CAR containing anti-GPC3 single-chain antibody

Abstract

To provide a CAR that is capable of specifically bonding to a GPC3 localized in a cell membrane, and includes a single chain antibody useful for cancer immunotherapy using a CAR-T cell, an immunocompetent cell that expresses the CAR, or the like.SOLUTION: When a gene encoding a CAR includes: a single chain antibody which includes specific heavy chains CDR1 to 3 and specific light chains CDR1 to 3 defined in Claim 1, and specifically binds to a human-derived GPC3 polypeptide; a cell membrane penetrating region fused to a carboxyl terminal of the single chain antibody; and an immunocompetent cell activation signaling region fused to a carboxyl terminal of the cell membrane penetrating region is fabricated, and introduced into an immunocompetent cell, a CAR expression immunocompetent cell having excellent cancer cytotoxic activity and interferon γ(IFN-γ) productivity can be fabricated.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a chimeric antigen receptor (hereinafter also referred to as "CAR") comprising a single-chain antibody that specifically binds to a polypeptide derived from human GPC3 (Glypican-3) consisting of the amino acid sequence shown in SEQ ID NO: 11, a transmembrane domain fused to the carboxyl (C) terminus of the single-chain antibody, and an immunocompetent cell activation signal transduction domain fused to the C terminus of the transmembrane domain; immunocompetent cells that express the CAR; anticancer agents comprising the immunocompetent cells; a CAR gene encoding the CAR; and a vector comprising the CAR gene. [Background technology]

[0002] CAR is an artificial chimeric protein that combines a single-chain antibody that recognizes cell surface antigens on cancer cells with a signaling domain that induces T cell activation. By introducing a gene encoding CAR into peripheral blood T cells (peripheral blood T lymphocytes) that do not show tumor reactivity, it is possible to produce large quantities of tumor-reactive CAR-expressing T cells (hereinafter also referred to as "CAR-T cells"). Tumor-reactive CAR-T cells have tumor cytotoxic activity independent of interaction with major histocompatibility complex (MHC).

[0003] Cancer immunotherapy using CAR-T cells, more specifically, a therapy in which T cells are collected from a patient, a gene encoding a CAR is introduced into the T cells, the cells are amplified, and the cells are then reintroduced into the patient (Non-Patent Document 1), is currently undergoing clinical trials around the world, and results have shown its effectiveness in treating hematopoietic malignancies such as leukemia and lymphoma.

[0004] In recent years, various studies on CAR-T cells have been conducted. For example, there are a pharmaceutical composition comprising modified autologous human T cells comprising a nucleic acid encoding a CAR consisting of a CD19 antigen-binding region, a transmembrane region, a 4-1BB costimulatory signal region, and a CD3ζ signal region (Patent Document 1), a T cell population expressing one or more therapeutically effective anti-tag chimeric antigen receptors (AT-CARs) that bind to a tagged protein and induce cancer cell death, which is administered to a subject simultaneously with or separately from a formulation of one or more tagged proteins that bind to cancer cells (Patent Document 2), a cell comprising a nucleic acid encoding a chimeric antigen receptor comprising the antigen-binding domain of human antibody 139, an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain (Patent Document 3), and a cell comprising a nucleic acid sequence encoding a chimeric antigen receptor, wherein the chimeric antigen receptor Proposed cell lines include cells in which the CD19 antigen receptor contains an antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, and a CD3ζ signaling domain containing a specific amino acid sequence (Patent Document 4); genetically engineered CD19-specific T cells that express and possess a CD19-specific chimeric receptor on the cell surface, the chimeric receptor comprising an intracellular signaling domain for immune cell effector function, at least one transmembrane domain, and at least one extracellular domain, the extracellular domain containing a CD19-specific receptor (Patent Document 5); and chimeric antigen receptor-expressing cells into which a nucleic acid encoding a chimeric antigen receptor containing the intracellular domain of the glucocorticoid-induced tumor necrosis factor receptor (GITR) has been introduced (Patent Document 6). Furthermore, the present inventors have recently reported that expression of interleukin-7 (IL-7) or chemokine ligand 19 (CCL19) in CAR-T cells results in superior immune induction and antitumor activity compared to conventional CAR-T cells (Patent Document 7).

[0005] On the other hand, GPC3 is an extracellular matrix protein expressed in fetal tissues, particularly the liver and kidney, and is involved in organogenesis. Although GPC3 is not expressed in adult tissues other than the placenta, it is expressed in various cancer tissues, including hepatocellular carcinoma, melanoma, ovarian clear cell carcinoma, and lung squamous cell carcinoma. Because GPC3 is expressed in fetal tissues, like proteins such as α-fetoprotein (AFP) and carcinoembryonic antigen (CEA), it is classified as a carcinoembryonic antigen. Therefore, GPC3 is not expressed in normal tissue cells, but is specifically expressed in cancer cells, making it a useful target molecule for cancer therapy, a tumor marker, and a diagnostic marker.

[0006] A representative existing anti-GPC3 antibody is the monoclonal antibody 1G12 sold by BioMosaic. This antibody was obtained by immunizing Balb / c mice with an antigen (a polypeptide of the C-terminal 70 residues of GPC3) designed to avoid the complex structure and localization of GPC3, producing hybridomas, and screening using the antigen. Antibodies GC33 and GC199, developed by a Japanese pharmaceutical manufacturer, are also monoclonal antibodies established based on a similar concept, and were obtained using a C-terminal partial fragment of GPC3 as an antigen (Patent Document 8).

