Chimeric target factor receptor

Abstract

The purpose of the present invention is to provide a chimeric target factor receptor which can activate an antigen presenting cell, e.g., a dendritic cell, in a target-factor-specific manner. A chimeric target factor receptor is provided, which contains a target factor binding domain, a transmembrane domain, and an intracellular domain comprising a TLR intracellular domain.

Description

[Technical Field]

[0001] This invention relates to chimeric target factor receptors, etc. [Background technology]

[0002] Chimeric antigen receptors bind to antigens according to the specificity of the antibody, thereby inducing damage to target cells, cytokine release, and post-stimulation T cell division. Specificity can be conferred to T cells through gene transfer, and cell preparation is generally easier than culturing and amplifying endogenous tumor-specific T cell receptor-positive T cells. Furthermore, therapies utilizing chimeric antigen receptors (CAR-T therapy) can exhibit higher cytotoxic activity compared to antibody therapy.

[0003] Dendritic cells are immune cells that recognize microbial-specific antigens such as bacteria and viruses and play a role in activating innate immunity. Focusing on this immune-activating ability, "dendritic cell vaccine therapy," which involves returning dendritic cells that have recognized cancer antigens back into the body, is also being attempted. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] US 2017 / 0151281 A1 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] CAR-DCs incorporating chimeric antigen receptors into dendritic cells have been reported in Patent Document 1. However, there are no reports that have examined the composition of the intracellular domain in detail.

[0006] The present invention aims to provide a chimeric target factor receptor that can specifically activate antigen-presenting cells such as dendritic cells based on target factors. [Means for solving the problem]

[0007] In view of the above problems, the inventors diligently conducted research and found that a chimeric target factor receptor comprising a target factor-binding domain, a transmembrane domain, and an intracellular domain including a TLR intracellular domain can solve the above problems. Based on this finding, further research led to the completion of the present invention. That is, the present invention encompasses the following aspects.

[0008] Item 1. A chimeric target factor receptor comprising a target factor-binding domain, a transmembrane domain, and an intracellular domain including a TLR intracellular domain.

[0009] Item 2. The chimeric target factor receptor according to Item 1, wherein the intracellular TLR domain is at least one selected from the group consisting of the TLR2 intracellular domain, the TLR3 intracellular domain, and the TLR9 intracellular domain.

[0010] Item 3. The chimeric target factor receptor according to claim 1 or 2, wherein the intracellular TLR domain is at least one selected from the group consisting of the TLR3 intracellular domain and the TLR9 intracellular domain.

[0011] Item 4. A chimeric target factor receptor according to any one of items 1 to 3, wherein the intracellular domain further comprises a CD40 intracellular domain.

[0012] Item 5. A chimeric target factor receptor according to any one of items 1 to 4, wherein the structure of the target factor-binding domain is a single-chain antibody structure.

[0013] Item 6. A chimeric target factor receptor according to any one of items 1 to 5, wherein the target factor is at least one selected from the group consisting of cancer antigens, cytokine receptors, and cell growth factor receptors.

[0014] Item 7. A polynucleotide encoding a chimeric target factor receptor as described in any of items 1-6.

[0015] Item 8. Cells containing the polynucleotides described in Item 7.

[0016] Item 9. The cell according to Item 8, which is an antigen-presenting cell.

[0017] Item 10. The cell according to Claim 8 or 9, which is a dendritic cell.

[0018] Item 11. A pharmaceutical composition comprising the cell according to any one of Items 8 to 10.

[0019] Item 12. The pharmaceutical composition according to Item 11, which is for the treatment, prevention, or amelioration of at least one selected from the group consisting of cancer, autoimmune diseases, allergic diseases, and infectious diseases.

[0020] Item 12A. A method for treating, preventing, or ameliorating at least one selected from the group consisting of cancer, autoimmune diseases, allergic diseases, and infectious diseases, comprising administering the cell according to any one of Items 8 to 10 to a subject or patient suffering from at least one selected from the group consisting of cancer, autoimmune diseases, allergic diseases, and infectious diseases.

[0021] Item 12B. The cell according to any one of Items 8 to 10, for use as a pharmaceutical composition for the treatment, prevention, or amelioration of at least one selected from the group consisting of cancer, autoimmune diseases, allergic diseases, and infectious diseases.

[0022] Item 12C. Use of the cell according to any one of Items 8 to 10 for producing a pharmaceutical composition for the treatment, prevention, or amelioration of at least one selected from the group consisting of cancer, autoimmune diseases, allergic diseases, and infectious diseases.

[0023] Item 13. The pharmaceutical composition according to Item 11 or 12, which is for the treatment, prevention, or amelioration of solid cancer.

[0024] Item 14. The pharmaceutical composition according to Item 13, wherein the TLR intracellular domain is at least one selected from the group consisting of a TLR3 intracellular domain and a TLR9 intracellular domain, and the cell is a dendritic cell.

[0025] Item 15. The pharmaceutical composition according to item 13 or 14, wherein the intracellular domain further comprises a CD40 intracellular domain, and the cell is a dendritic cell.

[0026] Item 16. A pharmaceutical composition according to any one of items 11 to 15, for use in combination with an immune checkpoint inhibitor. [Effects of the Invention]

[0027] According to the present invention, a chimeric target factor receptor can be provided that can specifically activate antigen-presenting cells such as dendritic cells with a target factor. Furthermore, according to the present invention, a polynucleotide encoding the chimeric target factor receptor, cells containing the polynucleotide, a pharmaceutical composition containing the cells, and the like can also be provided. [Brief explanation of the drawing]

