Chimeric antigen receptor

Abstract

The present invention addresses the problem of providing a CAR that targets CAF in which cytotoxic activity is enhanced.　The present invention provides a CAR capable of binding to FAP, the CAR comprising an intracellular domain, a transmembrane domain, and an extracellular domain including a humanized scFV having specific CDR sequences.

Description

Chimeric antigen receptor

[0001] The present invention relates to chimeric antigen receptors (CARs), nucleic acids encoding them, vectors, and cells, pharmaceutical compositions, and cell preparations containing them.

[0002] In recent years, there has been a surge in approvals for therapies using genetically modified T cells, including chimeric antigen receptor (CAR) T cells. In particular, research and development of CARs for cancer treatment are becoming increasingly active.

[0003] Active research is being conducted on targets for cancer-associated fibroblasts (CARs), and cancer-associated fibroblasts (CAFs), which are fibroblasts that proliferate in the stroma of cancer, are attracting attention as one of the promising targets. CAFs are present in the vicinity of cancer cells and, although they are normal cells, are known to contribute to the local invasion of cancer cells, extravascular migration, and metastatic colony formation.

[0004] To date, CARs targeting proteins expressed in CAFs have been developed. For example, Patent Document 1 describes a CAR based on a mouse single-chain variable region fragment (scFV) that targets fibroblast-activating protein α (FAP), a protein expressed in CAFs.

[0005] US2014 / 0099340

[0006] Despite the background described above, no CARs targeting FAP have yet been brought to market. The reason for this is their low cytotoxic activity. In fact, the cytotoxic activity of the CAR described in Patent Document 1 was found to be significantly lower than that of CARs targeting cancer cell surface antigens (such as CD19) that are already on the market.

[0007] Therefore, the object of the present invention is to provide a CAR that targets CAFs with enhanced cytotoxic activity.

[0008] To solve the above problems, the inventors diligently researched the design of the extracellular region, transmembrane region, and intracellular region including scFV. As a result, they developed a CAR in which the localization amount on the cell surface and the cytotoxic activity of the CAR were improved.

[0009] In other words, the present invention provides a CAR capable of binding to FAP, which includes an extracellular region, a transmembrane region, and an intracellular region, containing a humanized scFV having a specific CDR sequence.

[0010] The present invention provides a highly cytotoxic CAR capable of binding to FAP. By using this CAR, a highly efficient adoptive immunotherapy targeting CAF can be provided.

[0011] This figure shows the results of CAR expression levels in cells in Example 1. This figure shows the results of the proliferation rate of CAR-expressing cells in Example 2. This figure shows the results of the cytotoxic activity of CAR-T cells against FAP-expressing cells in Example 3. Error bars in the figure indicate the standard deviation. This figure shows the relationship between the cytotoxic activity of CAR-T cells against FAP-expressing cells and the E:T ratio in Example 4. Error bars in the figure indicate the standard deviation. This figure shows the results of the long-term cytotoxic activity (serial killing) of CAR-T cells against FAP-expressing cells in Example 5. This figure shows the relationship between the cytotoxic activity of CAR-T cells against synovial cells derived from rheumatoid arthritis patients and the E:T ratio in Example 6. Figure 6A shows the results for cell sample 1, and Figure 6B shows the results for cell sample 2. Error bars in the figure indicate the standard deviation. This figure shows the relationship between the cytotoxic activity of CAR-T cells against synovial cells derived from rheumatoid arthritis patients and the E:T ratio in Example 6. Figure 7A shows the results for cell sample 3, and Figure 7B shows the results for cell sample 4. Error bars in the figure indicate the standard deviation. This figure shows the relationship between the cytotoxic activity of CAR-T cells and the E:T ratio against synovial cells (cell sample 5) derived from rheumatoid arthritis patients in Example 6. Error bars in the figure indicate the standard deviation. This figure shows the vector map of an exemplary CAR expression plasmid vector. This figure shows the tumor reduction effect of CAR-expressing T cells in Example 7. In the figure, "1st" indicates the time when wild-type T cells or FAP CAR-expressing T cells were intravenously injected, and "2nd" indicates the time when Mesothelin CAR-expressing T cells were intravenously injected (day 14).