[0007] Recently, the present inventors have discovered an anti-GPC3 antibody that recognizes an epitope different from that of existing antibodies (e.g., GC33, GC199) and can specifically bind to GPC3 localized on the cell membrane even in the form of a single-chain antibody (PCT / JP2018 / 257). [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US Patent Application Publication No. 2014 / 0106449 [Patent Document 2] Special Publication No. 2014-504294 [Patent Document 3] Special Publication No. 2014-516510 [Patent Document 4] Special Publication No. 2014-507118 [Patent Document 5] Japanese Patent Application Laid-Open No. 2011-004749 [Patent Document 6] International Publication No. 2013 / 051718 Brochure [Patent Document 7] International Publication No. 2016 / 056228 Brochure [Patent Document 8] Patent No. 4011100 [Non-patent literature]

[0009] [Non-Patent Document 1] Hirozo Nakazawa Shinshu Medical Journal 61(4):197~203(2013) Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a CAR containing a single-chain antibody that can specifically bind to GPC3 localized on the cell membrane and is useful for cancer immunotherapy using CAR-T cells, as well as immunocompetent cells that express such a CAR. [Means for solving the problem]

[0011] The present inventors have been conducting intensive research to solve the above problems. In the course of this research, they produced an anti-GPC3 single-chain antibody based on an anti-GPC3 antibody (GC33 antibody) developed by a domestic pharmaceutical manufacturer, and discovered that when a vector expressing a CAR fused with such an anti-GPC3 single-chain antibody is introduced into T cells, CAR-T cells with excellent cancer cell cytotoxicity and interferon-γ (IFN-γ) production ability can be produced, which led to the completion of the present invention.

[0012] That is, the present invention is as follows. [1] A chimeric antigen receptor (CAR) comprising a single-chain antibody that specifically binds to a polypeptide derived from human GPC3 (Glypican-3) consisting of the amino acid sequence set forth in SEQ ID NO: 11, a transmembrane domain fused to the carboxyl terminus of the single-chain antibody, and an immunocompetent cell activation signaling domain fused to the carboxyl terminus of the transmembrane domain, The single chain antibody a heavy chain complementarity determining region (CDR) 1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6, The above-mentioned CAR (hereinafter referred to as "the CAR"). [2] The single-chain antibody is A heavy chain variable region consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a light chain variable region consisting of an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8, The CAR described in [1] above. [3] The single-chain antibody is an amino acid sequence having at least 80% sequence identity with the amino acid sequence set forth in SEQ ID NO: 12; or Contains an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, The CAR described in [1] or [2] above. [4] A CAR described in any one of [1] to [3] above, wherein the transmembrane domain and the immunocompetent cell activation signal transduction domain fused to the carboxyl terminus of the transmembrane domain comprise an amino acid sequence having at least 80% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 14 to 16. [5] An immunocompetent cell expressing the CAR described in any one of [1] to [4] above (hereinafter, sometimes referred to as the "immunocompetent cell of the present invention"). [6] The immunocompetent cells according to [5] above, which further express interleukin 7 (IL-7) and chemokine ligand 19 (CCL19). [7] An anticancer agent (hereinafter sometimes referred to as "the anticancer agent") containing the immunocompetent cells described in [5] or [6] above and a pharmaceutically acceptable additive. [8] A CAR gene encoding the CAR described in any one of [1] to [4] above (hereinafter sometimes referred to as "the CAR gene of the present invention"). [9] A vector comprising a promoter and a CAR gene encoding the CAR described in claim 8 operably linked downstream of the promoter (hereinafter sometimes referred to as "the CAR expression vector of the present invention").

[0013] Other embodiments of the present invention include a method for producing immunocompetent cells that express the present CAR, which includes a step of introducing the present CAR expression vector into immunocompetent cells; immunocompetent cells that express the present CAR, produced by introducing the present CAR expression vector into immunocompetent cells; and immunocompetent cells that express the present CAR and contain the present CAR gene (preferably immunocompetent cells that express the present CAR, as well as IL-7 and CCL19, which further contain the IL-7 gene and CCL19 gene). [Effects of the Invention]

[0014] According to the present invention, when the present CAR is expressed on the cell membrane of immunocompetent cells such as T cells, the immunocompetent cells exhibit excellent cancer cell cytotoxicity and IFN-γ production, making the present CAR useful in cancer immunotherapy using immunocompetent cells. [Brief explanation of the drawings]

[0015] [Figure 1]Figure 1A shows the results of flow cytometric analysis of CAR (horizontal axis) and CD8 (vertical axis) expression in T cells (Figure 1A) and CAR-T cells (Figure 1B). The boxed regions "Q1," "Q2," "Q3," and "Q4" in the figure represent the "CAR-negative CD8-positive T cell group," "CAR-positive CD8-positive T cell group," "CAR-positive CD8-negative T cell group," and "CAR-negative CD8-negative T cell group," respectively. The percentage (%) of the number of cells in each cell group relative to the total number of cells in these four cell groups is shown in each region. [Figure 2] Sk-HEP-1 cells ("mock" in the figure) and Sk-HEP-1 GPC3 cells ("GPC3" in the figure) were cultured in the presence of a T cell group ("T cells" in the figure) or a T cell group containing anti-GPC3scFv CAR-T cells ("CAR-T cells" in the figure). The number of CD45-negative cells (corresponding to the remaining Sk-HEP-1 GPC3 cells) (Figure 2A) and the ratio of these CD45-negative cells to all cells (Figure 2B) were analyzed. [Figure 3] This figure shows the results of analyzing the concentration of IFN-γ produced in the culture medium when Sk-HEP-1 cells ("mock" in the figure) and Sk-HEP-1 GPC3 cells ("GPC3" in the figure) were cultured in the presence of a T cell group ("T cells" in the figure) or a T cell group containing anti-GPC3scFv CAR-T cells ("CAR-T cells" in the figure). [Figure 4] These figures show the results of analyzing the concentrations of IL-7 (Fig. 4A) and CCL19 (Fig. 4B) produced in the culture medium when Sk-HEP-1 cells ("mock" in the figure) and Sk-HEP-1 GPC3 cells ("GPC3" in the figure) were cultured in the presence of a T cell group ("T cells" in the figure) or a T cell group containing anti-GPC3scFv CAR-T cells ("CAR-T cells" in the figure). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present CAR is a single chain antibody (single chain antibody) that specifically binds to a human GPC3-derived polypeptide (corresponding to a polypeptide consisting of glycine at position 537 to lysine at position 563 of human GPC3) consisting of the amino acid sequence of SEQ ID NO: 11, a heavy (H) chain complementarity-determining region (CDR) 1 consisting of the amino acid sequence of SEQ ID NO: 1, an H chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2, and an H chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3, and a light (L) chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, an L chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and an L chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6) consisting of the amino acid sequence of SEQ ID NO: 11. The present single-chain antibody is a polypeptide comprising at least i) to iii) the following: (i) a Fv; scFv) (hereinafter sometimes referred to as "the present single-chain antibody"); ii) a transmembrane domain fused to the C-terminus of the present single-chain antibody; and iii) an immunocompetent cell activation signaling domain fused to the C-terminus of the transmembrane domain; wherein the present single-chain antibody and the transmembrane domain, or the transmembrane domain and the immunocompetent cell activation signaling domain, may be fused via a peptide linker or an IgG4 hinge region. Furthermore, the present single-chain antibody typically comprises an H-chain variable (V) region comprising the above-mentioned H-chain CDRs 1 to 3, and an L-chain V region comprising the above-mentioned L-chain CDRs 1 to 3, and wherein the H-chain V region and the L-chain V region are typically linked via a peptide linker.