[0028] [Figure 1] The results of an in vitro dendritic cell activation test (Test Example 4, co-culture for 5 hours, indicator factor CD80) are shown. "With TLR" indicates the results when the intracellular domain of the TLR (shown in the upper left of the graph) was used, while "Without TLR" indicates the results when the intracellular domain was absent. The horizontal axis represents the CD80 expression level, and the vertical axis represents the cell number. [Figure 2] The results of the in vitro dendritic cell activation test (Test Example 4, co-culture for 24 hours, indicator factor CD80) are shown. The explanation of each notation is the same as in Figure 1. [Figure 3] The results of the in vitro dendritic cell activation test (Test Example 4, co-culture for 48 hours, indicator factor CD80) are shown. The explanation of each notation is the same as in Figure 1. [Figure 4] The results of the in vitro dendritic cell activation test (Test Example 4, co-culture for 5 hours, indicator factor CD86) are shown. The explanation of each notation is the same as in Figure 1. [Figure 5]The results of the in vitro dendritic cell activation test (Test Example 4, co-culture for 24 hours, indicator factor CD86) are shown. In the leftmost column, "With TLR" shows the results when the intracellular domain of the TLR, shown in the upper left of the graph, is used as the intracellular domain, and "Without TLR" shows the results when the intracellular domain is absent. In the middle and rightmost columns, "With TLR+CD40" shows the results when the intracellular domains of the TLR and CD40 (shown in the upper left of the graph, with the left side indicating the cell membrane side) are used as the intracellular domains, and "Without TLR+CD40" shows the results when the intracellular domain is absent. The explanation of the vertical and horizontal axes is the same as in Figure 1. [Figure 6] The results of an in vitro macrophage phagocytosis test (Test Example 5) are shown. The horizontal axis represents the intracellular domain, and Δ represents the case without the intracellular domain. The vertical axis represents the phagocytic activity of the macrophages. [Figure 7] The results of an antitumor efficacy evaluation test (Test Example 6, using MSLN-expressing cell (Pan02 / MSLN) transplanted mice) are shown. Empty represents the case where dendritic cells transducing an empty vector (pMXs-GFP) were administered, MSLN-TLR9 represents the case where chimeric target factor receptor-expressing dendritic cells (Test Example 2: target is MSLN, intracellular domain is the intracellular domain of TLR9) were administered, and Control represents the case where no cells were administered. The vertical axis shows tumor size, and the horizontal axis shows the number of days since the first administration. [Figure 8] The results of the antitumor efficacy evaluation test (Test Example 6, using MSLN-non-expressing cell (Pan02) transplanted mice) are shown. The explanation of each notation is the same as in Figure 7. [Figure 9] The results of the in vitro dendritic cell activation test (Test Example 7, indicator factor MHC class I) are shown. The explanation of each notation is the same as in Figure 1. [Figure 10] The results of the in vitro dendritic cell activation test (Test Example 7, indicator factor MHC class II) are shown. The explanation of each notation is the same as in Figure 1. [Figure 11]The results of the in vitro dendritic cell activation test (Test Example 8, indicator factor CD86) are shown. The explanation of each notation is the same as in Figure 1. [Figure 12] The results of the in vitro dendritic cell activation test (Test Example 8, indicator factor MHC class II) are shown. The explanation of each notation is the same as in Figure 5. [Figure 13] The results of an antitumor efficacy evaluation test (Test Example 9, using MSLN-expressing cell (Pan02 / MSLN) transplanted mice) are shown. "Control" represents the case without cell administration, while "Other" indicates the target of the chimeric target factor receptor (MSLN) and its intracellular domain (e.g., "TLR9" indicates the intracellular domain of TLR9; "Δ" indicates the absence of the intracellular domain). The vertical axis represents tumor size, and the horizontal axis represents the number of days since the first administration. [Figure 14] The results of an antitumor efficacy evaluation test (Test Example 11, using MSLN-expressing cell (Pan02 / MSLN) transplanted mice) are shown. "Control" represents the case without cell administration, while "Other" indicates the target of the chimeric target factor receptor (MSLN) and its intracellular domain (e.g., "TLR9" indicates the intracellular domain of TLR9). The vertical axis represents tumor size, and the horizontal axis represents the number of days since the first administration. [Modes for carrying out the invention]

[0029] 1.Definition In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0030] The "identity" of amino acid sequences refers to the degree of agreement between two or more comparable amino acid sequences. Therefore, the higher the agreement between two amino acid sequences, the higher their identity or similarity. The level of amino acid sequence identity can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on this BLAST algorithm. The specific methods for these analyses are publicly known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). Furthermore, the 'identity' of a nucleotide sequence is defined in accordance with the above.

[0031] In this specification, "conservative substitution" means that an amino acid residue is substituted for an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, are considered conservative substitutions. Other examples of conservative substitutions include amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0032] In this specification, "CDR" means C complementarity D petermining R CDR is an abbreviation for complementarity-determining region, also known as the complementarity-determining region. CDR is a region located in the variable region of an immunoglobulin or T cell receptor, and is deeply involved in the specific binding of an antibody or T cell receptor to an antigen. "Light chain CDR" refers to the CDR located in the light chain variable region of an immunoglobulin, while "heavy chain CDR" refers to the CDR located in the heavy chain variable region of an immunoglobulin. T cell receptors also have α-chain, β-chain, γ-chain, δ-chain, etc., and the same applies to their CDRs.

[0033] In this specification, the term "variable region" refers to the region containing CDR1 to CDR3 (hereinafter simply referred to as "CDRs1-3"). The arrangement order of these CDRs1-3 is not particularly limited, but preferably, it refers to a region arranged in the order of CDR1, CDR2, and CDR3, or in the reverse order, from the N-terminus to the C-terminus, either consecutively or via other amino acid sequences referred to as framework regions (FRs) described later. The term "heavy chain variable region" refers to the region in immunoglobulin where the above-mentioned heavy chain CDRs1-3 are arranged, and the term "light chain variable region" refers to the region in immunoglobulin where the above-mentioned light chain CDRs1-3 are arranged. T cell receptors also have α-chains, β-chains, γ-chains, δ-chains, etc., and the same applies to these CDRs.

[0034] The regions of each variable region other than CDR1-3 are referred to as framework regions (FRs) as described above. Specifically, the region between the N-terminus of the variable region and CDR1 is defined as FR1, the region between CDR1 and CDR2 is defined as FR2, the region between CDR2 and CDR3 is defined as FR3, and the region between CDR3 and the C-terminus of the variable region is defined as FR4.

[0035] 2. Chimeric target factor receptors In one embodiment, the present invention relates to a chimeric target factor receptor (which may be referred to herein as "the chimeric target factor receptor of the present invention") comprising a target factor-binding domain, a transmembrane domain, and an intracellular domain including a TLR intracellular domain. This is described below.