[0012] 1. Chimeric Antigen Receptor (CAR) 1-1. Overview The first aspect of the present invention is a chimeric antigen receptor (CAR). The CAR of the present invention comprises an extracellular domain containing a humanized single-chain variable region fragment (scFV), a transmembrane domain, and an intracellular domain, and the antigen-binding domain can bind to fibroblast-activating protein α (FAP). T cells into which the CAR of the present invention has cytotoxic activity against cells having FAP on their cell surface, such as cancer-associated fibroblasts (CAFs) and activated synovial fibroblasts in rheumatoid arthritis patients.

[0013] 1-2. Definitions The following terms, frequently used in this specification, are defined below. "Adoptive immunotherapy" refers to a treatment method that involves collecting cells, such as immune cells, from a donor, culturing, stimulating, manipulating, and proliferating them outside the body, and then introducing them into a recipient. For example, in cancer patients, it can enhance the toxic activity of immune cells against cancer cells. Examples of adoptive immunotherapy include tumor-infiltrating lymphocyte therapy, TCR gene-modified T-cell therapy, and chimeric antigen receptor-T (CAR-T) cell therapy. Adoptive immunotherapy may also include leukocyte apheresis. Adoptive immunotherapy mainly refers to autologous transplantation, where the donor providing the cells and the recipient into whom the cells are introduced are the same individual, but it also includes allogeneic transplantation (allogeneic or xenogeneic transplantation), where the donor and recipient are different individuals.

[0014] "Tumor-infiltrating lymphocyte therapy" is a treatment method that involves collecting tumor-infiltrating lymphocytes (TILs) from a donor, processing them outside the body through activation and amplification, and then introducing them into the recipient.

[0015] "TCR gene-transformed T-cell therapy" is a treatment method that involves introducing cancer antigen-specific T cell receptor (TCR) genes into immune cells such as T cells collected from the peripheral blood of a donor, and then introducing them into a recipient.

[0016] "Chimeric antigen receptor T-cell therapy (CAR-T cell therapy)" is a treatment method that involves creating CAR-T cells capable of recognizing and attacking cancer cells by introducing a chimeric antigen receptor (CAR) gene into immune cells such as T cells collected from the peripheral blood of a donor, and then introducing these CAR-T cells into the recipient through infusion or other means.

[0017] A "chimeric antigen receptor (CAR)" refers to a fusion protein containing an extracellular domain capable of binding to antigens such as cancer antigens, a transmembrane domain, and an intracellular domain with signal transduction activity. More specifically, it refers to a fusion protein containing a variable region (V) of the light and heavy chains derived from a monoclonal antibody that recognizes cell surface antigens. L and V H CARs are artificial antigen receptors created by combining a single-chain antibody (scFv) conjugated with CD3ζ (also known as CD247) with a transmembrane domain and a signaling domain of a co-stimulatory molecule involved in T cell activation. CARs can be broadly classified into first, second, and third generations based on the structure and type of signaling domain they contain. First-generation CARs consist solely of the CD3ζ (also known as CD247) signaling domain. Second-generation CARs contain one co-stimulatory molecule such as CD28 or CD137 (4-1BB) in addition to the CD3ζ signaling domain. Third-generation CARs contain multiple co-stimulatory molecules such as CD28 or CD137 (4-1BB) in addition to the CD3ζ signaling domain. Furthermore, CARs modified to enhance cellular function are sometimes called fourth-generation CARs. In T cells expressing CARs, the CAR recognizes tumor cells and activates T cells, thereby efficiently killing target cells. T cells into which the CAR gene has been introduced are called "chimeric antigen receptor T cells (CAR-T cells)."

[0018] Fibroblast-activating protein α (FAP) is a homodimeric intrinsic membrane gelatinase belonging to the serine protease family. FAP possesses a dipeptidyl peptidase 4N-terminal domain and a peptidase S9 prolyl oligopeptidase enzyme activity domain. An exemplary amino acid sequence of human FAP is shown in SEQ ID NO: 11. It is known to be expressed on the cell surface of activated fibroblasts, including cancer-associated fibroblasts, activated synovial fibroblasts, and activated cardiac fibroblasts.