[0017] The length of the peptide linker can be, for example, 1 to 100 amino acid residues, preferably 10 to 50. Specific examples of the peptide linker in the present antibody include those in which three consecutive amino acid sequences each consisting of one to four glycines (G) and one serine (S) are linked together.

[0018] The V region of the present single-chain antibody includes framework regions (FRs) other than CDR1 to CDR3. Examples of the H-chain FRs include H-chain FR1 linked to the N-terminus of H-chain CDR1, H-chain FR2 linked to the C-terminus of H-chain CDR1 (N-terminus of H-chain CDR2), H-chain FR3 linked to the C-terminus of H-chain CDR2 (N-terminus of H-chain CDR3), and H-chain FR4 linked to the C-terminus of H-chain CDR3. Examples of the L-chain FRs include L-chain FR1 linked to the N-terminus of L-chain CDR1, L-chain FR2 linked to the C-terminus of L-chain CDR1 (N-terminus of L-chain CDR2), L-chain FR3 linked to the C-terminus of L-chain CDR2 (N-terminus of L-chain CDR3), and L-chain FR4 linked to the C-terminus of L-chain CDR3.

[0019] Specific examples of the above-mentioned H chain FR1 include a polypeptide consisting of the 1st to 30th amino acid residues of the amino acid sequence shown in SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of said polypeptide.

[0020] Specific examples of the H chain FR2 include a polypeptide consisting of the 36th to 49th amino acid residues of the amino acid sequence shown in SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of the polypeptide.

[0021] Specific examples of the H chain FR3 include a polypeptide consisting of the 67th to 98th amino acid residues of the amino acid sequence shown in SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of the polypeptide.

[0022] Specific examples of the H chain FR4 include a polypeptide consisting of amino acid residues 105 to 114 of the amino acid sequence set forth in SEQ ID NO: 7, or a polypeptide consisting of an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of said polypeptide.

[0023] Specific examples of the above-mentioned L chain FR1 include a polypeptide consisting of the 1st to 23rd amino acid residues of the amino acid sequence shown in SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of said polypeptide.

[0024] Specific examples of the L chain FR2 include a polypeptide consisting of the 40th to 54th amino acid residues of the amino acid sequence shown in SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence having at least 80% sequence identity with the amino acid sequence of said polypeptide.

[0025] Specific examples of the L chain FR3 include a polypeptide consisting of the 62nd to 93rd amino acid residues of the amino acid sequence shown in SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of the polypeptide.

[0026] Specific examples of the L chain FR4 include a polypeptide consisting of amino acid residues 103 to 113 of the amino acid sequence shown in SEQ ID NO: 8, or a polypeptide consisting of an amino acid sequence that has at least 80% sequence identity with the amino acid sequence of said polypeptide.

[0027] The FRs of the present single-chain antibodies are preferably those of known human antibodies. Examples of such "known human antibody FRs" include FRs of human antibodies registered in known sequence databases such as GenBank, and FRs selected from consensus sequences derived from various human antibody subgroups (Human Most Homologous Consensus Sequences; Kabat, EA et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health and Human Services, 1991).

[0028] In the present single-chain antibody, the heavy chain CDR1 is typically located at positions H31 to H35 according to the Kabat numbering system (see Kabat, EA et al., (1991) NIH Publication No. 91-3242, Sequences of Proteins of Immunological Interest). The heavy chain CDR2 is typically located at positions H50 to H52, H52A, and H53 to H65 according to the Kabat numbering system. The heavy chain CDR3 is typically located at positions H95 to H100, H100A, H100B, H101, and H102 according to the Kabat numbering system. The light chain CDR1 is typically located at positions L24 to L34 according to the Kabat numbering system. The light chain CDR2 is typically located at positions L50 to L56 according to the Kabat numbering system. Furthermore, the L-chain CDR3 in the present single-chain antibody is generally located at positions L89 to L97 according to the Kabat numbering system.

[0029] A specific example of the H chain V region in the present single-chain antibody is an H chain V region consisting of an amino acid sequence that has at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and a specific example of the L chain V region in the present single-chain antibody is an L chain V region consisting of an amino acid sequence that has at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0030] Specific examples of the present single-chain antibody include those whose effects have been demonstrated in the present Example described below, namely, single-chain antibodies comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 12 and single-chain antibodies comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 13.

[0031] The above-mentioned cell transmembrane domain may be any peptide capable of penetrating a cell membrane, and examples thereof include all or part of the cell transmembrane domain derived from CD8, the α and β chains of the T cell receptor, CD3ζ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, EGFR (Epidermal Growth Factor Receptor), or GITR. A specific example is the human CD8 cell transmembrane domain consisting of amino acid residues 1 to 83 of the amino acid sequence set forth in SEQ ID NO: 14. Furthermore, the above-mentioned cell transmembrane domain may be a domain in which 1 to 10 amino acid residues, preferably 6 to 7 amino acid residues, have been deleted from the C-terminus of a peptide capable of penetrating a cell membrane. Examples of such domains include variant 1 of the human CD8 cell transmembrane domain, which consists of amino acid residues 1 to 77 of the amino acid sequence set forth in SEQ ID NO: 14, and variant 2 of the human CD8 cell transmembrane domain, which consists of amino acid residues 1 to 76 of the amino acid sequence set forth in SEQ ID NO: 14.

