), and was later classified as a GPI-linked heparan sulfate proteoglycan with a core protein of molecular weight 69 kDa, OCT, of the glypican family. -5 (Non-patent Document 2). It has been reported that glypican 3 forms a protein-protein complex with insulin-like growth factor-2 and regulates the activity of this growth factor (Non-Patent Document 3).
• TCR The T cell receptor
• TCR is a receptor used by T cells to recognize antigens
• TCR is composed of a dimer of an ⁇ chain and a ⁇ chain, or a ⁇ chain and a ⁇ chain.
• TCR forms a complex with CD3 molecules on the surface of T cells, recognizes antigens, and transmits stimulating signals to T cells.
• Each TCR chain has a variable region and a constant region; the constant region has a short cytoplasmic portion that penetrates the cell membrane, and the variable region exists outside the cell and binds to antigen-HLA (MHC) complexes.
• MHC antigen-HLA
• the three CDRs are called CDR1, CDR2 and CDR3, respectively.
• Non-Patent Document 4 cytotoxic T cell clones expressing various GPC3 144-152 peptide-specific TCRs into peripheral blood cells from patients vaccinated with the HLA-A02-restricted GPC3 144-152 peptide. were established from mononuclear cells (PBMC) (Non-Patent Document 4), but Non-Patent Document 1 does not disclose the TCR sequences of these CTL clones.
• PBMC mononuclear cells
• the present invention relates to a novel T cell receptor (TCR) that specifically recognizes glypican 3 (GPC3), regenerated T cells obtained by differentiating iPS cells introduced with the TCR that exhibits higher cytotoxicity, and It is an object of the present invention to provide a manufacturing method and a method for manufacturing regenerated T cells obtained by differentiating iPS cells into which the TCR has been introduced. Another object of the present invention is to provide a medicament for preventing or treating GPC3-expressing cancers and tumors using the TCR (for example, using cytotoxic T cells containing the TCR).
• GPC3 peptide EYILSLEEL, SEQ ID NO: 29
• PBMCs peripheral blood mononuclear cells
• GPC3 peptide HLA-A24-restricted GPC3 peptide
• TCRs are responsive to GPC3-expressing cancer cells, and that by introducing the gene into iPS cells and inducing differentiation, we can generate the GPC3 peptide (EYILSLEEL, SEQ ID NO: 29). ), and that these regenerated T cells exhibit extremely high cytotoxicity, leading to the completion of the present invention.
• the present invention provides the following.
• [1] As the complementarity determining region of the ⁇ chain One amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NOS: 1 to 13, One amino acid sequence selected from the group consisting of amino acid sequences in which one or several amino acids are deleted, substituted, or added in the amino acid sequences shown in SEQ ID NOs: 1 to 13, or the amino acids shown in SEQ ID NOs: 1 to 13 Contains one amino acid sequence selected from the group consisting of amino acid sequences having 90% or more identity with the sequence,
• As the complementarity determining region of the ⁇ chain One amino acid sequence selected from the group consisting of amino acid sequences shown in SEQ ID NOS: 14 to 28, One amino acid sequence selected from the group consisting of amino acid sequences in which one or several amino acids are deleted, substituted, or added in the amino acid sequences shown in SEQ ID NOs: 14 to 28, or the amino acids shown in SEQ ID NOs: 14 to 28
• a T cell receptor (TCR) comprising
• the complementarity determining region of the ⁇ chain contains one amino acid sequence selected from the group consisting of the amino acid sequences shown by SEQ ID NOs: 1 to 13, and the complementarity determining region of the ⁇ chain contains the amino acids shown by SEQ ID NOs: 14 to 28.
• the TCR according to [1] comprising one amino acid sequence selected from the group consisting of sequences.
• An expression vector comprising the nucleic acid described in [4].
• [6] A cell containing the nucleic acid according to [4].
• [7] A cell containing the vector described in [5].
• [8] The cell according to [6], wherein the cell is a lymphocyte or a pluripotent stem cell.
• the cell according to [7], wherein the cell is a CD8-positive cytotoxic T cell.
• the method for producing the cell according to [6] which comprises the step of introducing the nucleic acid according to [4] into the cell.
• [14] A T cell derived from a pluripotent stem cell, containing the nucleic acid according to [4].
• [15] T cells derived from pluripotent stem cells, containing the vector described in [5].
• [16] A medicament containing the cell according to any one of [6] to [11], [14] and [15].
• [18] [6] - [11], [14] and [15] A cell killer expressing glypican 3, which contains the cell according to any one of [14] and [15].
• a method for preventing or treating cancer in a mammal which comprises administering to the mammal an effective amount of the cells according to any one of [6] to [11], [14] and [15].
• a method for preventing or treating cancer in a mammal which comprises administering to the mammal an effective amount of the medicament according to [16].
• a method for preventing or treating cancer in a mammal which comprises administering to the mammal an effective amount of the killing agent according to [18].
• a method for preventing or treating cancer in a mammal which comprises administering to the mammal an effective amount of the CD8-positive cytotoxic T cells described in [26].
• a method for preventing or treating cancer in a mammal which comprises administering to the mammal an effective amount of the medicament according to [27].
• a method for producing CD8-positive cytotoxic T cells which comprises introducing TCR produced using the method described in [25].
• CD8-positive cytotoxic T cells produced using the method described in [33] for use in cancer prevention or treatment.
• Method of manufacturing TCR including the following steps: (1) Stimulating lymphocytes obtained from the peripheral blood of a patient administered GPC3 peptide with GPC3 peptide, (2) a step of single-cell sorting the CD137-positive activated T cells obtained by the stimulation; (3) producing PCR fragments of TCR ⁇ chain and TCR ⁇ chain genes from the single cell sorted CD137-positive activated T cells (secondary PCR); and (4) producing TCR ⁇ chain and TCR ⁇ chain genes highly responsive to GPC3 peptide.
• Step of selecting TCR ⁇ chain gene pairs [37]
• the T cell receptor of the present invention has the ability to bind to a GPC3 peptide (HLA-A24-restricted GPC3 peptide (EYILSLEEL, SEQ ID NO: 29)) or a complex between the peptide and an HLA-A molecule (HLA-A24). Furthermore, by using the method for producing T cell receptors of the present invention, T cell receptors can be produced more efficiently and in a shorter period of time than conventional methods. Furthermore, since the nucleic acid encoding the T cell receptor can impart cytotoxic activity to T cells against cells presenting HLA-A molecules and GPC3 peptides, it is useful for the prevention or treatment of cancers and tumors that express GPC3. Useful.
• FIG. 1 shows the frequency of GPC3 peptide-specific cytotoxic T cells (CTL) observed over time in liver cancer patients who received an HLA-A24-restricted GPC3 peptide vaccine.
• the frequency of GPC3 peptide-specific cytotoxic T cells (CTL) was measured by ELISPOT assay as the frequency of IFN ⁇ -producing cells after GPC3 peptide vaccine stimulation. Specimens at the points indicated by arrows (P0102, P0103, P0201 and P0202) were measured.
• Figure 2 shows the process of GPC3-responsive TCR isolation.
• FIG. 3 shows that PCR fragments of TCR ⁇ chain and TCR ⁇ chain genes with transcriptional activity were introduced into the NF-AT reporter-introduced Jurkat cell line, and the cells were co-cultured with COS cells expressing HLA-A24 to which GPC3 peptide had been added.
• the results of intensity analysis of antigen-specific TCR signals obtained by measuring the NF-AT reporter activity of the Jurkat cell line are shown below.
• GPC3 peptide-specific TCR signals were observed in regenerated T cells expressing P0103_TCR_10, P0103_TCR_12, P0103_TCR_18, P0103_TCR_20, and P0103_TCR_82.