[0036] The target factor-binding domain is a domain located extracellularly when the chimeric target factor receptor of the present invention is positioned on the cell membrane, and is not particularly limited as long as it is a domain that can recognize a target factor and bind to that target factor (preferably specifically). Examples of target factors include cancer antigens, various receptors (e.g., cytokine receptors such as interleukin receptors, cell growth factor receptors, etc.), cell adhesion molecules, and bacterial and viral membrane proteins. Specific examples of target factors include CD19, GD2, GD3, CD20, CD37, CEA, HER2, EGFR, type III mutant EGFR, CD38, BCMA, MUC-1, PSMA, WT1, cancer testis antigen (e.g., NY-ESO-1, MAGE-A4, etc.), mutation peptide (e.g., k-ras, h-ras, p53, etc.), hTERT, PRAM, TYRP1, mesothelin, PMEL, mucin, IL-12R, IL-4R, IL-13R, IL-6R, IL-23R, CTLA4, EGFR806, PSCA, and Claudin. Examples include 18.2, EpCAM, VEGFR2, Nectin4 / FAP, LewisY, Glypican-3, IL-13Rα2, CD171, MUC16, AFP, AXL, CD80 / 86, c-MET, DLL-3, DR5, EpHA2, FR-α, gp100, MAGE-A1 / 3 / 4, LMP1, etc. Examples of target factor-binding domains include domains containing antibody or T cell receptor CDRs, domains containing all or part of cytokines or cell growth factors (cytokine or cell growth factor domains), and domains containing all or part of cell adhesion factors (cell adhesion factor domains). A domain containing an antibody or T cell receptor CDR typically has one or more, preferably two, three, four, five or more, and more preferably all six, of the CDRs of the antibody or T cell receptor against the target factor (for example, if it is an antibody, heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3). A domain containing a CDR more preferably includes the variable region of the antibody or T cell receptor against the target factor (for example, in the case of an antibody, the heavy chain variable region and / or the light chain variable region).Examples of cytokine or cell growth factor domains include domains containing receptor-binding domains, such as IL-12, IL-4, IL-13, IL-6, IL-23, and GM-CSF.

[0037] When the target factor binding domain is a domain containing a CDR, a single-chain antibody structure is preferred, and an scFv structure is more preferred. When an antibody includes both a heavy chain variable region and a light chain variable region, such as in an scFv structure, the heavy chain variable region and the light chain variable region are usually linked via a linker. The linker is not particularly limited and is arbitrary, as long as the target factor binding ability is not significantly impaired. Preferably, the linker is glycine or a linker composed of glycine and serine (GGS linker, GS linker, GGG linker, etc.). The length of the linker is not particularly limited. The number of amino acid residues in the linker is, for example, 5 to 30. The arrangement of the heavy chain variable region and the light chain variable region is not particularly limited, and either the light chain variable region being at the N-terminus or the heavy chain variable region being at the N-terminus can be adopted.

[0038] The transmembrane domain is a domain located within the cell membrane when the chimeric target factor receptor of the present invention is positioned on the cell membrane, and is not particularly limited as long as it can constitute the chimeric target factor receptor. Examples of transmembrane domains that can be used include CD28, CD3ε, CD8α, CD3, CD4, 4-1BB, etc. The transmembrane domain is either publicly known or can be easily determined from known sequence information (for example, using a transmembrane domain prediction program). Alternatively, a transmembrane domain consisting of an artificially constructed polypeptide may be used. Mutations may be introduced into these transmembrane domains as appropriate, as long as they do not significantly inhibit the function of the chimeric target factor receptor.

[0039] Preferably, the transmembrane region of CD28 can be used as the transmembrane domain. Specific examples of the CD28 transmembrane region include the amino acid sequence described in (a) below or the amino acid sequence described in (b) below: (a) The amino acid sequence shown in Sequence ID No. 2, or (b) An amino acid sequence having 85% or more identity with the amino acid sequence shown in Sequence ID No. 2, and which can be positioned within the cell membrane when the chimeric target factor receptor of the present invention is positioned on the cell membrane. These are some examples.

[0040] In (b) above, the identity is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0041] An example of the amino acid sequence described in (b) above is, for example, (b') An amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted to the amino acid sequence shown in Sequence ID No. 3, and which can be positioned within the cell membrane when the chimeric target factor receptor of the present invention is positioned on the cell membrane. These are some examples.

[0042] In (b') above, "multiple" means, for example, 2 to 5, preferably 2 to 3, and more preferably 2.

[0043] The target factor-binding domain and the transmembrane domain are linked directly or via a spacer. The sequence of the spacer is not particularly limited, but for example, the hinge portion or a part thereof of IgG, preferably human IgG (e.g., subtype IgG1 or IgG4), the hinge portion and a part of CH2, or a part of a factor used in the transmembrane domain (e.g., CD28) can be used as the spacer. Furthermore, by using the sequence of the hinge portion or a part thereof, it is expected that a highly flexible spacer can be constructed.

[0044] The intracellular domain is the domain located inside the cell when the chimeric target factor receptor of the present invention is positioned on the cell membrane, and is a domain capable of transmitting signals necessary for the activation of antigen-presenting cells. The chimeric target factor receptor of the present invention has an intracellular domain that includes a TLR intracellular domain.

[0045] The intracellular domain of a TLR is a domain consisting of the amino acid sequence of the intracellular domain of a TLR (Toll-like receptor), and is not particularly limited in this respect.

[0046] As TLRs, various mammalian TLRs can be used, such as those derived from humans, monkeys, mice, rats, dogs, cats, and rabbits. Among these, TLRs derived from the target organism (e.g., humans) are preferred. The amino acid sequences of the intracellular domains of TLRs derived from various species are publicly known.

[0047] Examples of TLRs include TLR1 (the amino acid sequence of the intracellular domain is, for example, SEQ ID NO: 3), TLR2 (the amino acid sequence of the intracellular domain is, for example, SEQ ID NO: 4), TLR3 (the amino acid sequence of the intracellular domain is, for example, SEQ ID NO: 5), TLR4 (the amino acid sequence of the intracellular domain is, for example, SEQ ID NO: 6), and TLR9 (the amino acid sequence of the intracellular domain is, for example, SEQ ID NO: 7). Among these, TLR2, TLR3, and TLR9 are preferred from the viewpoint of antigen-presenting cell activation ability and the ability to activate antigen-presenting cells earlier and more sustained in response to target factors, more preferably TLR3 and TLR9 are preferred, and even more preferably TLR3. In a preferred embodiment of the present invention, the intracellular domain of the TLR is at least one selected from the group consisting of the TLR2 intracellular domain, the TLR3 intracellular domain, and the TLR9 intracellular domain.

[0048] The intracellular domain of the TLR may have mutations such as amino acid substitutions, deletions, additions, or insertions, as long as it retains its antigen-presenting cell activation effect. From the viewpoint of minimizing the impairment of the above effect, substitutions are preferred, and more preferably, conservative substitutions.

[0049] Preferred specific examples of amino acid sequences for the intracellular domain of TLRs include the amino acid sequences described in (c) below and the amino acid sequences described in (d) below: (c) The amino acid sequence shown in any of Sequence IDs 3 to 7, and (d) Amino acid sequences that have 85% or more identity with the amino acid sequences shown in any of Sequence IDs 3-7 and that constitute a protein having antigen-presenting cell activation activity. At least one selected from the group consisting of the following is mentioned.

[0050] In (d) above, the identity is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0051] An example of the amino acid sequence described in (d) above is, for example, (d') An amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence shown in Sequence ID No. 4, and which has an antigen-presenting cell-activating effect. These are some examples.