[0032] The immunocompetent cell activation signaling region may be any region capable of transmitting signals into immunocompetent cells upon binding of the present single-chain antibody to human GPC3. Preferably, the immunocompetent cell activation signaling region comprises at least one or more polypeptides selected from the group consisting of CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3ζ, and the intracellular domain of Fc Receptor-associated γ chain. More preferably, the immunocompetent cell activation signaling region comprises three polypeptides: CD28, 4-1BB, and CD3ζ. A specific example of such a CD28 intracellular domain polypeptide is a human CD28 intracellular domain polypeptide consisting of amino acid residues 85 to 124 of the amino acid sequence set forth in SEQ ID NO: 14. Furthermore, a specific example of the "4-1BB intracellular domain polypeptide" is a human 4-1BB intracellular domain polypeptide consisting of amino acid residues 125 to 170 of the amino acid sequence set forth in SEQ ID NO: 14. Furthermore, specific examples of the polypeptide of the intracellular region of CD3ζ include the polypeptide of the intracellular region of human CD3ζ consisting of amino acid residues 172 to 283 of the amino acid sequence shown in SEQ ID NO:14.

[0033] The arginine (Arg) at position 84 of the amino acid sequence shown in SEQ ID NO: 14, the arginine at position 78 of the amino acid sequence shown in SEQ ID NO: 15, and the arginine at position 77 of the amino acid sequence shown in SEQ ID NO: 16 are consensus sequences between the polypeptide of the human CD8-derived transmembrane domain and the polypeptide of the intracellular domain of human CD28. The leucine (Leu) at position 171 of the amino acid sequence shown in SEQ ID NO: 14, the leucine at position 165 of the amino acid sequence shown in SEQ ID NO: 15, and the leucine at position 164 of the amino acid sequence shown in SEQ ID NO: 16 are consensus sequences between the polypeptide of the intracellular domain of human 4-1BB and the polypeptide of the intracellular domain of human CD3ζ.

[0034] As used herein, the term "immunocompetent cells" refers to cells (preferably cells isolated from a living organism) that perform immune functions in a living organism. Examples of immunocompetent cells include lymphoid cells such as T cells, natural killer cells (NK cells), and B cells; antigen-presenting cells such as monocytes, macrophages, and dendritic cells; and granulocytes such as neutrophils, eosinophils, basophils, and mast cells. Specific examples include T cells derived from mammals such as humans, dogs, cats, pigs, and mice, with human T cells being particularly preferred. Furthermore, T cells can be obtained by isolating a cell population containing immunocompetent cells from body fluids such as blood and bone marrow fluid, tissues such as the spleen, thymus, and lymph nodes, or cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites. Furthermore, to increase the proportion of T cells in the cell population, the isolated cell population can be further isolated or purified by standard methods, as needed. Furthermore, T cells prepared from embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS cells) may also be used. Such T cells include alpha-beta T cells, gamma-delta T cells, and CD8 + T cells, CD4 + Examples of such immunocompetent cells include T cells, tumor-infiltrating T cells, memory T cells, naive T cells, and NKT cells. The origin of the immunocompetent cells may be the same as or different from the recipient. Furthermore, when the recipient is a human, the immunocompetent cells may be autologous cells collected from the patient themselves, or allogeneic cells collected from another person. That is, the donor and recipient may be matched or mismatched, but matching is preferred.

[0035] The subjects of administration are preferably mammals or mammalian cells, and among such mammals, more preferably humans, mice, dogs, rats, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, monkeys, and chimpanzees, with humans being particularly preferred.

[0036] The present CAR is preferably used in cancer treatment by expressing it ex vivo on the cell surface of immunocompetent cells collected from cancer patients. When T cells are used as the immunocompetent cells, a specific example of a peptide consisting of the transmembrane domain of the present CAR and an immunocompetent cell activation signal transduction domain fused to the C-terminus of the transmembrane domain is a peptide containing an amino acid sequence having at least 80% sequence identity with any of the amino acid sequences set forth in SEQ ID NOs: 14 to 16. Specific examples of the present CAR include those comprising the present single-chain antibody and a peptide linked to the C-terminus of the single-chain antibody, the peptide having an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 14; those comprising the present single-chain antibody and a peptide linked to the C-terminus of the single-chain antibody, the peptide having an amino acid sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 15; and those comprising the present single-chain antibody and a peptide linked to the C-terminus of the single-chain antibody, the peptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0037] The immunocompetent cells of the present invention may be any immunocompetent cells that express the present CAR. Because the present CAR does not normally exist in nature, the immunocompetent cells are immunocompetent cells that express an exogenous CAR, rather than an endogenous CAR. Preferably, the immunocompetent cells also express IL-7 and / or CCL19. When the immunocompetent cells are cells (e.g., T cells) that do not express IL-7 and / or CCL19, or when the immunocompetent cells are cells other than T cells that express low levels of IL-7 and / or CCL19, the immunocompetent cells preferably express exogenous IL-7 and / or CCL19. Here, immunocompetent cells that express exogenous IL-7 and / or CCL19 can be obtained by introducing an exogenous IL-7 gene and / or CCL19 gene (preferably, an exogenous IL-7 gene and / or CCL19 gene operably linked downstream of a promoter) into immunocompetent cells. Immunocompetent cells obtained by introducing an exogenous IL-7 gene and / or CCL19 gene contain the exogenous IL-7 gene and / or CCL19 gene in the immunocompetent cells, either integrated into the genome or not integrated into the genome (e.g., episomal). The immunocompetent cells also include cell cultures expressing the CAR obtained by culturing the immunocompetent cells.

[0038] The present immunocompetent cells can be produced by introducing the present CAR expression vector into immunocompetent cells. The introduction method may be any method for introducing DNA into immunocompetent cells, such as electroporation (Cytotechnology, 3, 133 (1990)), calcium phosphate method (JP Patent Publication No. 2-227075), lipofection (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), or viral infection. Examples of such viral infection methods include transfecting a CAR expression vector (WO 2016 / 056228) and a packaging plasmid into packaging cells such as GP2-293 cells (Takara Bio), Plat-GP cells (Cosmo Bio), PG13 cells (ATCC CRL-10686), and PA317 cells (ATCC CRL-9078) to produce a recombinant virus, and then infecting T cells with the recombinant virus. The immunocompetent cells obtained by introducing the CAR expression vector (the immunocompetent cells) contain the CAR gene either integrated into the genome or not integrated into the genome.