• FIG. 4 shows a schematic diagram (A) of a plasmid encoding a transposon vector and a list (B) showing the positions of each component.
• Figure 5 shows the expression of tracer gene CD19 in iPS cells.
• a piggyBac vector encoding the HLA-A24-restricted GPC3 peptide vaccine-responsive TCR ⁇ chain and TCR ⁇ chain was introduced into 1 ⁇ 10 6 FF-I01s04 iPS cells together with a plasmid vector encoding transposase using a gene introduction device (MaxCyte ATx). It was introduced using Gene introduction was carried out by suspending cells in 50 ⁇ L of gene transfer buffer, using a total DNA amount of 320 ⁇ g/mL, a piggyBac vector/transposase vector ratio of 1/3, and introduction conditions of Optimization 8.
• FIG. 5 shows the expression of the tracer gene CD19 in iPS cells 8 days after this gene introduction.
• #1 Control indicates CD19 expression of iPS cells into which no genes were introduced
• #2 Empty indicates CD19 expression of iPS cells into which piggyBac vector containing only dCD19, which does not incorporate TCR ⁇ chain and TCR ⁇ chain, was introduced.
• show. #3, #4, and #5 show CD19 expression in iPS cells transfected with piggyBac vectors incorporating the TCR ⁇ chain and TCR ⁇ chain of P0103_TCR_10 (TCR10), P0103_TCR_18 (TCR18), and P0103_TCR_82 (TCR82), respectively.
• FIG. 5 shows that the piggyBac vector encoding the TCR ⁇ chain and TCR ⁇ chain was efficiently introduced into iPS cells under the above conditions.
• FIG. 6 shows the method of Kaneko et al. (Kawai Y. et al., Microscopic images of embryoid body (EB) formation and hematopoietic stem cells induced according to Mol Ther. 2021;29:3027-3041) are shown.
• the embryoid body (EB) formed by culture and the blood cells (HC) that appeared around it are shown.
• Figure 7 shows the expression of T cell markers in differentiated T cells. iPS cells into which the TCR ⁇ chain and TCR ⁇ chain genes were introduced using the piggyBac vector were differentiated into T cells according to the method of Kaneko et al. (Kawai Y. et al., Mol Ther. 2021;29:3027-3041).
• FIG. 8 shows the results of a cytotoxicity test against target cells to which A24-restricted GPC3 epitope peptide was added using regenerated T cells that were differentiated into T cells after introducing P0103_TCR_10, P0103_TCR_18, and P0103_TCR_82 into iPS cells.
• the upper panel shows the cytotoxicity towards target cells when target cells with or without a fixed amount of GPC3 epitope peptide (EYILSLEEL, SEQ ID NO: 29) were co-cultured with different numbers of regenerated T cells.
• Regenerated T cells exhibited concentration-dependent cytotoxicity toward target cells to which GPC3 peptide was added. No cytotoxicity was shown against target cells to which the GPC3 peptide was not added. This indicates that regenerated T cells have antigen-specific cytotoxicity against the GPC3 epitope peptide. In the lower panel, cytotoxicity was verified by fixing the ratio of regenerated T cells to target cells at 10:1 and varying the concentration of the GPC3 epitope peptide added. From the obtained results, the cytotoxic EC50 of regenerated T cells expressing P0103_TCR_10, P0103_TCR_18, and P0103_TCR_82 was 10 nM, ⁇ 1 nM, and ⁇ 1 nM, respectively.
• FIG. 9 shows that P0103_TCR_10 (TCR#10), P0103_TCR_18 (TCR#18), and P0103_TCR_82 (TCR#82) were introduced into iPS cells and regenerated T cells differentiated into T cells were used for GPC3-expressing SK-Hep liver cancer.
• the results are shown in which the tumor growth over time was verified by measuring the chemiluminescence emitted by the tumor when the tumor was intraperitoneally administered six times to NOG mice with a tumor implanted in the peritoneal cavity.
• a cell mixture containing equal amounts of the three types of regenerated T cells described above was also administered and the progress was observed.
• mice The results of investigating the effect on the survival of NOG mice in which tumors were implanted into the peritoneal cavity when administered six times intraperitoneally are shown.
• a cell mixture containing equal amounts of the three types of regenerated T cells described above was also administered, and the progress was observed.
• Mice received regenerated T cells expressing TCR#18 and TCR#82, respectively, or a cell mixture containing equal amounts of regenerated T cells expressing TCR#18, TCR#82, and TCR#10.
• Significant survival prolongation was observed in mice. No prolonged survival was observed in mice receiving regenerated T cells expressing TCR#10.
• T Cell Receptors The present invention provides T cell receptors (also referred to as T cell receptors or TCRs) capable of binding to GPC3 peptides (HLA-A24-restricted GPC3 peptides) or complexes of the peptides and HLA-A24. . Furthermore, the TCR of the present invention may be isolated.
• T cell receptor is composed of a heterodimer of TCR chains ( ⁇ chain and ⁇ chain), and is composed of a heterodimer of TCR chains ( ⁇ chain and ⁇ chain), and is an antigen or the antigen-HLA (human leukocyte antigen) (MHC; major histocompatibility).
• Gene complex refers to a receptor that recognizes the complex and transmits a stimulating signal to T cells.
• Each TCR chain is composed of a variable region and a constant region, which are formed by somatic recombination of the V region, D region, and J region, which are arranged on the chromosome as multiple fragments. There are three complementarity determining regions (CDR1, CDR2 and CDR3).
• CDR1 and CDR2 are found in the variable (V) region of a polypeptide chain, with CDR3 comprising part of the V region, the diversity (D) region (heavy chain only) and the entire joining (J) region. Because most sequence changes associated with T cell receptors are found in the CDRs, these regions are sometimes referred to as "hypervariable regions.” Among these, CDR3 is the most variable. This is because CDR3 is encoded by VJ rearrangement in the case of a light chain, and by VDJ rearrangement in the case of a heavy chain.
• the TCR of the present invention includes not only those in which the ⁇ chain and ⁇ chain of TCR constitute a heterodimer, but also those in which the TCR constitutes a homodimer. Furthermore, the TCR of the present invention includes those in which part or all of the constant region is deleted, those in which the amino acid sequence is recombined, and those in which the TCR is made into a soluble TCR.
• soluble TCR refers to TCR that has been solubilized by chemical modification of TCR, binding to an Fc receptor, or removal of the cell membrane-spanning domain. It exists as a monodisperse heterodimer in saline solution (PBS) (2.7 mM KCl, 1.5 mM KH 2 PO 4 , 137 mM NaCl and 8 mM Na 2 PO 4 , pH 7.1-7.5) and its TCR 90% or more means the ability to remain as a monodisperse heterodimer after incubation at 25° C. for 1 hour.
• PBS saline solution
• a new disulfide bond may be artificially introduced between the constant regions of each chain in order to increase stability.
• Such a soluble TCR can be produced, for example, according to the method described in WO 2004/074322 pamphlet, Boulter et al., Clin Exp Immunol, 2005, 142(3):454-460.
• the concentration is not particularly limited as long as the TCR can bind to the antigen or the antigen-HLA complex, but for example, when used for in vitro tests, it may be 40 ⁇ g/mL or more. preferable.
• GPC3 peptide or "HLA-A24-restricted GPC3 peptide” means a peptide fragment of glypican 3 (GPC3) consisting of the amino acid sequence shown by SEQ ID NO: 29.
• the TCR of the present invention is capable of specifically recognizing and binding a complex of GPC3 peptide and HLA-A24.
• TCR of the present invention to specifically recognize and bind to the above complex can be confirmed by known methods. Suitable methods include, for example, dextramer assay or ELISPOT assay using HLA-A24 molecules and GPC3 peptide. By performing an ELISPOT assay, it can be confirmed that T cells expressing the TCR on the cell surface recognize target cells by TCR and that the signal is transmitted into the cells.