[0052] In (d') above, "multiple" means, for example, 2 to 5, preferably 2 to 3, and more preferably 2.

[0053] From the viewpoint of antigen-presenting cell activation ability and the ability to activate antigen-presenting cells more quickly and sustainably in response to target factors, the intracellular domain may preferably further include a CD40 intracellular domain.

[0054] The CD40 intracellular domain is a domain consisting of the amino acid sequence of the intracellular domain of the CD40 amino acids, and is not particularly limited in this respect.

[0055] CD40 can be derived from various mammals, such as humans, monkeys, mice, rats, dogs, cats, and rabbits. Among these, CD40 derived from the target organism (e.g., humans) is preferred.

[0056] The amino acid sequences of the intracellular domains of CD40 from various biological species are publicly known. Specifically, examples include the amino acid sequence shown in Sequence ID No. 8.

[0057] The intracellular domain of CD40 may have mutations such as amino acid substitutions, deletions, additions, or insertions, as long as it retains its antigen-presenting cell activation effect. From the viewpoint of minimizing the impairment of the above effect, substitutions are preferred, and more preferably, conservative substitutions.

[0058] Preferred specific examples of the amino acid sequence of the CD40 intracellular domain include the amino acid sequence described in (e) below and the amino acid sequence described in (f) below: (e) The amino acid sequence shown in Sequence ID No. 8, and (f) Amino acid sequences that have 85% or more identity with the amino acid sequence shown in Sequence ID No. 8 and that constitute a protein having antigen-presenting cell activation activity. At least one selected from the group consisting of the following is mentioned.

[0059] In (f) above, the identity is preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more.

[0060] An example of the amino acid sequence described in (f) above is, for example, (f') An amino acid sequence in which one or more amino acids are substituted, deleted, added, or inserted into the amino acid sequence shown in Sequence ID No. 8, and which has an antigen-presenting cell-activating effect. These are some examples.

[0061] In (f') above, "multiple" means, for example, 2 to 5, preferably 2 to 3, and more preferably 2.

[0062] When the intracellular domain includes a CD40 intracellular domain, the arrangement of the TLR intracellular domain and the CD40 intracellular domain is not particularly limited, and either the configuration in which the N-terminal side is the TLR intracellular domain or the configuration in which the N-terminal side is the CD40 intracellular domain can be adopted. The TLR intracellular domain and the CD40 intracellular domain are preferably directly linked or linked via a linker. The linker is not particularly limited and can be any. Preferably, the linker is glycine or a linker composed of glycine and serine (GGS linker, GS linker, GGG linker, etc.). The length of the linker is not particularly limited. The number of amino acid residues of the linker is, for example, 1 to 20, preferably 1 to 10, and more preferably 1 to 5.

[0063] The chimeric target factor receptor of the present invention may include other regions besides those described above. Other regions include, for example, intracellular domains such as CD3ζ and FcεRIγ, leader sequences (signal peptides) to promote the transport of CARs onto the cell membrane (e.g., the leader sequence of the GM-CSF receptor), and spacers / linkers (e.g., between the transmembrane domain and the intracellular signaling domain, and between each domain within the intracellular domain).

[0064] The chimeric target factor receptor of the present invention has a target factor-binding domain, a transmembrane domain, and an intracellular domain arranged in this order.

[0065] The chimeric target factor receptor of the present invention may be a molecule composed of one polypeptide, or a molecule composed of a complex of two or more polypeptides. Furthermore, the chimeric target factor receptor of the present invention may be a molecule composed of a polypeptide or a complex thereof, or it may be a molecule in which another substance (e.g., a fluorescent substance, a radioactive substance, inorganic particles, etc.) is linked to a polypeptide or a complex thereof.

[0066] The chimeric target factor receptor of the present invention may be chemically modified. The polypeptide constituting the chimeric target factor receptor of the present invention has a carboxyl group (-COOH) at the C-terminus, a carboxylate (-COO)- ), which may be either an amide (-CONH2) or an ester (-COOR). Here, examples of R in the ester include C such as methyl, ethyl, n-propyl, isopropyl, n-butyl and the like 1-6 alkyl group; for example, C such as cyclopentyl, cyclohexyl and the like 3-8 cycloalkyl group; for example, C such as phenyl, α-naphthyl and the like 6-12 aryl group; for example, phenyl-C such as benzyl, phenethyl and the like 1-2 alkyl group; α-naphthyl-C such as α-naphthylmethyl and the like 1-2 C such as alkyl group and the like 7-14 aralkyl group; pivaloyloxymethyl group and the like are used. In the polypeptide constituting the chimeric target factor receptor of the present invention, a carboxyl group (or carboxylate) other than the C-terminus may be amidated or esterified. As the ester in this case, for example, the aforementioned C-terminal ester and the like are used. Furthermore, in the polypeptide constituting the chimeric target factor receptor of the present invention, the amino group of the N-terminal amino acid residue may have a protecting group (for example, C such as formyl group, acetyl group and the like 1-6 C such as alkanoyl and the like 1-6 acyl group and the like), those obtained by pyroglutamine oxidation of the N-terminal glutamine residue that can be generated by cleavage in vivo, and those in which a substituent on the side chain of an intramolecular amino acid (for example, -OH, -SH, amino group, imidazole group, indole group, guanidino group and the like) is protected with an appropriate protecting group (for example, C such as formyl group, acetyl group and the like 1-6 C such as alkanoyl group and the like 1-6 acyl group and the like) are also encompassed.

[0067] The chimeric target factor receptor of the present invention may have a known protein or peptide such as a protein tag, a signal sequence and the like added thereto. Examples of the protein tag include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, fluorescent protein tag and the like.

[0068] The chimeric target factor receptor of the present invention may be in the form of a pharmaceutically acceptable salt with an acid or a base. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and either an acidic salt or a basic salt can be used. For example, examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and p-toluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0069] The chimeric target factor receptor of the present invention may also be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples include water, ethanol, glycerol, acetic acid, etc.

[0070] Techniques for producing chimeric target factor receptors and cells expressing them are publicly known. These can be produced in accordance with or in accordance with known methods.

[0071] 3. Polynucleotides and their uses In one embodiment, the present invention relates to a polynucleotide (which may be referred to herein as "the polynucleotide of the present invention") encoding the chimeric target factor receptor of the present invention. This will be described below.

[0072] The polynucleotide of the present invention may contain sequences other than the coding sequence for the chimeric target factor receptor of the present invention. Preferably, the polynucleotide of the present invention contains the chimeric target factor receptor of the present invention in a state capable of expression. Examples of other sequences include promoter sequences, enhancer sequences, repressor sequences, insulator sequences, replication bases, reporter protein (e.g., fluorescent protein) coding sequences, and drug resistance gene coding sequences.