[0019] A "signal peptide" is an extracellular translocation signal necessary for the secretion of proteins biosynthesized by gene expression into the extracellular space, and is also called a leader peptide. Signal peptides may contain a region composed of hydrophobic amino acids. After translation, signal peptides are cleaved and removed by signal peptidases before translocation to the extracellular space. Signal peptide sequences are located at the N-terminus of many secretory proteins and membrane proteins, and are, for example, 15 to 30 amino acids long. The species from which the signal peptide is derived may be any species. The species may be human or non-human, for example, insect cells or viruses, but human is preferred. The type of protein from which the signal peptide is derived is not particularly limited, but examples include CD8α (SEQ ID NO: 21), immunoglobulins, Oncostatin M, CD3ε, etc.

[0020] A "linker" is a peptide that can be inserted between fused regions in a fusion protein such as a CAR of the present invention in order for each fused region to perform its intended function. The length of linkers and hinges is not limited, but typically they are 3 to 100 amino acids long, preferably 5 to 50 amino acids long. Typically, linkers are peptides that contain many amino acids with relatively small side chains, such as serine or glycine. Hereafter in this specification, when simply referred to as a "linker," it refers to the linker between the variable regions of an scFV unless otherwise specified. In particular, the linker between the antigen-binding domain and the transmembrane region is called a "hinge" to distinguish it from the linker between the variable regions of an scFV.

[0021] In this specification, "immune cells" include cell types that can function as part of the immune system, as well as undifferentiated cells and progenitor cells (e.g., immune progenitor cells) that can differentiate into such cell types. Specific examples of immune cells include lymphocytes, granulocytes, dendritic cells, macrophages, and monocytes. While the term "immune cells" may not strictly include stem cells, in this specification, it also includes stem cells that can differentiate into lymphocytes (e.g., hematopoietic stem cells).

[0022] Examples of lymphocytes include T cells, B cells, and natural killer cells (NK cells). Lymphocytes may also be tumor-infiltrating lymphocytes. T cells include killer T cells (cytotoxic T cells), helper T cells, and regulatory T cells. T cells may be either CD8-positive T cells or CD4-positive T cells. T cells can also be classified into naive T cells, memory T cells, and effector T cells, or any of these. Naive T cells and memory T cells are known to be abundant in peripheral blood, while effector T cells are hardly present in peripheral blood. Examples of granulocytes include neutrophils, eosinophils, and basophils.

[0023] In this specification, "multiple" means two or more integers, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3 integers. Also, in this specification, "a few" means, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

[0024] In this specification, "amino acid identity (amino acid sequence identity)" refers to the percentage of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides being compared are aligned by inserting gaps as needed into one or both of them to maximize the number of matching amino acid residues. "Base identity (base sequence identity)" can be determined in the same manner.

[0025] In this specification, "amino acid substitution" refers to substitutions between the 20 amino acids that make up natural proteins. Amino acid substitutions are preferably within a group of conserved amino acids that have similar properties such as charge, side chain, polarity, and aromaticity. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), the group of branched-chain amino acids (Leu, Val, Ile), the group of neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), the group of neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), the group of acidic amino acids (Asp, Glu), the group of basic amino acids (Arg, Lys, His), and the group of aromatic amino acids (Phe, Tyr, Trp).

[0026] 1-3. Structure The chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In the CAR of the present invention, the "extracellular domain" includes an antigen-binding domain that binds to fibroblast-activating protein α (FAP).

[0027] In this specification, "capable of binding to fibroblast-activating protein α (FAP)" means capable of binding to the extracellular domain of FAP. The extracellular domain of FAP is not particularly limited, as long as it is located extracellularly when localized on the cell membrane and is a position to which the scFV of the present invention can bind. Specifically, it is a region consisting of several tens of amino acid residues located at the C-terminus of FAP, and examples include any partial sequence of the extracellular domain consisting of the amino acid sequence shown in SEQ ID NO: 12, or more specifically, the epitope of the F19 antibody.

[0028] The antigen-binding domain included in the extracellular region of the CAR of the present invention includes (a) a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1 or 35, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2 or 36, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and (b) a light chain variable region comprising LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0029] In particular, the antigen-binding domain contained in the extracellular region of the CAR of the present invention includes: (a-1) a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, or (a-2) a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 35, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 36, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3, and (b) a light chain variable region comprising LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 6.