• the term "capable of binding” means “having an ability to bind" to one or more other molecules in a non-covalent manner. Refers to the ability to form binding complexes.
• the complex of the present invention include a complex between a GPC3 peptide and an HLA molecule (eg, HLA-A24), or a complex between a GPC3 peptide and a TCR.
• HLA-A24 HLA-A24
• Another example of a complex of the invention is a complex between TCR and GPC3 peptide, which itself forms a complex with HLA.
• the binding is usually with high affinity, with an affinity measured by a KD value of preferably less than 1 ⁇ M, more preferably less than 100 nM, even more preferably less than 10 nM, even more preferably 1 Less than nM, even more preferably less than 100 pM, even more preferably less than 10 pM, even more preferably less than 1 pM.
• KD or "KD value” refers to the equilibrium dissociation constant as known in the art. In the context of the present invention, these terms relate to the equilibrium dissociation constant of a TCR for a particular antigen of interest (e.g., a peptide of GPC3 as defined herein, or the respective complex of a peptide and an HLA). obtain.
• the equilibrium dissociation constant is a measure of the tendency of a complex (eg, TCR-peptide-HLA complex) to reversibly dissociate into its components (eg, TCR and peptide-HLA complex).
• Methods for determining KD values are known in the art and include, for example, surface plasmon resonance.
• isolated means a state in which a specific component (eg, TCR) is identified, separated, or recovered from components in its natural environment.
• a specific component eg, TCR
• amino acids refers to, for example, 1, 2, 3, 4, or 5 amino acids (e.g., 1 to 4 amino acids, 1 to 3 amino acids, or 1 to 2 amino acids). amino acid).
• one or several preferably means 1, 2 or 3 amino acids.
• one or several preferably means 1 to 5, 1 to 4 or 1 to 3, especially 1, 2 or 3 amino acids.
• % identity means, for example, 90% or more (e.g. 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more) high) meaning sameness.
• Amino acid sequence identity was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) (https ://blast.ncbi.nlm.nih.gov/Blast.cgi). It is understood that to determine % identity, a sequence of the invention over its entire length is compared to another sequence. In other words, % identity in the present invention excludes comparisons of short fragments (eg 1-3 residues) of a sequence of the present invention with another sequence, or vice versa.
• the complementarity determining region of the ⁇ chain of the TCR of the present invention comprises each amino acid sequence of CDR3 shown in SEQ ID NOS: 1 to 13, respectively, and the complementarity determining region of the ⁇ chain of the TCR of the present invention includes: It contains each amino acid sequence of CDR3 shown in SEQ ID NOs: 14 to 28, respectively.
• the above amino acid sequence may contain one to several (for example, two or three) amino acids, as long as the TCR containing the CDR3 amino acid sequence has the ability to bind to the GPC3 peptide or the complex of the peptide and HLA-A24. may be deleted, substituted or added.
• the TCR of the present invention comprises a TCR ⁇ chain comprising each amino acid sequence of CDR3 shown in SEQ ID NOs: 1 to 13, respectively, and a TCR ⁇ chain comprising each amino acid sequence of CDR3 shown as SEQ ID NOs: 14 to 28, respectively.
• the ⁇ and ⁇ chains of the TCR form a heterodimer.
• the ⁇ chain of the TCR of the present invention preferably has SEQ ID NOS: 1 to one amino acid sequence among the amino acid sequences shown by SEQ ID NOs: 13, 1 or several (for example, 2, 3, 4 or 5) amino acids are deleted in the amino acid sequences shown by SEQ ID NOS: 1 to 13, 90% or more (for example, 91%, 92%, 93%, 94%, 95%, 96%) of one of the substituted or added amino acid sequences, or the amino acid sequence shown in SEQ ID NOS: 1 to 13. , 97%, 98% or 99% or more).
• the ⁇ chain of the TCR of the present invention preferably has SEQ ID NO: 14 to 28, one or several (for example, 2, 3, 4 or 5) amino acids are deleted in the amino acid sequences shown in SEQ ID NOs: 14 to 28, 90% or more (for example, 91%, 92%, 93%, 94%, 95%, 96%) of one of the substituted or added amino acid sequences, or the amino acid sequence shown in SEQ ID NOs: 14 to 28. , 97%, 98% or 99% or more) identity).
• the TCR of the present invention comprises a TCR ⁇ chain comprising the amino acid sequence shown in SEQ ID NOs: 1 to 13, and a TCR ⁇ chain comprising one of the amino acid sequences shown in SEQ ID NOs: 14 to 28, The ⁇ chain and ⁇ chain of the TCR form a heterodimer.
• the TCR of the present invention comprises a TCR ⁇ chain comprising one of the amino acid sequences shown in SEQ ID NOs: 1-13, and one of the amino acid sequences shown in SEQ ID NOs: 1-13. The ⁇ and ⁇ chains of the TCR form a heterodimer.
• the TCR of the present invention can be produced by genetic engineering using the nucleic acid or vector of the present invention described below. For example, by introducing both a nucleic acid encoding the ⁇ chain and a nucleic acid encoding the ⁇ chain of the TCR of the present invention into cells to express the TCR ⁇ chain and ⁇ chain polypeptides, the present invention can be applied to the cells.
• the TCR can be expressed and isolated by a method known per se.
• nucleic acid of the present invention provides a nucleic acid encoding the TCR of the present invention described above (hereinafter abbreviated as "nucleic acid of the present invention").
• the nucleic acids of the present invention may be isolated.
• the nucleic acid of the present invention may be a nucleic acid encoding the ⁇ chain of TCR, a nucleic acid encoding the ⁇ chain of TCR, or a nucleic acid encoding both ⁇ chain and ⁇ chain of TCR.
• the invention also relates to nucleic acids encoding any or more of the CDRs, variable regions and/or constant regions described herein.
• the invention also encompasses nucleic acids that are capable of hybridizing under stringent conditions to the complement of any of the nucleic acids defined herein.
• the hybridizable nucleic acids encode CDR, variable region or constant region amino acid sequences having the functions described herein.
• the hybridizable nucleic acid encodes an amino acid sequence such that a TCR containing the amino acid sequence has the ability to bind to the GPC3 peptide or the complex of the peptide and HLA-A24.
• the nucleic acid encoding the TCR ⁇ chain of the present invention may be any nucleic acid as long as it encodes the TCR ⁇ chain defined above. Examples include nucleic acids. Further, the nucleic acid encoding the TCR ⁇ chain of the present invention may be any nucleic acid as long as it encodes the TCR ⁇ chain defined above, and for example, the polypeptide shown by SEQ ID NOS: 14 to 28. Examples include nucleic acids encoding.
• the nucleic acid of the present invention may be DNA, RNA, or a DNA/RNA chimera, but is preferably DNA. Further, the nucleic acid may be double-stranded or single-stranded. If it is double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. When the nucleic acid is RNA, T in the sequence listing shall be read as U for the RNA sequence.
• the nucleic acids of the present invention may also include natural nucleotides, modified nucleotides, nucleotide analogs, or mixtures thereof, as long as they can express the polypeptide in vitro or in cells.
• the nucleic acid of the present invention can be constructed by a method known per se.
• a DNA chain can be chemically synthesized based on the TCR amino acid sequence or nucleic acid sequence listed in the sequence listing, or synthesized partially overlapping oligo DNA short chains can be synthesized using the PCR method or Gibson Assembly method. It is possible to construct a DNA encoding the full length or a part of the TCR of the present invention by connecting the TCR of the present invention.
• the nucleic acids of the invention can be incorporated into expression vectors. Therefore, the present invention provides an expression vector (hereinafter abbreviated as "vector of the present invention") containing any of the above-described nucleic acids of the present invention.