[0073] The polynucleotide of the present invention preferably comprises an expression cassette for the chimeric target factor receptor of the present invention. The expression cassette comprises a promoter and a coding sequence for the chimeric target factor receptor of the present invention under the control of the promoter. The coding sequence is usually placed downstream of the promoter so that it is under the control of the promoter. Examples of promoters that can be used in the expression cassette include CMV-IE (cytomegalovirus early gene-derived promoter), SV40ori, retroviral LTP, SRα, EF1α, and β-actin promoter. The promoter is operably ligated to the coding sequence. Here, "the promoter is operably ligated to the coding sequence" is synonymous with "the coding sequence is placed under the control of the promoter," and usually the coding sequence is ligated directly or via another sequence to the 3' end of the promoter. A poly-A addition signal sequence is placed downstream of the coding sequence. Transcription is terminated by using the poly-A addition signal sequence. Examples of poly-A addition signal sequences that can be used include the poly-A addition sequence of SV40 and the poly-A addition sequence of the bovine growth hormone gene.

[0074] The expression cassette may also include detection genes (reporter genes, cell or tissue-specific genes, selection marker genes, etc.), enhancer sequences, WRPE sequences, etc. Detection genes are used to determine the success and efficiency of introducing the expression cassette, to detect or determine the expression efficiency of CAR genes, and to select and separate cells that express CAR genes. On the other hand, the use of enhancer sequences can improve expression efficiency. The genes used for detection include the neo gene, which confers resistance to neomycin; the npt gene (Herrera Estrella, EMBO J. 2 (1983), 987-995) and the nptII gene (Messing & Vierra. Gene 1 9:259-268 (1982)), which confers resistance to kanamycin, etc.; the hph gene (Blochinger & Diggl mann, Mol Cell Bio 4:2929-2931), which confers resistance to hygromycin; and the dhfr gene (Bourouis et al., EMBO J.2 (7)), which confers resistance to metatrexate (all of these are marker genes). Also used is the luciferase gene (Giacomin, P1. Sci. 116 (1996), 59-72; Scikantha, J. Bact. Genes such as the β-glucuronidase (GUS) gene, the GFP (Gerdes, FEBS Lett. 389 (1996), 44-47) and its variants (EGFP, d2EGFP, etc.) genes for fluorescent proteins (all of which are reporter genes), and the epidermal growth factor receptor (EGFR) gene lacking an intracellular domain can be used. The detection gene is linked to the chimeric target factor receptor gene via, for example, a bicistronic regulatory sequence (e.g., an intra-ribosome recognition sequence (IRES)) or a sequence encoding a self-cleaving peptide. An example of a self-cleaving peptide is the 2A peptide (T2A) derived from Thosea asigna virus, but it is not limited to this. Known self-cleaving peptides include 2A peptide (F2A) derived from foot-of-foot disease virus (FMDV), 2A peptide (E2A) derived from equine rhinitis A virus (ERAV), and 2A peptide (P2A) derived from porcine teschovirus (PTV-1).

[0075] The polynucleotide of the present invention may be a linear polynucleotide or a cyclic polynucleotide (such as a vector). The vector may be a plasmid vector or a viral vector. The vector may also be, for example, a cloning vector or an expression vector. Examples of expression vectors include vectors for prokaryotic cells such as Escherichia coli or actinomycetes, or vectors for eukaryotic cells such as yeast cells, insect cells, or mammalian cells. More specifically, viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, Sendai virus vectors, etc., while examples of non-viral vectors include various plasmid vectors, liposome vectors, positively charged liposome vectors (Felgner, PL, Gadek, TR, Holm, M. et al., Proc. Natl. Acad. Sci., 84:7413-7417, 1987), YAC vectors, and BAC vectors.

[0076] The polynucleotides of the present invention include not only DNA and RNA, but also those that have been chemically modified as shown below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residue of each nucleotide can be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. The hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may also be replaced with -OR (where R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at position 5 of the pyrimidine base, or by substituting the carbonyl group at position 2 with a thiocarbonyl group. Furthermore, examples include, but are not limited to, those in which the phosphate or hydroxyl portion is modified with, for example, biotin, amino groups, lower alkylamine groups, or acetyl groups. Also, the term "polynucleotide" encompasses not only natural nucleic acids but also BNA (Bridged Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), etc.

[0077] In one embodiment of the present invention, antigen-presenting cells that are activated in response to a target factor can be obtained by a method comprising introducing the polynucleotide of the present invention into antigen-presenting cells. These cells express the chimeric target factor receptor of the present invention. Examples of antigen-presenting cells into which the polynucleotide of the present invention is introduced include dendritic cells, macrophages, and their precursor cells. Various cell populations can be used as long as they include these cells.

[0078] 4.Cells In one embodiment, the present invention relates to cells (which may be referred to herein as "cells of the present invention") containing the polynucleotides of the present invention. This will be described below.

[0079] The cells from which the cells of the present invention originate are not particularly limited. If the purpose is to use the cells of the present invention in the purification of the chimeric target factor receptor of the present invention, suitable cells for origination include cells that can be used for protein expression (e.g., insect cells, eukaryotic cells, mammalian cells, etc.).

[0080] The cells of the present invention are preferably antigen-presenting cells (e.g., dendritic cells, macrophages, etc., particularly preferably dendritic cells). In one embodiment, the cells of the present invention may also be stem cells that can be differentiated into antigen-presenting cells, such as ES cells and iPS cells. These cells are preferably cells that express the chimeric target factor receptor of the present invention, and in a more specific embodiment, these cells express the target factor receptor of the present invention on the cell membrane, and preferably the target factor receptor of the present invention is expressed with its target factor-binding domain exposed outside the cell membrane.

[0081] Antigen-presenting cells expressing chimeric target factor receptors recognize target factors with their target factor-binding domains, then transmit the recognition signal to the intracellular space, activating the cells themselves. This regulates the activity of T cells (e.g., Th1 cells, Th2 cells, Th17 cells, cytotoxic T cells, etc.), B cells, etc., thereby exhibiting anticancer activity, immunomodulatory activity, etc. Therefore, cells (especially antigen-presenting cells) containing polynucleotides encoding the chimeric target factor receptor of the present invention are useful as active ingredients in pharmaceutical compositions.

[0082] 5. Pharmaceutical Compositions In one embodiment, the present invention relates to a pharmaceutical composition (which may also be referred to as "the pharmaceutical composition of the present invention" in this specification) containing the cells of the present invention. This will be described below.