[0030] The antigen-binding domain contained in the extracellular region of the CAR of the present invention can specifically bind to a peptide identical to the epitope of, for example, the extracellular domain consisting of the amino acid sequence shown in SEQ ID NO: 12 of FAP. Further, for example, the antigen-binding domain contained in the extracellular region of the CAR of the present invention competes with the F19 antibody for binding to the extracellular domain consisting of the amino acid sequence shown in SEQ ID NO: 12 of FAP.

[0031] In the present specification, "specifically binds" is not particularly limited, but the binding between an antigen and an antibody has a KD value of 10 -8 M or less, preferably 10 -9 M or less, more preferably 10 -10 M or less, which may mean having a binding affinity. Examples of the method for measuring the binding affinity include, in addition to surface plasmon resonance, biolayer interferometry, etc. For example, biolayer interferometry can be preferably used.

[0032] The scFV in this specification is a humanized scFV humanized from a mouse scFV derived from F19. When an scFV is "humanized", it means replacing the CDRs in a human antibody with the CDRs in an antibody derived from a non-human mammal. The variable region (V region) of an immunoglobulin molecule is composed of four framework regions (FRs) (FR1, FR2, FR3, and FR4) and three complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3) linked in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminal side. Usually, in a humanized scFV, all or part of the four FRs have the amino acid sequence in a human antibody, and all three CDRs have the amino acid sequence derived from a non-human mammal. As the humanized scFV of the present invention, for example, an scFV in which all four FRs have the amino acid sequence derived from a human antibody can be preferably used.

[0033] As a method for humanizing scFV, any method known in the art can be used and is not particularly limited. For example, the CDR grafting method, veneering or resurfacing method, chain shuffling method, optimization method by machine learning, computational models represented by the CUMAb method, or combinations thereof can be used.

[0034] The method for identifying the position of the FR is not particularly limited. For example, any method can be used to identify the CDR, and the sequence in between can be identified as the FR. The identification of the CDR can be performed using any method known in the art, and the specific method is not particularly limited. For example, the Kabat method or the like can be used.

[0035] For the scFV of the present invention, additional treatments such as directed evolution can be performed as needed.

[0036] In one embodiment, the antigen-binding domain included in the extracellular region of the CAR of the present invention includes a heavy chain variable region consisting of the amino acid sequence shown in (ia) SEQ ID NO: 7 or 9, and / or a light chain variable region consisting of the amino acid sequence shown in (ib) SEQ ID NO: 8 or 10. Alternatively, the light chain variable region included in the antigen-binding domain included in the extracellular region of the CAR of the present invention may be a light chain variable region consisting of an amino acid sequence in the amino acid sequence shown in (ic) SEQ ID NO: 8, where the amino acid at position 2 and the amino acid at position 29 are independently leucine or isoleucine, respectively.

[0037] In one embodiment, the antigen-binding domain (i) included in the extracellular region of the CAR of the present invention includes (ia) and (ib).

[0038] In another embodiment, the antigen-binding domain included in the extracellular region of the CAR of the present invention includes a heavy chain variable region consisting of an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with respect to the amino acid sequence shown in (iia) SEQ ID NO: 7 or 9, excluding the CDR sequence portion, and / or a light chain variable region consisting of an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% or more sequence identity with respect to the amino acid sequence shown in (iib) SEQ ID NO: 8 or 10, excluding the CDR sequence portion.

[0039] In another embodiment, the antigen-binding domain (ii) included in the extracellular region of the CAR of the present invention comprises (iia) and (iib).

[0040] For example, the antigen-binding domain included in the extracellular region of the CAR of the present invention includes (iia) and (ib) or (ic). It also includes, for example, (ia) and (iib).

[0041] In yet another embodiment, the antigen-binding domain included in the extracellular region of the CAR of the present invention includes (iiia) a heavy chain variable region consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted, excluding the CDR sequence portion, in the amino acid sequence shown in SEQ ID NO: 7 or 9, and / or (iiib) a light chain variable region consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted, excluding the CDR sequence portion, in the amino acid sequence shown in SEQ ID NO: 8 or 10.