• the vector of the present invention may be a vector that does not integrate into the genome of the target cell. In one embodiment, a vector that is not integrated into the genome is capable of replicating outside the genome of the target cell. The vector may exist in multiple copies outside the genome of the target cell. In a further embodiment of the invention, the vector is integrated into the genome of the target cell. In a preferred embodiment, the vector integrates into the genome of the target cell at a predetermined location.
• promoters used in the vector of the present invention include ubiquitin promoter, EF1 ⁇ promoter, CAG promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV ( Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, TCR V ⁇ gene promoter, TCR V ⁇ gene promoter, etc. are used.
• ubiquitin promoter, EF1 ⁇ promoter, CAG promoter, MoMuLV LTR, CMV promoter, SR ⁇ promoter, etc. are preferred.
• the vector of the present invention may optionally contain transcriptional and translational regulatory sequences, ribosome binding sites, enhancers, origins of replication, polyA addition signals, selection marker genes, and the like.
• the selectable marker gene include a dihydrofolate reductase gene, a neomycin resistance gene, a puromycin resistance gene, and the like.
• an expression vector containing a nucleic acid encoding the ⁇ chain and a nucleic acid encoding the ⁇ chain of the TCR of the present invention described above is introduced into a target cell, and the TCR is introduced into the target cell or on the cell surface.
• a heterodimer of an ⁇ chain and a ⁇ chain can be formed.
• the nucleic acid encoding the ⁇ chain and the nucleic acid encoding the ⁇ chain of TCR may be incorporated into separate expression vectors, or may be incorporated into one expression vector.
• these two types of nucleic acids are preferably integrated via a sequence that enables polycistronic expression.
• Sequences that enable polycistronic expression include, for example, the T2A sequence of foot-and-mouth disease virus (see PLoS ONE3, e2532, 2008, Stem Cells 25, 1707, 2007), the internal ribosome entry site (IRES) (U.S. Patent No. 4,937,190), but from the viewpoint of uniform expression level, the T2A sequence is preferred.
• Expression vectors that can be used in the present invention are not particularly limited as long as they can express TCR for a period sufficient to prevent or treat diseases when introduced into cells, and include viral vectors and plasmid vectors.
• viral vectors include retrovirus vectors (including lentivirus vectors and pseudotype vectors), adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, Sendai virus, episomal vectors, and the like.
• a transposon expression system may be used.
• plasmid vectors include animal cell expression plasmids (eg, pa1-11, pXT1, pRc/CMV, pRc/RSV, and pcDNAI/Neo).
• the present invention provides a cell containing the nucleic acid or vector of the present invention (in other words, a cell having the nucleic acid or vector of the present invention) (hereinafter abbreviated as "cell of the present invention").
• the nucleic acid of the present invention is preferably introduced into desired cells in the form of a vector of the present invention.
• the invention also encompasses introducing the nucleic acids of the invention into the host genome by genome editing (eg, CRISPR system, TALEN system, etc.).
• a preferred embodiment of the cell of the present invention includes, but is not limited to, a cell into which both a nucleic acid encoding a TCR ⁇ chain and a nucleic acid encoding a TCR ⁇ chain have been introduced.
• Confirmation that the cells of the present invention have cytotoxic activity may be performed using known methods, and preferred methods include, for example, measuring cytotoxic activity against HLA-A24-positive target cells, such as a chromium release assay.
• the cells of the invention are human cells.
• lymphocytes examples include lymphocytes and lymphocyte progenitor cells including pluripotent stem cells.
• lymphocytes refers to one of the subtypes of white blood cells in the immune system of vertebrates, and lymphocytes include T cells, B cells, and natural killer cells (NK cells). Since T cell receptors play an important role in antigen recognition by T cells, T cells are preferred as cells into which the nucleic acid or vector of the present invention is introduced.
• a "T cell” is a type of white blood cell found in lymphoid organs or peripheral blood, and is a lymphocyte that differentiates and matures mainly in the thymus gland and expresses a T cell receptor (TCR).
• TCR T cell receptor
• T cells include cytotoxic T cells (CTL), which are CD8-positive cells, helper T cells, regulatory T cells, and effector T cells, which are CD4-positive cells. , preferably cytotoxic T cells.
• CD4/CD8 both positive cells are also included in T cells.
• T cells expressing the TCR of the present invention can be obtained by introducing the nucleic acid or vector of the present invention into T cells collected from a living body.
• T cells expressing the TCR of the present invention can be derived from lymphoid progenitor cells (e.g., pluripotent stem cells) into which the nucleic acid or vector of the present invention has been introduced. cells) can be obtained.
• lymphoid progenitor cells e.g., pluripotent stem cells
• the cell of the present invention eg, cytotoxic T cell
• the cells of the present invention differ from cells collected from living bodies.
• the lymphocytes can be collected from, for example, peripheral blood, bone marrow, and umbilical cord blood of humans or non-human mammals.
• the cell population is preferably collected from the person to be treated or a donor whose HLA type matches the HLA type of the person to be treated.
• Preferred subjects or donors are humans.
• lymphocyte precursor cells including pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic tumor cells (EC cells). ), embryonic germ stem cells (EG cells), hematopoietic progenitor cells including hematopoietic stem cells, multipotent progenitors (MMPs) that have lost self-renewal ability, myelolymphoid common progenitor cells (MLPs), myeloid lineage Examples include progenitor cells (MP), granulocyte mononuclear progenitors (GMP), macrophage-dendritic cell progenitors (MDP), and dendritic cell progenitors (DCP).
• ES cells embryonic stem cells
• iPS cells induced pluripotent stem cells
• EC cells embryonic tumor cells
• EG cells embryonic germ stem cells
• hematopoietic progenitor cells including hematopoietic stem cells include multipotent progeni
• Any cell derived from a human embryo, particularly an ES cell may be a cell produced by destroying the embryo, or a cell produced without destroying the embryo. From an ethical standpoint, iPS cells, EC cells, EG cells, hematopoietic progenitor cells, MMP, MLP, MP, GMP, MDP, DCP, and ES cells produced without destroying embryos are preferred.
• iPS cells can be produced by introducing specific reprogramming factors in the form of DNA or proteins into somatic cells, and have properties almost equivalent to ES cells, such as pluripotency and the ability to proliferate through self-renewal.
• Artificial stem cells derived from somatic cells e.g., Takahashi K, Yamanaka S. Cell, 126;663-676 (2006): Takahashi K. et al. Cell, 131;861-872 (2007): Yu J. et al. Science, 318;1917-1920 (2007): Nakagawa M. et al. Nat. Biotechnol. 26;101-106 (2008)).
• the iPS cells When using iPS cells, the iPS cells may be produced from somatic cells by a method known per se, or already established and stocked iPS cells may be used. Although there are no restrictions on the somatic cells from which the iPS cells used in the present invention are derived, cells derived from peripheral blood or umbilical cord blood are preferred. There is no restriction on the animal from which the pluripotent stem cells are derived, and examples include mammals such as mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, and humans, with humans being preferred.
• hematopoietic progenitor cells refer to CD34-positive cells, preferably CD34/CD43 dual-positive (DP) cells.
• the origin of the hematopoietic progenitor cells used in the present invention is not limited, and for example, hematopoietic progenitor cells obtained by inducing differentiation of pluripotent stem cells by the method described below may be used. Hematopoietic progenitor cells isolated by the method described above may also be used.
• nucleic acid or vector of the present invention there are no particular limitations on the method for introducing the nucleic acid or vector of the present invention into cells, and known methods can be used.
• introducing a nucleic acid or a plasmid vector it can be carried out, for example, by a calcium phosphate coprecipitation method, a PEG method, an electroporation method, a microinjection method, a lipofection method, or the like.