[0083] The pharmaceutical composition of the present invention can be widely used as a cell preparation for the treatment, prevention, or improvement of tumors / cancers expressing target factors, as well as for the treatment, prevention, or improvement of autoimmune diseases, allergic diseases, etc., and for the treatment, prevention, or improvement of infectious diseases. Cancer includes solid tumors and hematological cancers. Examples of cancers include various B-cell malignant lymphomas (B-cell acute lymphoblastic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin lymphoma, etc.), myeloproliferative disorders, myelodysplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, multiple myeloma, lung cancer, colorectal cancer, ovarian cancer, breast cancer, brain tumors, gastric cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, etc. "Treatment" includes alleviating (reducing the severity of) the symptoms or associated symptoms characteristic of the target disease, and preventing or delaying the worsening of symptoms. "Prevention" means preventing or delaying the onset / appearance of a disease (disorder) or its symptoms, or reducing the risk of onset / appearance. On the other hand, "improvement" means that a disease (disorder) or its symptoms are alleviated (reduced), improve, go into remission, or are cured (including partial cure).

[0084] The pharmaceutical composition of the present invention contains a therapeutically effective amount of the cells of the present invention. For example, 1 × 10⁻¹⁶ cells per single dose. 4 pieces~1×10 10 It is possible to include individual cells in the cell preparation. For the purpose of protecting the cells, dimethyl sulfoxide (DMSO) and serum albumin may be included, for the purpose of preventing bacterial contamination, antibiotics may be included, and various components (vitamins, cytokines, growth factors, steroids, etc.) may be included for the purpose of activating, proliferating, or inducing differentiation of cells.

[0085] The administration route of the pharmaceutical composition of the present invention is not particularly limited. For example, it may be administered by intravenous injection, intra-arterial injection, intra-portal injection, intradermal injection, subcutaneous injection, intramuscular injection, or intraperitoneal injection. It may also be administered locally rather than systemically. As an example of local administration, direct injection into the target tissue, organ, or system can be used. The administration schedule should be created considering the gender, age, weight, and condition of the subject (patient). In addition to a single dose, it may be administered multiple times consecutively or regularly.

[0086] The pharmaceutical composition and cells of the present invention can be used in combination with other drugs. In particular, the pharmaceutical composition and cells of the present invention can improve antitumor effects when used in combination with immune checkpoint inhibitors (e.g., anti-PD-1 antibodies (e.g., nivolumab and pembrolizumab), anti-PD-L1 antibodies (e.g., Tecentriq, durvalumab and avelumab), anti-CTLA-4 antibodies (e.g., ipilimumab), etc.). From this viewpoint, the pharmaceutical composition and cells of the present invention can be used in combination with immune checkpoint inhibitors, and the pharmaceutical composition of the present invention may contain immune checkpoint inhibitors. The timing of combination administration is not particularly limited; for example, the pharmaceutical composition and cells of the present invention and the immune checkpoint inhibitor may be administered simultaneously or on the same day, or the other may be administered one or more days (e.g., 2 to 14 days) before or after the administration of one. [Examples]

[0087] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0088] Test Example 1. Creation of Chimeric Target Factor Receptor Constructs From the N-terminus, • GMCSFR signal peptide (amino acid sequence: SEQ ID NO: 19, nucleotide sequence: SEQ ID NO: 20) • Anti-Her2 scFv domain (amino acid sequence: SEQ ID NO: 1, nucleotide sequence: SEQ ID NO: 9), • CD28 transmembrane domain (amino acid sequence: SEQ ID NO: 2, nucleotide sequence: SEQ ID NO: 10) • Intracellular domain: (a) TLR1 intracellular domain (amino acid sequence: SEQ ID NO: 3, nucleotide sequence: SEQ ID NO: 11) (b) TLR2 intracellular domain (amino acid sequence: SEQ ID NO: 4, nucleotide sequence: SEQ ID NO: 12) (c) TLR3 intracellular domain (amino acid sequence: SEQ ID NO: 5, nucleotide sequence: SEQ ID NO: 13) (d) TLR4 intracellular domain (amino acid sequence: SEQ ID NO: 6, nucleotide sequence: SEQ ID NO: 14) (e) TLR9 intracellular domain (amino acid sequence: SEQ ID NO: 7, nucleotide sequence: SEQ ID NO: 15) (f) TLR2 intracellular domain - CD40 intracellular domain (amino acid sequence: SEQ ID NO: 8, nucleotide sequence: SEQ ID NO: 16) (g) TLR3 intracellular domain - CD40 intracellular domain (h)TLR9 intracellular domain - CD40 intracellular domain (i) CD40 intracellular domain - TLR2 intracellular domain (j) CD40 intracellular domain - TLR3 intracellular domain, (k) CD40 intracellular domain - TLR9 intracellular domain We designed a chimeric target factor receptor construct arranged in the following order.

[0089] Furthermore, from the N-terminus, • GMCSFR signal peptide (amino acid sequence: SEQ ID NO: 19, nucleotide sequence: SEQ ID NO: 20) • Anti-mesoserin (MSLN) scFv domain (amino acid sequence: SEQ ID NO: 17, nucleotide sequence: SEQ ID NO: 18) • CD28 transmembrane domain, • Intracellular domain: (A) TLR1 intracellular domain (B) TLR2 intracellular domain (C)TLR3 intracellular domain (D)TLR4 intracellular domain (E)TLR9 intracellular domain (F)TLR2 intracellular domain - CD40 intracellular domain, (G)TLR9 intracellular domain - CD40 intracellular domain (H)CD40 intracellular domain (I) CD40 intracellular domain - TLR9 intracellular domain (J) None We designed a chimeric target factor receptor construct arranged in the following order.

[0090] The coding sequences of the above-mentioned chimeric target factor receptor and the chimeric target factor receptor in which the intracellular domain was deleted were inserted into a pMXs-GFP vector (Cosmo Bio Co., Ltd.) to obtain a pMXs-CAR-GFP vector.

[0091] Test Example 2. Generation of dendritic cells expressing chimeric target factor receptors. Retrovirus-prepared Plat-A cells (Cell Biolabs Inc.) were introduced with the pMXs-CAR-GFP vector (Test Example 1) using TransIT-293 Reagent (Mirus). After 48 hours, the culture supernatant was collected. The culture supernatant was added to THP-1 cells (obtained from the JCRB cell bank) and infected with the virus. After 48 hours of infection, GFP-positive cells were collected using BD FACS Aria. Human GM-CSF (BioLegend) and human IL-4 (BioLegend) were added to the obtained cells, and they were cultured for 5 days to differentiate into dendritic cells, thereby obtaining chimeric target factor receptor-expressing dendritic cells.