[0042] In yet another embodiment, the antigen-binding domain (iii) included in the extracellular region of the CAR of the present invention includes (iiia) and (iiib).

[0043] In the following, antigen-binding domains (ii) and (iii) may be collectively referred to as antigen-binding domains homologous to antigen-binding domain (i).

[0044] The scFV can be composed of the heavy-chain variable region and light-chain variable region described above, connected by a flexible linker. The specific linker used is not particularly limited, but for example, a Whitlow sequence (sequence number 19), a Yol-tag sequence (sequence number 17), an L1 linker (sequence number 18), or a GS linker (sequence number 16) can be used, and more specifically, a Whitlow sequence, a Yol-tag sequence, or an L1 linker can be preferably used. Furthermore, the arrangement of the heavy-chain variable region and the light-chain variable region is not particularly limited, and either may be located on the N-terminal side or the C-terminal side.

[0045] In the CAR of the present invention, the "transmembrane region" is a region for stably incorporating the CAR of the present invention into a lipid membrane, and is usually composed mainly of hydrophobic amino acid residues. As the transmembrane region, a transmembrane domain contained in any membrane protein or an artificial transmembrane domain mainly containing hydrophobic amino acid residues can be used. The transmembrane domain is not limited to the following, but includes the α or β chain of the T cell receptor, CD8, CD28, CD3ε, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1, ICOS, 4-1BB, GITR, CD40L, BAFFR, HVEM, SLAMF7, NKp80, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, Examples of transmembrane domains derived from IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1, SLAMF4, CD84, CD96, CEACAM1, CRTAM, Ly9, CD160, PSGL1, CD100, SLAMF6, SLAM, BLAME, SELPLG, LTBR, ​​or PAG / Cbp are cited. Preferred transmembrane domains include the transmembrane domain of CD28 and the transmembrane domain of CD8α. The specific amino acid sequences are not particularly limited, but examples include: amino acid sequences consisting of or containing the amino acid sequences shown in SEQ ID NOs. 23 and 28; amino acid sequences consisting of or containing amino acid sequences that have 90% or more, 95% or more, 98% or more, or 99% or more identity with the amino acid sequences shown in SEQ ID NOs. 23 and 28; and amino acid sequences consisting of or containing amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequences shown in SEQ ID NOs. 23 and 28.

[0046] In some embodiments, the CAR may have two or more transmembrane domains, which may be repeats of the same transmembrane domain or different transmembrane domains.

[0047] In the CAR of the present invention, the "transmembrane region" may be connected to the extracellular region via a hinge. The type of hinge in this case is not particularly limited, but for example, a hinge derived from the protein exemplified for the transmembrane region can be used. Specifically, for example, a hinge derived from CD8α, CD8β, CD28, or CD4 can be used, and more specifically, a hinge derived from CD8α or CD28 can be preferably used.

[0048] In the CAR of the present invention, the "intracellular domain" refers to the region that transmits an activation signal to the inside of the cell expressing the CAR after the CAR has bound to an antigen molecule on a target cell in its extracellular domain. The intracellular domain of the CAR of the present invention includes one or more protein signaling domains. The protein from which these signaling domains originate is not particularly limited. For example, CD3ζ, CD28, 4-1BB / CD137, OX-40, CD27, CD40L, GITR, ICOS, CD30, PD-1 (CD279), LFA-1, LIGHT, CD2, CD7, NKG2C (CD94), NK cell activator receptor, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD247, CD29, CD3δ, CD3ε, CD3γ, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD8, CD8α, CD8β, CD9 6 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, CD83-specific ligand, LTBR, ​​Ly9 (CD229), Ly108, MHC Examples of signal transduction regions include class 1 molecules, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), PAG / Cbp, PSGL1, SELPLG (CD162), SLAM, SLAMF1, SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, VLA6 and / or B7-H3 (CD276).

[0049] In one embodiment, the intracellular region of the CAR of the present invention includes a CD3ζ signaling domain. In this case, the intracellular region of the CAR of the present invention may consist only of the CD3ζ signaling domain, or it may include one or more co-stimulatory domains in addition to the CD3ζ signaling domain.