• a calcium phosphate coprecipitation method for example, a calcium phosphate coprecipitation method, a PEG method, an electroporation method, a microinjection method, a lipofection method, or the like.
• the nucleic acid of the present invention is introduced into an appropriate packaging cell (e.g., Plat-E cells) or a complementary cell line (e.g., 293 cells) to obtain the virus produced in the culture supernatant.
• the vector can be collected and introduced into cells by infecting the cells with the vector using an appropriate method depending on each virus vector. For example, specific methods for using retrovirus vectors as vectors are described in International Publication No. 2007/69666 pamphlet, Takahashi K, Yamanaka S. Cell, 126, 663-676 (2006) and Takahashi K, et al. Cell, 131, Disclosed in reports such as 861-872 (2007).
• a retrovirus vector when using a retrovirus vector, highly efficient gene transfer into various cells is possible by using the recombinant fibronectin fragment CH-296 (manufactured by Takara Bio).
• a transposon vector such as a piggyBac vector may be used.
• the nucleic acid of the present invention may also be directly introduced into cells in the form of RNA and used to express TCR within the cells.
• a method for introducing RNA a known method can be used, and for example, lipofection, electroporation, etc. can be suitably used.
• endogenous TCR ⁇ originally expressed by the T cells should be and TCR ⁇ chain expression may be suppressed by siRNA.
• the base sequence of the nucleic acid encoding the TCR is replaced with siRNA that suppresses the expression of the endogenous TCR ⁇ chain and TCR ⁇ chain. It is preferable to use a sequence (codon conversion type sequence) different from the base sequence corresponding to the acting RNA.
• the above-mentioned base sequence can be produced by introducing a silent mutation into a naturally obtained TCR-encoding nucleic acid or by chemically synthesizing an artificially designed nucleic acid.
• part or all of the constant region of the nucleic acid encoding the TCR of the present invention may be replaced with a constant region derived from an animal other than humans, such as a mouse.
• TCR genes are associated with the administration of HLA-A24-restricted GPC3 peptide vaccine to liver cancer patients conducted at the National Cancer Center Hospital East. Obtained through clinical research.
• the GPC3 peptide vaccine was administered to two patients (P01 first administration date: January 18, 2012, P02 first administration date: February 24, 2008) continuously every two weeks. Later, blood was collected and stored at the center.
• samples at the two time points shown in Figure 1 were obtained and the TCR gene was isolated.
• Isolation of the TCR gene was performed according to the method described in Figure 2. First, peripheral blood was collected from a patient to whom GPC3 peptide was administered according to the GPC3 peptide administration conditions in the above clinical study, and lymphocytes were purified using the obtained peripheral blood. Thereafter, the purified lymphocytes were stimulated with GPC3 peptide under in vitro conditions.
• CD137-positive activated T cells were subjected to single cell sorting using a cell sorter, and the TCR ⁇ chain and TCR ⁇ were determined by PCR from the single-cell sorted CD137-positive activated T cells. Isolation of gene pairs (primary PCR) was performed. Furthermore, from this isolation step, PCR fragments of TCR ⁇ chain and TCR ⁇ chain genes having transcriptional activity were prepared (secondary PCR).
• TCR ID is the ID of the TCR obtained from the sample
• TRAV is the TCRV ⁇ segment
• TRAJ is the TCRJ ⁇ segment
• CDR3A is the CDR3 ⁇ segment
• TRBV indicates the TCRV ⁇ segment
• TRBJ indicates the TCRJ ⁇ segment
• TRBD indicates the TCRD ⁇ segment
• CDR3B indicates the CDR3 ⁇ segment.
• amino acids were indicated by conventional one-letter abbreviations.
• the present invention also provides a method for producing cells of the present invention (hereinafter abbreviated as "the production method of the present invention"), which includes a step of introducing the nucleic acid or vector of the present invention into cells. . 4. Cells into which the nucleic acid or vector of the present invention is introduced and the introduction method. As described in .
• hematopoietic progenitor cells in one embodiment, (1) differentiating pluripotent stem cells into which the nucleic acid or vector of the present invention has been introduced into hematopoietic progenitor cells, and (2) differentiating the hematopoietic progenitor cells into T cells.
• a method of producing a T cell including the steps.
• Step (1) Step of differentiating pluripotent stem cells into hematopoietic progenitor cells (step (1))
• the method for differentiating pluripotent stem cells into hematopoietic progenitor cells is not particularly limited as long as it can be differentiated into hematopoietic progenitor cells, but examples include International Publication No. 2013/075222 pamphlet, International Publication No. 2016/076415 pamphlet, and Liu S.
• examples include a method of culturing pluripotent stem cells in a medium for inducing hematopoietic progenitor cells.
• the medium for inducing hematopoietic progenitor cells is not particularly limited, but a medium used for culturing animal cells can be prepared as a basal medium.
• the basal medium include Iscove's Modified Dulbecco's Medium (IMDM) medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's s medium , Neurobasal Medium (Life Technologies), or a mixed medium thereof.
• the medium may contain serum or may be serum-free. If necessary, the basal medium may contain, for example, vitamin C (e.g.
• ascorbic acid albumin
• insulin transferrin
• selenium fatty acids
• trace elements 2-mercaptoethanol
• thiolglycerol lipids
• amino acids L-glutamine.
• non-essential amino acids vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines, etc.
• vitamin C refers to L-ascorbic acid and derivatives thereof, and L-ascorbic acid derivatives refer to those that become vitamin C through an enzymatic reaction in vivo.
• derivatives of ascorbic acid used in the present invention include vitamin C phosphate, ascorbyl glucoside, ascorbyl ethyl, vitamin C ester, ascobyl tetrahexyldecanoate, ascobyl stearate, and ascorbic acid-2-phosphate-6-palmitic acid.
• vitamin C phosphate for example, phosphate-L-ascorbate salts such as Na phosphate-L-ascorbate or Mg phosphate-L-ascorbate.
• a preferred basal medium used in step (1) is an IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, L-glutamine, and ascorbic acid.
• the culture solution used in step (1) is at least one type selected from the group consisting of BMP4 (Bone morphogenetic protein 4), VEGF (vascular endothelial growth factor), SCF (Stem cell factor), and FLT-3L (Flt3 Ligand). Cytokines may be further added. More preferred is a culture medium supplemented with VEGF, SCF and FLT-3L.
• vitamin C is preferably added (supplemented) separately every 4 days, every 3 days, every 2 days, or every 1 day, and preferably added every 1 day.
• the amount of vitamin C in the culture solution is equivalent to 5 ng/mL to 500 ng/mL (e.g. 5 ng/mL, 10 ng/mL, 25 ng/mL, 50 ng/mL, 100 ng/mL). , 200 ng/mL, 300 ng/mL, 400 ng/mL or 500 ng/mL).
• the concentration of BMP4 in the culture medium is not particularly limited, but is between 10 ng/mL and 100 ng/mL (e.g. 10 ng/mL, 20 ng/mL, 30 ng/mL). , 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL or 100 ng/mL), preferably 20 ng/mL to 40 ng/mL. ml is more preferable.
• the concentration of VEGF in the culture medium is not particularly limited, but is between 10 ng/mL and 100 ng/mL (e.g. 10 ng/mL, 20 ng/mL, 30 ng/mL). , 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL or 100 ng/mL), especially 20 ng/mL. preferable.
• the concentration of SCF in the culture medium is not particularly limited, but is between 10 ng/mL and 100 ng/mL (e.g. 10 ng/mL, 20 ng/mL, 30 ng/mL). , 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL or 100 ng/mL), and 30 ng/mL is particularly preferred. preferable.