[0092] Experiment Example 3. Creation of Chimeric Target Factor Receptor-Expressing Macrophages Retrovirus-prepared Plat-A cells (Cell Biolabs Inc.) were introduced with the pMXs-CAR-GFP vector (Test Example 1) using TransIT-293 Reagent (Mirus). After 48 hours, the culture supernatant was collected. The culture supernatant was added to U937 cells (obtained from the JCRB cell bank) and infected with the virus. 48 hours after infection, GFP-positive cells were collected using BD FACS Aria to obtain chimeric target factor receptor-expressing macrophages.

[0093] Test Example 4. In vitro dendritic cell activation test 1 Her2-expressing cells (SKOV3 cells: obtained from ATCC) were combined with chimeric target factor receptor-expressing dendritic cells (Test Example 2: target factor is Her2), cultured for 5, 24, and 48 hours, and the expression of dendritic cell activation indicator factors (CD80 and CD86) was examined by FACS.

[0094] The results are shown in Figures 1-5. As shown in Figures 1-3, it was found that by using the intracellular domain of TLRs as the intracellular domain, the expression of CD80 can be increased in response to the target factor. In particular, when the intracellular domains of TLR3 and 9 were used, CD80 expression could be increased at a relatively early stage (5 hours, Figure 1), and when the intracellular domains of TLR2, 3, and 9 were used, CD80 expression could be increased more sustainedly (48 hours, Figure 3). Furthermore, as shown in Figure 4, it was found that by using the intracellular domain of TLRs as the intracellular domain, CD86 expression could also be increased in response to the target factor. In addition, it was found that by using the intracellular domain of CD40 in addition to the intracellular domain of TLRs as the intracellular domain, CD86 expression could be increased more sustainedly (24 hours, Figure 5).

[0095] Test Example 5. In vitro macrophage phagocytosis test Her2-expressing cells (SKOV3 cells: obtained from ATCC) were combined with chimeric target factor receptor-expressing macrophages (Test Example 2: target factor is Her2), cultured for 48 hours, and phagocytosis activity was examined using a phagocytosis assay kit (Cayman).

[0096] The results are shown in Figure 6. As shown in Figure 6, it was found that by adopting the intracellular domain of a TLR as the intracellular domain, macrophages can also be activated in response to the target factor.

[0097] Test Example 6. Evaluation Test of Antitumor Effect 1 <Test Example 6-1. Preparation of MSLN-expressing mouse tumor cells Pan02 / MSLN> The human MSLN gene was introduced into a pMXs-GFP vector. The resulting vector and the pCL-Eco vector (Addgene) were introduced into G3T-hi cells (Takara Bio) using TransIT-293 Reagent (Mirus). After 48 hours, the culture supernatant was collected. The culture supernatant was added to mouse pancreatic cancer cells (Pan02 cells), and the cells were infected with the virus. 48 hours after infection, GFP-positive cells were collected using BD FACS Aria.

[0098] <Example 6-2. Evaluation of antitumor effect> Pan02 or Pan02 / MSLN (Test Example 6-1) was transplanted subcutaneously into mice, and dendritic cells (Empty: pMXs-GFP introduced) or chimeric target factor receptor-expressing dendritic cells (Test Example 2: target is MSLN, intracellular domain is (E) of Test Example 1) were administered. The antitumor effects of each were examined using tumor growth inhibition as an indicator. Tumors, cut into 2 mm squares, were transplanted subcutaneously into the flanks of mice using a tumor transplantation needle. The tumor size was 100 mm. 3 The cells were divided into groups once they reached a certain size, and cell administration was started. The cells (dendritic cells (Empty) or chimeric target factor receptor-expressing dendritic cells (MSLN-TLR9)) were divided into 1 x 10⁻¹⁶ cells. 5 The drug was administered once into the tumor (Day 0). Tumor size was measured twice a week to evaluate the antitumor effect.

[0099] The results are shown in Figures 7 and 8. As shown in Figures 7 and 8, it was found that by adopting the intracellular domain of TLR as the intracellular domain, an antitumor effect can be exerted in response to the target factor (MSLN).

[0100] Test Example 7. In vitro dendritic cell activation test 2 Her2-expressing cells (SKOV3 cells: obtained from ATCC) were combined with chimeric target factor receptor-expressing dendritic cells (Test Example 2: target factor is Her2), cultured for 24 hours, and the expression of dendritic cell activation indicators (MHC class I and MHC class II) was examined by FACS.

[0101] The results are shown in Figures 9-10. It was found that by using the intracellular domain of a TLR as the intracellular domain, the expression of MHC class I and MHC class II could be increased in response to the target factor. In particular, the use of TLR2, 3, and 9 resulted in a stronger degree of activation (Figure 9).

[0102] Test Example 8. In vitro dendritic cell activation test 3 <Test Example 8-1. Preparation of MSLN-expressing mouse tumor cells Panc-1 / MSLN> The human MSLN gene was introduced into the pMXs-rCD2 vector. The resulting vector was introduced into Plat-A cells (Cell Biolabs Inc.) using TransIT-293 Reagent (Mirus). After 48 hours, the culture supernatant was collected. The culture supernatant was added to human pancreatic cancer cells (Panc-1 cells, obtained from ATCC) and infected with the virus. 48 hours after infection, rCD2-positive cells were collected using BD FACS Aria.

[0103] <Test Example 8-2. Evaluation Test for Activation Potential> Panc-1 / MSLN (Test Example 8-1) was combined with chimeric target factor receptor-expressing dendritic cells (Test Example 2: target factor is MSLN), cultured for 24 hours, and the expression of indicator factors for dendritic cell activation (CD86 and MHC class II) was examined by FACS.

[0104] The results are shown in Figures 11-12. It was found that by adopting the intracellular domain of TLRs as the intracellular domain, the expression of CD86 and MHC clas II could be increased even when different target factors were used than those in Test Examples 4 and 8. Furthermore, in this test example, it was found that by using the intracellular domain of CD40 in addition to the intracellular domain of TLRs as the intracellular domain, the degree and duration of dendritic cell activation could be enhanced.

[0105] Test Example 9. Evaluation Test of Antitumor Effect 2 Pan02 or Pan02 / MSLN (Test Example 6-1) was transplanted subcutaneously into mice, and dendritic cells (Empty: pMXs-GFP introduced) or chimeric target factor receptor-expressing dendritic cells were administered. The antitumor effects of each were examined using tumor growth suppression as an indicator. The target of the administered chimeric target factor receptor-expressing dendritic cells (Test Example 2) was MSLN, and the intracellular domain of the chimeric target factor receptor was one of (A)~(E), (H), and (J) from Test Example 1. Tumors, finely shredded into 2 mm squares, were transplanted subcutaneously into the flanks of mice using a tumor transplantation needle. Seven days after transplantation, the mice were divided into groups, and cell administration was started. Cells (dendritic cells (Empty) or chimeric target factor receptor-expressing dendritic cells) 1 x 10 5 The drug was administered once into the tumor (Day 0). Tumor size was measured twice a week to evaluate the antitumor effect.