[0050] In this specification, "CD3ζ signaling domain" means a domain containing the activation motif ITAM, which triggers various phosphorylation signals in the CD3ζ subunit of the T cell receptor / CD3 complex when MHC and antigen peptides bind to the T cell receptor. The specific amino acid sequence of the CD3ζ signaling domain is not particularly limited, but examples include an amino acid sequence consisting of or containing the amino acid sequence shown in SEQ ID NO: 25; an amino acid sequence consisting of or containing an amino acid sequence having 90% or more, 95% or more, 98% or more, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 25; an amino acid sequence consisting of or containing an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 25. Further examples include a domain in which one or more ITAM motifs are deleted in any CD3ζ signaling domain containing the activation motif ITAM. In addition, there is no limit to the number of CD3ζ signaling domains in the intracellular region of the CAR of the present invention, and there may be one or more. For example, the function of CAR can be enhanced by tandem linking two CD3ζ signaling domains.

[0051] In a further embodiment, the intracellular region of the CAR of the present invention further comprises at least one co-stimulatory domain in addition to the CD3ζ signaling domain.

[0052] In this specification, "co-stimulatory domain" means a domain derived from a co-stimulatory molecule (co-stimulatory molecule) that enhances effector function, cytotoxic activity, cytokine production, proliferation, and / or viability based on the intracellular region including the signaling domain of CD3ζ. As an example of a co-stimulatory domain, any of the signaling regions exemplified with respect to the signaling domain can be used.

[0053] Preferred co-stimulatory domains include, for example, the signal transduction domains of CD28 and 4-1BB / CD137. These specific amino acid sequences are not particularly limited, but examples include: amino acid sequences consisting of or containing the amino acid sequences shown in SEQ ID NOs. 24 and 29, respectively; amino acid sequences consisting of or containing amino acid sequences having 90% or more, 95% or more, 98% or more, or 99% or more identity with the amino acid sequences shown in SEQ ID NOs. 24 and 29; and amino acid sequences consisting of or containing amino acid sequences in which one or more amino acids are deleted, substituted, or added to the amino acid sequences shown in SEQ ID NOs. 24 and 29.

[0054] In the intracellular region of the CAR of the present invention, the positions of the above-mentioned signal transduction domain and co-stimulatory domain are not particularly limited. For example, the intracellular region may include one or more co-stimulatory domains on its N-terminal side and a signal transduction domain on its C-terminal side.

[0055] The composition of the intracellular domain in the CAR of the present invention is not particularly limited, but for example, the signal transduction domain may be derived from CD3ζ and the co-stimulatory domain may be derived from CD28 and / or 4-1BB / CD137.

[0056] The combination of the co-stimulatory domain and transmembrane region in the CAR of the present invention is not particularly limited, but for example, if the co-stimulatory domain is derived from CD28, the transmembrane region of CD28 can be used, and if the co-stimulatory domain is derived from 4-1BB / CD137, the transmembrane region of CD8α can be used.

[0057] The CAR of the present invention may optionally contain additional peptides. For example, it may contain an additional linker between the transmembrane domain and the intracellular domain, or it may be a fusion protein in which a labeled protein is fused to the C-terminal region, etc. The type of labeled protein is not particularly limited, but examples include fluorescent proteins.

[0058] In some embodiments, the CAR of the present invention includes a signal peptide at its N-terminus. For example, the CAR of the present invention includes, in order from the N-terminus, a signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain.

[0059] The combination of regions in the CAR of the present invention is not particularly limited. For example, it includes a signal peptide derived from CD8α; a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7 or 9 or an amino acid sequence homologous thereto; a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 or 10 or an amino acid sequence homologous thereto; an antigen-binding domain including a Whitlow sequence, a Yol-tag sequence, an L1 linker or a GS linker; and an extracellular region including a hinge derived from CD8α or CD28; a transmembrane region derived from CD8α or CD28; a co-stimulatory domain including signal transduction domains for CD28 and 4-1BB / CD137; and a signal transduction domain for CD3ζ. Furthermore, for example, it includes a signal peptide derived from CD8α; a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence homologous thereto; a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence homologous thereto; an antigen-binding domain including a Whitlow sequence, a Yol-tag sequence, or an L1 linker sequence (e.g., a Whitlow sequence); an extracellular region including a hinge derived from CD28; a transmembrane region derived from CD28; a co-stimulatory domain including the signal transduction domain of CD28; and a signal transduction domain of CD3ζ.