• the concentration of FLT-3L in the culture medium is not particularly limited, but may range from 1 ng/mL to 100 ng/mL (e.g. 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL or 100 ng/mL), particularly preferably 10 ng/mL.
• the pluripotent stem cells may be cultured by adhesive culture or suspension culture.
• adhesive culture the pluripotent stem cells may be cultured using a culture container coated with a coating agent.
• Co-culture may be used.
• Other co-cultured cells include C3H10T1/2 (Takayama N. et al. J Exp Med. 2010;207(13):2817-2830) and heterologous stromal cells (Niwa A. et al. J Cell Physiol. 2009;221(2):367-377).
• An example of the coating agent is Matrigel (Niwa A, et al. PLoS One. 2011;6(7):e22261). In suspension culture, Chadwick K. et al.
• the culture temperature conditions are not particularly limited, but are preferably about 37°C to 42°C, and about 37°C to 39°C, for example. Furthermore, a person skilled in the art can appropriately determine the culture period while monitoring the number of hematopoietic progenitor cells.
• the number of days is not particularly limited as long as hematopoietic progenitor cells are obtained, but for example, at least 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, or It is 14 days or more, preferably 14 days.
• hypoxic conditions include oxygen concentrations of 15%, 10%, 9%, 8%, 7%, 6%, 5%, or lower.
• Step (2) Step of differentiating hematopoietic progenitor cells into T cells
• the method for differentiating hematopoietic progenitor cells into T cells is not particularly limited as long as hematopoietic progenitor cells can be differentiated into T cells, but for example, as described in WO 2016/076415 pamphlet, etc.
• (2- Examples include a method comprising: 1) inducing CD4/CD8 dual-positive T cells from hematopoietic progenitor cells; and (2-2) inducing CD8-positive T cells from CD4/CD8 dual-positive T cells.
• hematopoietic progenitors be isolated in advance from the cell population obtained in step (1) using a hematopoietic progenitor cell marker.
• the marker includes at least one selected from the group consisting of CD43, CD34, CD31, and CD144.
• step (2-1) Step of inducing CD4/CD8 double positive T cells from hematopoietic progenitor cells (step (2-1))
• the method for differentiating into CD4/CD8 dual-positive T cells includes, for example, a method of culturing hematopoietic progenitor cells in an induction medium for CD4/CD8 dual-positive T cells.
• the medium for inducing differentiation into both CD4/CD8 positive T cells is not particularly limited, but a medium used for culturing animal cells can be prepared as a basal medium.
• the medium may contain serum or may be serum-free.
• the basal medium may contain, for example, vitamin C, albumin, insulin, transferrin, selenium, fatty acids, trace elements, 2-mercaptoethanol, thiolglycerol, lipids, amino acids, L-glutamine, non-essential amino acids, vitamins. , growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, cytokines, and the like.
• the preferred basal medium used in step (2-1) is ⁇ MEM medium containing serum, transferrin, serine, and L-glutamine.
• the vitamin C is the same as in step (1).
• the culture solution used in step (2-1) may further contain the cytokines FLT-3L and/or IL-7, and more preferably, the culture solution is supplemented with FLT-3L and IL-7. be.
• the concentration of IL-7 in the culture solution is 1 ng/mL to 50 ng/mL (e.g. 1 ng/mL, 2 ng/mL, 3 ng/mL).
• FLT-3L when FLT-3L is used in step (2-1), FLT-3L can be used in the same manner as in step (1) above.
• hematopoietic progenitor cells may be cultured in an adherent manner or in suspension.
• a culture vessel may be coated, or they may be co-cultivated with feeder cells or the like.
• feeder cells to be co-cultured is bone marrow stromal cell line OP9 cells (available from RIKEN BioResource Center).
• the OP9 cells are preferably OP-DL1 cells that constitutively express Dll1 (Holmes R, Zuniga-Pflucker JC. Cold Spring Harb Protoc. 2009(2):pdb.prot5156).
• Dll1 when using OP9 cells as feeder cells, this can also be done by appropriately adding Dll1 or a fusion protein of Dll1 and Fc, etc. prepared separately to the culture medium.
• Dll1 includes a protein encoded by a gene having the nucleotide sequence listed as NCBI accession number NM#005618 in the case of human and NM#007865 in the case of mouse, and a protein with high sequence identity thereto. Naturally occurring variants having the same functionality (for example, 90% or more) and equivalent functions are included.
• feeder cells are used to produce CD4/CD8 double-positive T cells, it is preferable to replace the feeder cells as appropriate during culturing. Replacement of feeder cells can be performed by transferring target cells in culture onto previously seeded feeder cells. The exchange may occur every 5 days, every 4 days, every 3 days, or every 2 days.
• the culture temperature conditions are not particularly limited, but are preferably about 37°C to 42°C, and about 37°C to 39°C, for example.
• the culture period can be appropriately determined by those skilled in the art while monitoring the number of CD4/CD8 double-positive T cells.
• the number of days of the culture period is not particularly limited as long as hematopoietic progenitor cells are obtained, but for example, at least 10 days or more, 12 days or more, 14 days or more, 16 days or more, 18 days or more, 20 days or more, 22 days or more, or 23 days or more. 23 days or more, preferably 23 days.
• the culture period is preferably 90 days or less, more preferably 42 days or less.
• step (2-2) Step of inducing CD8 positive T cells from CD4/CD8 double positive (DP) T cells (step (2-2)) By subjecting the CD4/CD8 DP cells obtained in step (2-1) to a step of inducing differentiation into CD8 single positive (SP) cells, they can be induced to differentiate into CD8 single positive (SP) cells.
• the basal medium and medium used in step (2-2) include those similar to the basal medium and medium described in step (1).
• the medium may contain an adrenal corticosteroid.
• adrenal corticosteroids include glucocorticoids and derivatives thereof; examples of the glucocorticoids include cortisone acetate, hydrocortisone, fludrocortisone acetate, prednisolone, triamcinolone, methylprednisolone, dexamethasone, betamethasone, Examples include beclomethasone propionate. Among them, dexamethasone is preferred.
• the concentration of dexamethasone in the culture medium is 1 nM to 100 nM (e.g. 1 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM , 70 nM, 80 nM, 90 nM or 100 nM), and 10 nM is especially preferred.
• the medium may contain antibodies (eg, anti-CD3 antibody, anti-CD28 antibody, and anti-CD2 antibody), cytokines (eg, IL-7, IL-2, and IL-15), and the like.
• antibodies eg, anti-CD3 antibody, anti-CD28 antibody, and anti-CD2 antibody
• cytokines eg, IL-7, IL-2, and IL-15
• the anti-CD3 antibody is not particularly limited as long as it is an antibody that specifically recognizes CD3, and includes, for example, an antibody produced from an OKT3 clone.
• the anti-CD3 antibody may be bound to magnetic beads or the like, and instead of adding the anti-CD3 antibody to the medium, the T Stimulation may be provided by culturing the lymphocytes for a certain period of time.
• the concentration of anti-CD3 antibody in the culture medium is 10 ng/mL to 1000 ng/mL (e.g. 10 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 300 ng/mL, 400 ng/mL).
• mL 500 ng/mL, 600 ng/mL, 700 ng/mL, 800 ng/mL, 900 ng/mL, 1000 ng/ml), and 500 ng/mL is particularly preferable.
• concentrations of other antibodies can also determine the concentrations of other antibodies as appropriate based on culture conditions and the like.
• the concentration of IL-2 in the medium is 10 U/mL to 1000 U/mL (e.g. 10 U/mL, 20 U/mL, 30 U/mL , 40 U/mL, 50 U/mL, 60 U/mL, 70 U/mL, 80 U/mL, 90 U/mL, 100 U/mL, 200 U/mL, 500 U/mL or 1000 U/mL ) is preferred, and 100 U/mL is particularly preferred.