[0039] Alternatively, the immunocompetent cells may be produced by incorporating nucleotides encoding the CAR into the genome of the cells so that the CAR can be expressed under the control of an appropriate promoter using known gene editing techniques, such as techniques using endonucleases such as zinc finger nucleases, TALENs (transcription activation-like effector nucleases), and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas systems.

[0040] The present anticancer agent may be a composition containing the present immunocompetent cells and a pharmaceutically acceptable additive, the use of which is specified as "anticancer (i.e., suppressing cancer growth)." Examples of pharmaceutically acceptable additives include saline, buffered saline, cell culture medium, dextrose, water for injection, glycerol, ethanol, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonicity agents, fillers, and lubricants. The additives may be a single additive or a mixture containing multiple (at least two) additives.

[0041] The present anticancer agent can be administered to a subject (cancer patient) who requires inhibition of cancer growth (in other words, cancer treatment) using methods known to those skilled in the art. Examples of administration methods include intravenous, intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarticular, intramedullary, intracardiac, intraarticular, intrasynovial, intracranial, intrathecal, subarachnoid (spinal fluid) injection, and administration to the tumor site using a catheter.

[0042] The number of the immunocompetent cells contained in the anticancer agent can be appropriately selected based on the type, location, and severity of cancer, as well as the age, weight, and condition of the subject receiving treatment. For example, 1 × 10 4 ~1×10 10 pieces, preferably 1 x 10 5 ~1×10 9 pieces, more preferably 5 x 10 6 ~5×10 8 There are individuals.

[0043] The anticancer drug may be administered, for example, four times, three times, two times, or once a day, and the administration interval may be, for example, every other day, every two days, every three days, every four days, every five days, every six days, every seven days, every eight days, every nine days, or every month.

[0044] Cancers to which the present anticancer agent can be administered include, for example, colorectal cancer (colon cancer or rectal cancer); gastric cancer; liver cancer; brain tumor; lung cancer (adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma); esophageal cancer; duodenal cancer; small intestine cancer; skin cancer; breast cancer; prostate cancer; bladder cancer; vaginal cancer; cervical cancer; uterine cancer; kidney cancer; pancreatic cancer; spleen cancer; tracheal cancer; bronchial cancer; head and neck cancer; gallbladder cancer; bile duct cancer; sperm cancer. Examples of cancer include follicular carcinoma; ovarian cancer; cancers in bone tissue, cartilage tissue, adipose tissue, muscle tissue, nerve tissue, vascular tissue, or hematopoietic tissue (specifically, sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma; germ cell tumors; lymphomas; and leukemia).

[0045] The present anticancer agent can also be used in combination with other anticancer agents, such as alkylating agents such as cyclophosphamide, bendamustine, iosfamide, and dacarbazine, antimetabolites such as pentostatin, fludarabine, cladribine, methotrexate, 5-fluorouracil, 6-mercaptopurine, and enocitabine, molecular targeted drugs such as rituximab, cetuximab, and trastuzumab, and kinase inhibitors such as imatinib, gefetinib, erlotinib, afatinib, dasatinib, sunitinib, and trametinib. anticancer antibiotics such as idarubicin, doxorubicin, and mitomycin C; plant alkaloids such as irinotecan and etoposide; platinum preparations such as cisplatin, oxaliplatin, and carboplatin; hormone therapy drugs such as tamoxifen and bicaludamide; and immunosuppressants such as interferon, nivolumab, and pembrolizumab.

[0046] The above-mentioned method of "using the present anticancer agent in combination with another anticancer agent" includes a method of treating with another anticancer agent and then using the present anticancer agent, a method of using the present anticancer agent and another anticancer agent simultaneously, and a method of treating with the present anticancer agent and then using another anticancer agent. Furthermore, when the present anticancer agent is used in combination with another anticancer agent, the cancer treatment effect is further improved, and by reducing the number of administrations or the dosage of each, it is possible to reduce the side effects caused by each agent.

[0047] The CAR gene of the present invention is not particularly limited as long as it is an antibody gene (nucleotide) that encodes a polypeptide comprising at least i) the present single-chain antibody, ii) a transmembrane domain fused to the C-terminus of the present single-chain antibody, and iii) i) to iii) an immunocompetent cell activation signal transduction domain fused to the C-terminus of the transmembrane domain. Specific examples of the antibody gene encoding the present single-chain antibody include an H chain V region gene consisting of a nucleotide sequence that has at least 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 9 (a gene encoding an H chain V region consisting of an amino acid sequence that has at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 7), and an L chain V region gene consisting of a nucleotide sequence that has at least 80% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 10 (a gene encoding an L chain V region consisting of an amino acid sequence that has at least 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 8).

[0048] As used herein, the term "identity" refers to the degree of polypeptide or polynucleotide sequence similarity, as determined by the match between a query sequence and another sequence, preferably of the same type (nucleic acid or protein sequence). Preferred computer program methods for calculating and determining "identity" include, for example, GCG BLAST (Basic Local Alignment Search Tool) (Altschulet et al., J. Mol. Biol. 1990, 215:403-410; Altschulet et al., Nucleic Acids Res. 1997, 25:3389-3402; Devereux et al., Nucleic Acids Res. 1984, 12:387), and BLASTN 2.0 (Gish W., http: / / blast.Wustl.edu, 1996-2002), and FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 1988, 85:2444-2448), and GCG BLASTN 2.0 (Gish W., http: / / blast.Wustl.edu, 1996-2002), which determines and aligns the longest overlapping pair of contigs. Mention may be made of GelMerge (Wibur and Lipman, SIAM J. Appl. Math. 1984, 44:557-567; Needleman and Wunsch, J. Mol. Biol. 1970, 48:443-453).

[0049] As used herein, "at least 80% identity" means identity of 80% or more, preferably 85% or more, more preferably 88% or more, even more preferably 90% or more, still more preferably 93% or more, particularly preferably 95% or more, especially more preferably 98% or more, and most preferably 100% identity.