[0106] The results are shown in Figure 13. It was found that by using the intracellular domain of TLR3 or TLR9 as the intracellular domain, a significantly higher antitumor effect could be achieved in response to the target factor (MSLN) compared to using the intracellular domain of other TLRs.

[0107] Test Example 10. Evaluation Test of Antitumor Effect 3 Pan02 or Pan02 / MSLN (Test Example 6-1) was transplanted subcutaneously into mice, and dendritic cells (Empty: pMXs-GFP introduced) or chimeric target factor receptor-expressing dendritic cells were administered. The antitumor effects of each were examined using tumor growth inhibition as an indicator. The target of the administered chimeric target factor receptor-expressing dendritic cells (Test Example 2) was MSLN, and the intracellular domain of the chimeric target factor receptor was (C) or (E) in Test Example 1. Tumors, cut into 2 mm squares, were transplanted subcutaneously into the flanks of mice using a tumor transplantation needle. Seven days after transplantation, the mice were divided into groups, and administration of cells and / or drugs (PD-1 antibody) was started. In the cell administration group, 1 x 10⁶ cells (chimeric target factor receptor-expressing dendritic cells (MSLN-TLR9)) were administered. 5 The drug was administered intratumor twice (Day 1 and Day 8). In the PD-1 antibody administration group, 250 μg of PD-1 antibody was administered intraperitoneally twice (Day 0 and Day 7). Tumor size was measured twice a week.

[0108] Tables 1 and 2 show the mean tumor size measurements for the group administered only cells (TLR3 group or TLR9 group) and the group administered only PD-1 antibody (αPD-1 group), the mean tumor size measurements (O-FTV) for the group administered both cells and PD-1 antibody, the expected tumor size when administered in combination (E-FTV (=mean measurement for group 1 × mean measurement for group 2)) based on the measurements for the TLR3 group or TLR9 group and the αPD-1 group, and the value obtained by dividing E-FTV by O-FTV (E / O). If E / O is greater than 1, it indicates that a synergistic effect was achieved by the combined administration of cells and PD-1 antibody. Table 1 shows the results when the intracellular domain of the chimeric target factor receptor is the intracellular domain of TLR-3, and Table 2 shows the results when the intracellular domain of the chimeric target factor receptor is the intracellular domain of TLR-9. In the tables, the "Day" column indicates the number of days elapsed, with Day 0 being 7 days after transplantation.

[0109] [Table 1]

[0110] [Table 2]

[0111] As shown in Tables 1 and 2, it was found that the antitumor effect was synergistically enhanced when chimeric target factor receptor-expressing cells having an intracellular TLR domain were used in combination with a PD-1 antibody.

[0112] Test Example 11. Evaluation Test of Antitumor Effect 4 Pan02 or Pan02 / MSLN (Test Example 6-1) was transplanted subcutaneously into mice, and dendritic cells (Empty: pMXs-GFP introduced) or chimeric target factor receptor-expressing dendritic cells were administered. The antitumor effects of each were examined using tumor growth suppression as an indicator. The target of the administered chimeric target factor receptor-expressing dendritic cells (Test Example 2) was MSLN, and the intracellular domain of the chimeric target factor receptor was (E), (F), or (I) from Test Example 1. Tumors, finely shredded into 2 mm squares, were transplanted subcutaneously into the flanks of mice using a tumor transplantation needle. Seven days after transplantation, the mice were divided into groups, and cell administration was started. Cells (dendritic cells (Empty) or chimeric target factor receptor-expressing dendritic cells) 1 x 10 5 The drug was administered once into the tumor (Day 0). Tumor size was measured twice a week to evaluate the antitumor effect.

[0113] The results are shown in Figure 14. It was found that the antitumor effect was improved by using the intracellular domain of CD40 in addition to the intracellular domain of TLR as the intracellular domain.

Claims

1. Antigen-presenting cells or their progenitor cells that are responsible for activating innate immunity, comprising a polynucleotide encoding a chimeric target factor receptor, including a target factor-binding domain, a transmembrane domain, and an intracellular domain including a TLR intracellular domain, Antigen-presenting cells or their progenitor cells, wherein the intracellular TLR domain is at least one selected from the group consisting of the TLR2 intracellular domain, the TLR3 intracellular domain, and the TLR9 intracellular domain.

2. The antigen-presenting cell or its progenitor cell according to claim 1, wherein the intracellular TLR domain is at least one selected from the group consisting of the TLR3 intracellular domain and the TLR9 intracellular domain.

3. The antigen-presenting cell or its progenitor cell according to claim 1 or 2, wherein the intracellular domain further comprises a CD40 intracellular domain.

4. The antigen-presenting cell or its progenitor cell according to any one of claims 1 to 3, wherein the structure of the target factor-binding domain is a single-chain antibody structure.

5. The antigen-presenting cell or its progenitor cell according to any one of claims 1 to 4, wherein the target factor is at least one selected from the group consisting of cancer antigens, cytokine receptors, and cell growth factor receptors.

6. A dendritic cell, which is an antigen-presenting cell or a precursor cell thereof according to any one of claims 1 to 5.

7. A pharmaceutical composition containing antigen-presenting cells or their precursor cells as described in any one of claims 1 to 6.

8. The pharmaceutical composition according to claim 7, for the treatment, prevention, or improvement of at least one condition selected from the group consisting of cancer, autoimmune diseases, allergic diseases, and infectious diseases.

9. The pharmaceutical composition according to claim 8, for the treatment, prevention, or improvement of solid tumors.

10. The pharmaceutical composition according to claim 9, wherein the intracellular TLR domain is at least one selected from the group consisting of the TLR3 intracellular domain and the TLR9 intracellular domain, and the antigen-presenting cell is a dendritic cell.

11. The pharmaceutical composition according to claim 9 or 10, wherein the intracellular domain further comprises a CD40 intracellular domain, and the antigen-presenting cell is a dendritic cell.

12. A pharmaceutical composition according to any one of claims 7 to 11, for use in combination with an immune checkpoint inhibitor.

13. A composition used for introduction into antigen-presenting cells or their precursor cells that are responsible for activating innate immunity, It comprises a polynucleotide encoding a chimeric target factor receptor, including a target factor-binding domain, a transmembrane domain, and an intracellular domain including a TLR intracellular domain, A composition wherein the TLR intracellular domain is at least one selected from the group consisting of a TLR2 intracellular domain, a TLR3 intracellular domain, and a TLR9 intracellular domain.

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