• the concentration of IL-7 or IL-15 used in step (2-2) in the medium is 1 ng/mL to 100 ng/mL (e.g.
• ng/mL 1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 30 ng/mL, 40 ng/mL, 50 ng/mL, 60 ng/mL, 70 ng/mL, 80 ng/mL, 90 ng/mL or 100 ng/mL), and among them, 10 ng/mL is preferred.
• the culture temperature conditions are not particularly limited, but are preferably about 37°C to 42°C, more preferably about 37°C to 39°C.
• the culture period can be appropriately determined by those skilled in the art while monitoring the number of CD8-positive T cells.
• the number of days is not particularly limited as long as CD8-positive T cells are obtained, but is preferably 1 day or more, 3 days or more, or 7 days or more, preferably 60 days or less, and more preferably 35 days or less.
• Medicinal product containing the nucleic acid, vector, or cell of the present invention provides a pharmaceutical product containing the nucleic acid, vector, or cell of the present invention as an active ingredient (hereinafter abbreviated as "medicine of the present invention").
• Cells containing the nucleic acids of the invention can exhibit cytotoxic activity against cells presenting HLA-A24 molecules and GPC3 peptides. Therefore, a medicament containing the nucleic acid, vector, or cell of the present invention can be used for the prevention or treatment of diseases in which GPC3 is expressed, and can be used, for example, in mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs). , cows, sheep, monkeys and humans), preferably humans.
• Diseases that express GPC3 include, but are not particularly limited to, cancers and tumors that express GPC3. Accordingly, in a preferred embodiment of the present invention, anticancer agents for the prevention or treatment of cancers and tumors expressing GPC3 are provided.
• liver cancer e.g. hepatocellular carcinoma
• ovarian cancer e.g. ovarian clear cell adenocarcinoma
• lung cancer e.g. squamous cell carcinoma, small cell lung cancer
• testicular cancer e.g. nonseminoma germ cell tumor
• soft tissue tumor e.g. liposarcoma, malignant fibrous histiocytoma
• uterine cancer e.g.
• cervical intraepithelial neoplasia cervical squamous cell carcinoma
• melanoma adrenal tumors (e.g. adrenal adenoma), neurological tumors (e.g. Schwannoma), gastric cancer (e.g. adenocarcinoma of the stomach), kidney cancer (e.g. Gravitz tumor), breast cancer (e.g. invasive lobular carcinoma, mucinous carcinoma), thyroid cancer (e.g. medullary carcinoma), laryngeal cancer (e.g. squamous cell carcinoma), bladder cancer (e.g. invasive transitional cell carcinoma), etc.
• these include, but are not limited to: Among these, from the viewpoint of the expression level of GPC3, liver cancer, ovarian cancer, pediatric cancer, and lung cancer are preferable, and among these, liver cancer, particularly hepatocellular carcinoma, is preferable.
• a nucleic acid or a vector as an active ingredient of the medicament of the present invention, known pharmaceutically acceptable carriers (excipients, diluents, fillers, binders, lubricants, flow aids, disintegrating agents, It is preferable to prepare a pharmaceutical composition by mixing the pharmaceutical composition with conventional additives (including agents, surfactants, etc.) or conventional additives.
• Excipients are well known to those skilled in the art and include, for example, phosphate buffered saline (e.g., 0.01 M phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), hydrochloride, hydrogen bromide.
• Aqueous solutions containing mineral acid salts such as acid salts, phosphates, sulfates, physiological saline solutions, solutions such as glycol or ethanol, and salts of organic acids such as acetates, propionates, malonates, benzoates, etc.
• mineral acid salts such as acid salts, phosphates, sulfates, physiological saline solutions, solutions such as glycol or ethanol, and salts of organic acids such as acetates, propionates, malonates, benzoates, etc.
• Auxiliary agents such as wetting agents or emulsifying agents, and pH buffering agents can also be used.
• formulation aids such as suspending agents, preservatives, stabilizers, and dispersants may also be used.
• the pharmaceutical compositions may also be in dry form for reconstitution with a suitable sterile liquid before use.
• the pharmaceutical composition can be prepared in any form (oral administration such as tablets, pills, capsules, powders, granules, syrups, emulsions, and suspensions; injections, infusions, external preparations, and suppositories).
• Parenteral administration can be performed systemically or locally depending on the type of drug (parenteral administration), etc.
• parenteral administration intravenous administration, intradermal administration, subcutaneous administration, rectal administration, transdermal administration, etc. are possible.
• acceptable buffers, solubilizing agents, isotonic agents, and the like may be added.
• the dosage is, for example, in the range of 0.001 mg to 10 mg per kg of body weight at a time.
• the dose is administered in the range of 0.001 to 50 mg to a patient weighing 60 kg.
• the active ingredient is a virus vector particle
• the dose is administered to a subject weighing 60 kg at a time, for example, in a range of about 1 x 10 3 pfu to 1 x 10 15 pfu in terms of virus titer. .
• the above dosage is just an example, and the dosage can be appropriately selected depending on the type of nucleic acid or vector used, the route of administration, the age, body weight, symptoms, etc. of the subject or patient.
• the cells When using the cells of the present invention as an active ingredient of the medicament of the present invention, the cells may be cultured and/or stimulated using an appropriate medium and/or stimulating molecules before administration to a subject. good.
• Stimulatory molecules include, but are not limited to, cytokines, appropriate proteins, other components, and the like. Examples of cytokines include IL-2, IL-7, IL-12, IL-15, and IFN- ⁇ , and preferably IL-2 can be used.
• the concentration of IL-2 in the medium is not particularly limited, but is, for example, preferably 0.01 to 1 ⁇ 10 5 U/mL, more preferably 1 to 1 ⁇ 10 4 U/mL.
• suitable proteins include, for example, CD3 ligand, CD28 ligand, and anti-IL-4 antibody.
• lymphocyte stimulating factors such as lectins can also be added.
• serum or plasma may be added to the medium.
• the amount of these added to the medium is not particularly limited, but 0% to 20% by volume is exemplified, and the amount of serum or plasma used can be changed depending on the culture stage. For example, serum or plasma concentrations can be reduced stepwise.
• the serum or plasma may be derived from either autologous or non-autologous sources, but from the viewpoint of safety, autologous sources are preferred.
• the medicament containing the cells of the present invention as an active ingredient is preferably administered to a subject parenterally.
• Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration.
• the dosage is appropriately selected depending on the condition, body weight, age, etc. of the subject, but the number of cells is usually 1 x 10 6 to 1 x 10 10 cells per dose for a subject weighing 60 kg.
• the number of doses is preferably 1 ⁇ 10 7 to 1 ⁇ 10 9 , more preferably 5 ⁇ 10 7 to 5 ⁇ 10 8 .
• it may be administered once or multiple times.
• the medicament of the present invention can be in a known form suitable for parenteral administration, such as an injection or an infusion.
• the medicament of the present invention may optionally contain pharmacologically acceptable excipients.
• Pharmacologically acceptable excipients include those described above.
• the medicament of the present invention may contain physiological saline, phosphate buffered saline (PBS), a medium, etc. to stably maintain cells.
• the medium is not particularly limited, and examples include, but are not limited to, RPMI, AIM-V, and X-VIVO10.
• a pharmaceutically acceptable carrier eg, human serum albumin
• preservative etc.
• the cells of the present invention can kill cells that express GPC3, they can be used as a killing agent for cells that express GPC3.
• a killing agent can be produced and used in the same manner as the above-mentioned pharmaceuticals.
• the TCR of the present invention can also be used as a fusion protein, for example, by combining TCR with a single chain antibody fragment (scFv) of an anti-CD3 antibody (or a similar antibody fragment that binds to T cells and activates a T cell response). You can also do that.