[0050] As used herein, "an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: X" refers to an amino acid sequence in which 0, 1, or several amino acid residues have been deleted, substituted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: X, and which has the same function as the amino acid sequence shown in SEQ ID NO: X. Here, "an amino acid sequence in which one or several amino acid residues have been deleted, substituted, inserted, and / or added" refers to an amino acid sequence in which, for example, 1 to 30 amino acid residues have been deleted, substituted, inserted, and / or added, preferably 1 to 20 amino acid residues, more preferably 1 to 15 amino acid residues, even more preferably 1 to 10 amino acid residues, even more preferably 1 to 5 amino acid residues, even more preferably 1 to 3 amino acid residues, and even more preferably 1 to 2 amino acid residues have been deleted, substituted, inserted, and / or added. Mutation of these amino acid residues can be carried out by any method known to those skilled in the art, such as chemical synthesis, genetic engineering, or mutagenesis.

[0051] As used herein, "a gene operably linked downstream of a promoter" means that promoter DNA and gene DNA are functionally linked so that the promoter can initiate transcription of the gene.

[0052] The promoter in the present CAR expression vector may be any region that initiates transcription of the mRNA encoded by the present CAR gene located downstream of the promoter, and the promoter usually includes a transcription start site (TSS).

[0053] The type of promoter and vector in the present CAR expression vector can be appropriately selected depending on the type of host cell (immunocompetent cell) to be introduced, and examples of promoters include the cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 early promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, SRα promoter, NFAT promoter, HIF promoter, etc. Furthermore, examples of vectors include retrovirus vectors such as the pMSGV vector (Tamada k et al., Clin Cancer Res 18:6436-6445 (2002)) and the pMSCV vector (Takara Bio Inc.), as well as vectors derived from such vectors.

[0054] The CAR expression vector may further include the nucleotide sequence of an enhancer region or ribosome binding site (RBS) to further enhance gene expression efficiency, or may further include a drug resistance gene appropriate for the type of immunocompetent cell (e.g., spectinomycin resistance gene, chloramphenicol resistance gene, tetracycline resistance gene, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, hygromycin resistance gene, blasticidin resistance gene, geneticin resistance gene, etc.) for screening of the immunocompetent cell. Such an enhancer region is typically located upstream of the promoter, and the RBS is typically located between the promoter and the CAR gene. The nucleotide sequence of the CAR gene in the CAR expression vector may have its codon sequence optimized to suit the immunocompetent cell to be expressed. The CAR, the CAR expression vector, and the CAR gene can be produced by known methods using genetic recombination techniques.

[0055] The CAR expression vector may further comprise a gene other than the CAR gene, such as the IL-7 gene, the CCL19 gene, or a gene encoding a polypeptide with apoptotic activity (a suicide gene) (preferably, the IL-7 gene, the CCL19 gene, or the suicide gene are operably linked downstream of a promoter). Examples of such suicide genes include the gene encoding herpes simplex virus thymidine kinase (HSV-TK) and the gene encoding inducible caspase 9. By incorporating a suicide gene into the CAR expression vector, it is possible to kill unnecessary immunocompetent cells in the body by administering a drug that activates the function of the polypeptide encoded by the suicide gene (e.g., ganciclovir in the case of HSV-TK, or AP1903, a chemical induction of dimerization (CID) compound in the case of inducible caspase-9) depending on the course of cancer treatment, for example, when cancer cells are eliminated (Cooper LJ, et al. Cytotherapy. 2006;8(2):105-17, Jensen MC et al. Biol Blood Marrow Transplant. 2010 Sep;16(9):1245-56, Jones BS. Front Pharmacol. 2014 Nov 27;5:254, Minagawa K., Pharmaceuticals (Basel). 2015 May 8;8(2):230-49, Bole-Richard E., Front Pharmacol. 2015 Aug 25;6:174).

[0056] All references cited herein, including journal articles, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0057] The present invention will be described in more detail below with reference to the following examples, but the technical scope of the present invention is not limited to these examples. In the following examples, the GP2-293 packaging cell line (manufactured by Takara Bio Inc.) was cultured in a CO2 incubator (37°C) in the presence of DMEM (Dulbecco's Modified Eagle's Medium) culture medium containing 10% fetal bovine serum (FBS), penicillin G, and streptomycin sulfate. Human T cells were cultured in a CO2 incubator (37°C) in the presence of GT-T551 (manufactured by Takara Bio Inc.) culture medium containing penicillin G (100 Units / mL) and other ingredients. [Example]

[0058] 1. Material Preparation [Construction of retroviral vectors expressing CAR, hIL-7, hCCL19, and HSV-TK] The H chain V region (V) of the anti-GPC3 antibody (GC33 antibody) developed by a domestic pharmaceutical manufacturer H ) and L chain V region (V L Based on the amino acid sequence of the anti-GPC3 human scFv (V H -Linker-V L [a polypeptide consisting of the amino acid sequence of SEQ ID NO: 12], and V L -Linker-V H [A polypeptide consisting of the amino acid sequence of SEQ ID NO: 13] was designed (see Table 1). The linker used was composed of 15 amino acid residues, with the polypeptide "GGGGS" repeated three times.

[0059] [Table 1] In the table, double squares indicate linkers, and single underlines indicate V H and double underline indicates V L Shows.

[0060] Of the two anti-GPC3 human scFvs designed, V H -Linker-V L(a polypeptide consisting of the amino acid sequence of SEQ ID NO: 12) was selected as an example, and a fusion peptide (amino acid residues 243 to 525 of the amino acid sequence of SEQ ID NO: 17) consisting of a human CD8-derived transmembrane domain and a human CD28 / 4-1BB / CD3zeta-derived immunocompetent cell activation signaling domain was linked downstream to design an anti-GPC3scFv CAR consisting of the amino acid sequence of SEQ ID NO: 17 (see Table 2).

[0061] [Table 2] In the table, single underlines indicate anti-GPC3 human scFv, and double underlines indicate fusion peptides consisting of a human CD8-derived transmembrane domain and a human CD28 / 4-1BB / CD3zeta-derived immunocompetent cell activation signaling domain.