[0060] 1-4. Effects CAR-T cells into which the CAR of the present invention has been introduced can induce antigen-specific cytotoxic activity against cells expressing FAP. FAP, which is targeted by the CAR of the present invention, is also expressed in cancer-associated fibroblasts (CAFs) that contribute to the invasion and migration of tumor cells. Therefore, it is possible to suppress the progression of cancer regardless of the surface antigen of the cancer cells. Furthermore, FAP is also expressed in activated synovial fibroblasts at the inflammatory sites of rheumatoid arthritis, an immune system disease. Therefore, by removing the cells that induce inflammation, the progression of rheumatoid arthritis can be suppressed. In addition, FAP is also expressed in activated fibroblasts (activated cardiac fibroblasts in the case of cardiac fibrosis) in fibroses such as cardiac fibrosis. Therefore, by removing the cells that cause fibrosis, the progression of fibrosis can be suppressed.

[0061] 2. Nucleic acids encoding CAR and CAR expression vectors 2-1. Overview A second aspect of the present invention is a nucleic acid encoding CAR and a vector containing the same.

[0062] 2-2. Composition The "car-encoding nucleic acid" can be any car-encoding nucleic acid described in the first embodiment. The base sequence of such nucleic acid is not particularly limited. For example, codon-optimized base sequences and base sequences with a start codon (ATG) added to the 5' end are examples.

[0063] The CAR vector of the present invention comprises a nucleic acid encoding CAR. In addition to the nucleic acid component, the CAR vector may optionally include components such as a promoter, a labeled gene (selection marker), an enhancer, a terminator, an origin of replication, and a polyA signal. When the CAR vector of the present invention further includes a promoter and is a gene expression vector capable of expressing CAR in cells, it is specifically referred to as a CAR expression vector.

[0064] In this specification, "gene expression vector" means a vector that contains a gene or gene fragment (hereinafter referred to as "gene etc.") in an expressible state and includes an expression unit that can control the expression of such gene etc. Gene expression vectors may also be plasmid vectors or viral vectors.

[0065] In this specification, the "expressible state" means that a gene or the like to be expressed is arranged in the downstream region of a promoter under the control of the promoter. Known vectors include plasmid vectors, viral vectors, etc., and any of these vectors can be used. Usually, a plasmid vector that is easy to use for genetic recombination operations or a viral vector that can easily introduce a gene into immune cells may be used.

[0066] The plasmid vector may be, for example, a commercially available expression vector for mammalian cells such as the pCI vector or pSI vector of Promega, or a shuttle vector that can replicate between mammalian cells and bacteria such as Escherichia coli.

[0067] As the viral vector, for example, retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotype vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors can be used. A viral vector lacking replication ability so as not to self-replicate in infected cells may also be used.

[0068] When a retroviral vector is used, a suitable packaging cell and a packaging signal sequence can be selected based on the LTR sequence to produce retroviral particles. Examples of packaging cells include PG13 (ATCC (R) CRL-10686 TM ), PA317 (ATCC (R) CRL-9078 TMThese include GP+E-86 and GP+envAm-12 (U.S. Patent No. 5,278,056), and Psi-Crip (1988; doi:10.1073 / pnas.95.21.12516), among others. Retroviral particles can also be produced using 293 cells or 293T cells with high transfection efficiency. Many types of viral vectors produced based on packaging cells that can be used for packaging retroviruses and retroviral vectors are commercially available from many companies.

[0069] In this specification, "promoter" refers to a gene expression regulatory region that can control the expression of a gene located downstream (at the 3' end) in a cell into which a gene expression vector has been introduced. Promoters can be classified into ubiquitous promoters (systemic promoters) and site-specific promoters based on the location in which they express a gene under expression control. A ubiquitous promoter is a promoter that controls the expression of a target gene (target gene, etc.) throughout the entire host organism. A site-specific promoter is a promoter that controls the expression of a target gene, etc. only in specific cells or tissues. The promoter included in the gene expression vector of the present invention may be either a ubiquitous promoter or a site-specific promoter, but it is preferable that it can induce expression in immune cells.