• a fusion protein a stable, soluble, high-affinity TCR, a new disulfide bond may be artificially introduced between each constant region of the polypeptides of the two TCR chains.
• the scFv of the fusion protein is preferably fused to the constant region of the ⁇ chain of TCR.
• Such fusion proteins are described, for example, in US Patent No. 7,569,664 and in the report of Oates J, Jakobsen BK. OncoImmunology. 2013; 2(2):e22891.
• the medicament containing the fusion protein and the nucleic acid encoding this protein can also be used for the prevention or treatment of diseases that express GPC3, similar to the medicament containing the nucleic acid or cells of the present invention.
• it can be prepared in the same manner as described above for the HLA-A24-restricted GPC3 peptide.
• SEQ ID NOs in the sequence listing of this specification are: SEQ ID NOS: 1 to 13 are the amino acid sequences of CDR3A of the TCR chain, SEQ ID NOs: 14 to 28 are the amino acid sequences of CDR3B of the TCR chain, and SEQ ID NO: 29 is the HLA-A24-restricted amino acid sequence.
• the amino acid sequence of GPC3 peptide is shown. Amino acids are indicated by conventional one-letter abbreviations.
• HLA Human Leukocyte Antigen HIV: human immunodeficiency virus
• ELISPOT Enzyme-Linked ImmunoSpot
• Example 1 For patients with advanced hepatocellular carcinoma, an antigen (HLA-A*24:02-restricted GPC3 peptide (hereinafter abbreviated as "GPC3 peptide") was synthesized according to Good Manufacturing Practice guidelines. ) (EYILSLEEL, SEQ ID NO: 29; American Peptide Company) was mixed with ICFA and emulsified and administered intradermally, and peripheral blood mononuclear cells (PBMC) were collected over time after the administration (see Figure 1). ).A specific method for isolating GPC3-responsive CD8 T cells is described below (see Figure 2).
• Test example 1 ELISPOT assay
• an ELISPOT assay was performed using the collected PBMC samples.
• a cancer cell line (SK-Hep- 1 /hGPC3) in which PBMC (1 x 10 cells per well) was forced to express GPC3 at 37°C in the presence of 5% CO2 or its mock control cancer cell line. (SK-Hep-1/vec) for 20 hours.
• the PBMC used in this test had the ability to produce interferon- ⁇ in response to cancer cells expressing GPC3.
• the obtained PBMCs were stimulated with GPC3 peptide in a test tube, and culture was continued. On the 12th day after stimulation, restimulation with GPC3 peptide was performed, and the resulting CD137-positive cells were isolated as GPC3 peptide-responsive cells, and single-cell sorting was performed using a cell sorter to obtain single-cell-sorted CD137-positive activated T cells.
• a pair of TCR ⁇ chain and TCR ⁇ chain genes was isolated from the cells by PCR (primary PCR). Furthermore, from the isolation step, PCR fragments of TCR ⁇ chain and TCR ⁇ chain genes having transcriptional activity were prepared (secondary PCR).
• Example 2 Decoding the TCR sequence 1. Sequence decoding The TCR sequence obtained from CD137-positive T cells after single cell sorting was analyzed by the following method. That is, total RNA of T cells was extracted using RNeasy Mini Kit (QIAGEN), amplified using Multiplex One-step RT-PCR, and analyzed using Sanger sequencing (see Table 1 and Sequence Listing).
• Test example 2 Analysis of antigen specificity of TCR using Jurkat cell line introduced with TCR1. Preparation of TCR Expression PCR Fragment Based on the above TCR ⁇ chain PCR product and TCR ⁇ chain PCR product, PCR fragments of TCR ⁇ chain and TCR ⁇ chain genes having transcriptional activity were prepared (secondary PCR).
• TCR gene introduction and antigen specificity analysis 1.
• the PCR fragment obtained was introduced into the NF-AT reporter and CD8-introduced TCR-negative Jurkat cell line, and luciferase was used to target A24-positive COS cells pulsed with GPC3 peptide for antigen-specific TCR activation. Reporter analysis was performed (see Figure 3).
• Example 3 Preparation of an expression vector incorporating the gene encoding the TCR of the present invention and iPS cell-derived T cells expressing the TCR 1.
• Preparation of an expression vector incorporating the gene encoding the TCR of the present invention 1) Preparation of piggyBac transposon vector incorporating the TCR ⁇ chain and TCR ⁇ chain Using the pIRII-IRES-dCD19 vector provided by Dr. Yozo Nakazawa of Shinshu University, P0103_TCR_10 , P0103_TCR_18 and P0103_TCR_82 were constructed (see Table 1).
• the piggyBac vector encoding the TCR ⁇ chain and the TCR ⁇ chain was introduced together with a plasmid vector encoding transposase into 1 ⁇ 10 6 FF-I01s04 iPS cells using a gene transfer device (MaxCyte ATx). Gene transfer was carried out by suspending cells in 50 ⁇ L of gene transfer buffer, using a total DNA amount of 320 ⁇ g/mL, a piggyBac vector/transposase vector ratio of 1/3, and introducing conditions under Optimization 8. Expression of the tracer gene CD19 was observed in the iPS cells 8 days after this gene introduction.
• T cell differentiation of TCR-introduced iPS cells The above-mentioned TCR-introduced iPS cells were differentiated into T cells according to the method described in the pamphlet of International Publication No. 2017/221975, and the expression of various markers in the cells after differentiation was The study was conducted using FACS Aria III. The results are shown in FIG. By subjecting TCR gene-transfected iPS cells to the above-described differentiation procedure, it was revealed that a complex of TCR ⁇ chain and TCR ⁇ chain can induce T cells expressed on the cell membrane surface.
• Test example 3 Cytotoxicity test of iPS cell-derived T cells expressing TCR of the present invention Cytotoxicity of iPS cell-derived T cells expressing TCR was measured. Specifically, the target cells were HLA-*24:02-positive lymphoblastoid cell lines (LCL) with or without the addition of the GPC3 antigen peptide. Cytotoxicity was measured based on the amount of intracellularly localized enzymes released from injured target cells. Effector cells (E) were added to target cells (T) at the ratio shown in Figure 8, and reacted at 37°C for 3 hours. Cytotoxic activity (% lysis) was calculated from the dye released in the supernatant after the reaction based on the following formula.
• LCL lymphoblastoid cell lines
• the minimum release value is the amount of intracellular localized enzyme released in a well to which no effector cells are added, and indicates the amount of intracellular localized enzyme naturally released from the target cells.
• the maximum release value indicates the amount of intracellular localized enzyme released when target cells are lysed by adding 1% Triton X-100.
• Example 4 Efficacy in mice of iPS cell-derived T cells expressing TCR of the present invention
• Regenerated T cells introduced into cells and differentiated into T cells were intraperitoneally administered six times to NOG mice in which GPC3-expressing SK-Hep liver cancer tumors were implanted into the peritoneal cavity.
• the results of measuring the chemiluminescence emitted by the tumors are shown in FIG. 9, and the survival curve of the mice is shown in FIG. 10.
• a cell mixture containing equal amounts of the three types of regenerated T cells described above was also administered to mice, and the progress was observed.
• the present invention provides T cell receptors capable of binding to GPC3 peptides (HLA-A24-restricted GPC3 peptides or complexes of the peptides and HLA-A molecules (HLA-A24) and nucleic acids encoding them). Furthermore, by using the method for producing the T cell receptor, it is possible to produce the T cell receptor with high efficiency in a shorter period of time than with conventional methods. Nucleic acids are useful for preventing or treating diseases that express GPC3 because they can impart cytotoxic activity to T cells against cells presenting HLA-A molecules and GPC3 peptides.

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