[0062] Furthermore, a human immunoglobulin H chain-derived signal sequence (amino acid residues 1 to 19 of SEQ ID NO: 18) was linked upstream of the designed anti-GPC3 scFv CAR, and further, a 2A self-cleaving peptide (amino acid residues 551 to 575 of the amino acid sequence of SEQ ID NO: 18), human (h)IL-7 (amino acid residues 577 to 753 of the amino acid sequence of SEQ ID NO: 18), a 2A self-cleaving peptide (amino acid residues 754 to 778 of the amino acid sequence of SEQ ID NO: 18), hCCL19 (amino acid residues 779 to 876 of the amino acid sequence of SEQ ID NO: 18), a 2A self-cleaving peptide (amino acid residues 877 to 901 of the amino acid sequence of SEQ ID NO: 18), and herpes simplex virus thymidine kinase (HSV-TK) (amino acid residues 902 to 1277 of the amino acid sequence of SEQ ID NO: 18) were linked downstream thereof, in this order, to form a fusion polypeptide (anti-GPC3 scFv) consisting of the amino acid sequence of SEQ ID NO: 18. CAR+hIL-7+hCCL19+HSV-TK) was designed (see Table 3).

[0063] [Table 3] In the table, a single box indicates a signal sequence derived from human immunoglobulin H chain, a double box indicates a 2A self-cleaving peptide, a single underline indicates CAR, a double underline indicates hIL-7, a dashed underline indicates hCCL19, and a wavy underline indicates HSV-TK.

[0064] Then, a gene DNA (a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 19, see Table 4) encoding the above fusion polypeptide (CAR + hIL-7 + hCCL19 + HSV-TK) and consisting of a nucleotide sequence optimized for human codons was incorporated into the pMSGV retroviral vector (Tamada k et al., Clin Cancer Res 18:6436-6445(2002)) to prepare a retroviral vector for expressing CAR, hIL-7, hCCL19, and HSV-TK (hereinafter referred to as "retroviral vector for expressing anti-GPC3scFv CAR, etc.") (see WO 2016 / 056228 pamphlet).

[0065] [Table 4] TIFF0007737740000005.tif230154TIFF0007737740000006.tif58155In the table, a single box indicates a gene encoding a human immunoglobulin H chain-derived signal sequence, a double box indicates a gene encoding a 2A self-cleaving peptide, a single underline indicates a gene encoding CAR, a double underline indicates a gene encoding hIL-7, a dashed underline indicates a gene encoding hCCL19, and a wavy underline indicates a gene encoding HSV-TK.

[0066] [Production of recombinant retroviruses] The prepared retroviral vector for expressing anti-GPC3 scFv CAR, etc. was transfected into the GP2-293 packaging cell line (Takara Bio Inc.) using Lipofectamine 3000 (Life Technologies). 48 hours after transfection, the culture supernatant containing the produced recombinant retrovirus (retrovirus for expressing anti-GPC3 scFv CAR, etc.) was collected.

[0067] [Infection of recombinant retroviruses into T cells] To generate anti-GPC3 scFv CAR-expressing T cells, T cells were infected with a recombinant retrovirus (anti-GPC3 scFv CAR expression retrovirus) to introduce the anti-GPC3 scFv CAR expression retroviral vector into the T cells. Specifically, peripheral blood mononuclear cells were isolated from the peripheral blood of healthy individuals according to standard methods, and then activated and cultured for 48 hours in the presence of immobilized anti-CD3 monoclonal antibody clone (OKT3) (5 μg / mL), RetroNectin (registered trademark; Takara Bio Inc., 25 μg / mL), and IL-2. A virus adsorption plate was prepared by adding culture supernatant containing the recombinant retrovirus for expression of anti-GPC3 scFv CAR to a RetroNectin-coated plate and centrifuging at 4°C and 2000 g for 2 hours. 1 × 10 human T cells were then placed on the prepared virus adsorption plate. 5 The cells were seeded at 1000 cells / mL and cultured overnight in the presence of IL-2. The next day, the T cells were collected and seeded on a new virus-adsorbed plate for a second round of viral infection. After 4 hours of culture, the cells were collected, three times the volume of culture medium was added, and the cells were seeded on a cell culture plate and cultured for 3 days.

[0068] 2. Method [Flow cytometry analysis] Flow cytometry analysis was performed to confirm that human T cells infected with retroviruses expressing anti-GPC3 scFv CARs expressed anti-GPC3 scFv CARs. Specifically, human T cell populations infected with retroviruses expressing anti-GPC3 scFv CARs (including anti-GPC3 scFv CAR-T cells) were incubated on ice for 30 minutes in the presence of biotinylated recombinant GPC3 protein (R&D). After adding FACS Buffer (1% BSA / PBS solution) and centrifuging, the supernatant was removed. After adding APC-labeled anti-CD8 antibody (Biolegend) and BV421-labeled streptavidin (Biolegend), the mixture was incubated on ice for 30 minutes. Detection of APC and BV421 and measurement of their fluorescence levels were performed using a flow cytometer (BD LSR Fortessa) (BD Biosciences). As a negative control, a human T cell group (T cell group) not infected with a retrovirus for expressing anti-GPC3 scFv CAR, etc. was also subjected to flow cytometry analysis in the same manner.

[0069] [Cytotoxicity against GPC3-expressing cancer cells] To confirm the cytotoxic activity of anti-GPC3 scFv CAR-T cells, GPC3-expressing cancer cells were cultured in the presence of T cells containing anti-GPC3 scFv CAR-T cells. Specifically, the T cells containing anti-GPC3 scFv CAR-T cells were cultured at a 1:1 ratio (1 × 10) with a hepatocellular carcinoma-derived cell line (Sk-HEP-1 cells) (purchased from ECACC) or Sk-HEP-1 cells expressing GPC3 (Sk-HEP-1GPC3 cells). 5The cells were mixed at a concentration of 1000 cells / well and cultured in a 24-well plate. After 48 hours, the cells were harvested and incubated on ice for 30 minutes in the presence of a phycoerythrin-labeled anti-CD45 antibody (Biolegend). FACS buffer (1% BSA / PBS solution) was added, the mixture was centrifuged, and the supernatant was removed. Phycoerythrin detection and fluorescence level analysis were performed using a flow cytometer (BD LSR Fortessa) (BD Biosciences). The number and percentage of CD45-positive cells (corresponding to T cells) and CD45-negative cells (corresponding to remaining Sk-HEP-1GPC3 cells) were measured.

[0070] [IFN-γ production ability of anti-GPC3 scFv CAR-T cells] We analyzed whether anti-GPC3 scFv CAR-T cells have the ability to produce interferon-γ (IFN-γ). Specifically, a T cell group containing anti-GPC3 scFv CAR-T cells ("CAR-T cells" in Figure 3) or a gene-untransfected T cell group ("T cells" in Figure 3) was cocultured with Sk-HEP-1 cells or Sk-HEP-1GPC3 cells at a 1:1 ratio (1 × 10 5 The mixture was mixed at a concentration of 1000 μg / well and cultured in a 24-well plate for 48 hours, and the concentration of IFN-γ produced in the culture supernatant was measured by ELISA (Biolegend).

[0071] [IL-7 and CCL19 production ability of anti-GPC3 scFv CAR-T cells] We analyzed whether anti-GPC3 scFv CAR-T cells have the ability to produce IL-7 and CCL19. Specifically, a T cell group containing anti-GPC3 scFv CAR-T cells ("CAR-T cells" in Figure 4) or a non-transfected T cell group ("T cells" in Figure 4) was cocultured with Sk-HEP-1 cells or Sk-HEP-1GPC3 cells at a 1:1 ratio (1 × 10 5The cells were mixed at a concentration of 1 / well and cultured in a 24-well plate for 48 hours, and the concentrations of IL-7 and CCL19 produced in the culture supernatant were measured by ELISA (R&D). As a negative control, a group of T cells not transfected with the CAR gene (T cells) and Sk-HEP-1 or Sk-HEP-1GPC3 cells were cultured under the same conditions.

[0072] 3.Results [Flow cytometry analysis] Of the human T cell group infected with retrovirus for expressing anti-GPC3 scFv CAR, etc., 48.1% of the cells were shown to be CAR-positive, with BV421 detected (see Figure 1B). On the other hand, almost no CAR-positive cells were observed in the human T cell group not infected with the retrovirus (see Figure 1A). These results indicate that of the human T cell group infected with retrovirus for expressing anti-GPC3 scFv CAR, etc., approximately 50% of the cells were T cells expressing anti-GPC3 scFv CAR (anti-GPC3 scFv CAR-T cells).

[0073] [Cytotoxicity against GPC3-expressing cancer cells] Compared to T cell groups not infected with anti-GPC3 scFv CAR-T retrovirus, T cell groups containing anti-GPC3 scFv CAR-T cells exhibited significantly higher cytotoxicity against Sk-HEP-1 GPC3 cells ("GPC3" in Figure 2), killing almost all Sk-HEP-1 GPC3 cells (see Figure 2). On the other hand, T cell groups containing anti-GPC3 scFv CAR-T cells, like the T cell group, showed almost no cytotoxicity against Sk-HEP-1 cells ("mock" in Figure 2) (see Figure 2). These results demonstrate that anti-GPC3 scFv CAR-T cells exert cytotoxic activity specifically against cancer cells expressing GPC3.

[0074] [IFN-γ production ability of anti-GPC3 scFv CAR-T cells] In the presence of Sk-HEP-1 GPC3 cells, the T cell group containing anti-GPC3 scFv CAR-T cells exhibited significantly higher IFN-γ production than the T cell group not infected with retrovirus for expressing anti-GPC3 scFv CAR (see Figure 3). On the other hand, in the presence of Sk-HEP-1 cells, the T cell group containing anti-GPC3 scFv CAR-T cells showed almost no IFN-γ production, similar to the T cell group (see Figure 3). This result indicates that the anti-GPC3 scFv in the anti-GPC3 scFv CAR-T cells bound to the GPC3 surface antigen on cancer cells, activating them and resulting in the production of IFN-γ.

[0075] [IL-7 and CCL19 production ability of anti-GPC3 scFv CAR-T cells] T cell groups containing anti-GPC3 scFv CAR-T cells produced low levels of IL-7 and CCL19 in the presence of Sk-HEP-1 cells, but produced even higher levels of IL-7 and CCL19 in the presence of Sk-HEP-1 GPC3 cells (see Figure 4). In T cell groups not infected with retroviruses expressing anti-GPC3 scFv CARs, IL-7 and CCL19 production was barely observed in either the presence of Sk-HEP-1 or Sk-HEP-1 GPC3 cells. These results demonstrate that anti-GPC3 scFv CAR-T cells produce IL-7 and CCL19. [Industrial Applicability]

[0076] The present invention contributes to the field of cancer immunotherapy using immunocompetent cells.

Claims

1. expressing chimeric antigen receptor (CAR), interleukin 7 (IL-7), and chemokine ligand 19 (CCL19); the CAR comprises a single chain antibody that specifically binds to a polypeptide derived from human GPC3 (Glypican-3), which consists of the amino acid sequence shown in SEQ ID NO: 11, a transmembrane domain fused to the carboxyl terminus of the single chain antibody, and an immunocompetent cell activation signal transduction domain fused to the carboxyl terminus of the transmembrane domain, the single-chain antibody comprises a heavy chain complementarity-determining region (CDR) 1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6; the single-chain antibody comprises a heavy chain variable region consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, The single-chain antibody comprises a light chain variable region consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

8. immunocompetent cells.

2. Single chain antibodies an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 12; or An amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 13, The immunocompetent cell according to claim 1.

3. The immunocompetent cell according to claim 1 or 2, wherein the transmembrane domain and the immunocompetent cell activation signal transduction domain fused to the carboxyl terminus of the transmembrane domain comprise an amino acid sequence having at least 90% or more sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 14 to 16.

4. An anticancer agent comprising the immunocompetent cells according to any one of claims 1 to 3 and a pharmaceutically acceptable additive.

5. a promoter; a CAR gene encoding a CAR operably linked downstream of the promoter; a gene encoding IL-7; and a gene encoding CCL19; the CAR comprises a single chain antibody that specifically binds to a polypeptide derived from human GPC3 (Glypican-3), which consists of the amino acid sequence shown in SEQ ID NO: 11, a transmembrane domain fused to the carboxyl terminus of the single chain antibody, and an immunocompetent cell activation signal transduction domain fused to the carboxyl terminus of the transmembrane domain, the single-chain antibody comprises a heavy chain complementarity-determining region (CDR) 1 consisting of the amino acid sequence shown in SEQ ID NO: 1, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6; the single-chain antibody comprises a heavy chain variable region consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, The single-chain antibody comprises a light chain variable region consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

8. vector.

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