[0070] Furthermore, promoters are classified into constitutively active promoters, expression-inducing promoters, or time-specific active promoters based on the timing of expression. Constitutively active promoters can constitutively express target genes, etc., within cells. Expression-inducing promoters can induce the expression of target genes, etc., within cells at any desired time. Time-specific active promoters can induce the expression of target genes, etc., within cells only at specific stages of development. All of these promoters can be understood as overexpression promoters because they can lead to the overexpression of target genes within host cells. The promoter included in the gene expression vector of the present invention is preferably a constitutively active promoter, which enables long-term persistence of therapeutic effects.

[0071] In the gene expression vector of this embodiment, the promoter is a promoter that can induce the expression of the nucleic acid encoding CAR in cells such as immune cells. Since the target cells into which the gene expression vector of the present invention is introduced are, in principle, mammalian cells, particularly human-derived cells, such as human-derived immune cells, any promoter that can express downstream genes in those cells is acceptable. Examples include the SFFV promoter, CMV promoter (CMV-IE promoter), SV40 initial promoter, RSV promoter, EF1α promoter, Ub promoter, β-actin promoter, 5' LTR promoter, etc. In the case of retroviral vectors, the nucleic acid encoding CAR can be placed downstream of the 5' LTR promoter to induce its gene expression.

[0072] In this specification, "labeled gene" refers to a gene that encodes a labeled protein, also called a selection marker or reporter protein. "Labeled protein" refers to a peptide whose activity allows for the determination of whether or not a labeled gene is expressed. Detection of activity may be by directly detecting the activity of the labeled protein itself, or by indirectly detecting it through metabolites generated by the activity of the labeled protein, such as dyes. Detection may be any of the following: biological detection (including detection by binding of peptides or nucleic acids such as antibodies and aptamers), chemical detection (including enzymatic detection), physical detection (including behavioral analysis detection), or sensory detection by the detector (including detection by sight, touch, smell, hearing, and taste).

[0073] The type of labeled protein encoded by a labeled gene is not particularly limited, as long as its activity can be detected by methods known in the field. Labeled proteins that are less invasive to the transformant during detection are preferred. Examples include tag peptides, drug resistance proteins, pigment proteins, fluorescent proteins, and luminescent proteins.

[0074] In this specification, "enhancer" is not particularly limited as long as it can enhance the expression efficiency of a gene or fragment thereof within a vector.

[0075] In this specification, "terminator" is a sequence that can terminate the transcription of a gene or the like expressed by the activity of the promoter. The type of terminator is not particularly limited. Preferably, it is a terminator derived from the same species as the promoter. In a single gene expression regulatory system, a terminator paired with the promoter on the genome is particularly preferred.

[0076] 3. Host Cells 3-1. Overview A third aspect of the present invention is a host cell. The host cell of this aspect includes a nucleic acid encoding a CAR or a vector containing the same. The host cell of this aspect is, for example, a CAR-T cell.

[0077] 3-2. Composition The host cell of this embodiment includes the nucleic acid and / or vector described in the second embodiment as an essential component.

[0078] The type of host cell in this embodiment is not particularly limited. Examples of host cells include immune cells, peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells, skin keratinocytes, mesenchymal stem cells, hematopoietic stem cells, various cancer cell lines, and neural stem cells, iPS cells, and ES cells. Preferred host cells are immune cells. Suitable immune cells include, for example, T cells, NK cells, macrophages, or combinations thereof. Naive T cells and memory T cells are preferred among T cells. Host cells such as immune cells may be cells derived from living organisms, immortalized cell lines, or cells differentiated from ES cells or induced pluripotent stem cells (iPS cells). Suitable host cells in this embodiment are cells that do not express FAP localized on the cell membrane.

[0079] The CARs contained in the CAR-T cells of the present invention may be first-generation CARs, second-generation CARs, third-generation CARs, fourth-generation CARs, or fifth-generation or later CARs.

[0080] The host cells of this embodiment may contain, in addition to CAR, cytokines, chemokines, cytokine receptors, chemokine receptors, transcription factors, epigenetic factors, and / or immune checkpoint molecules, and may also contain nucleic acids and / or vectors encoding these. For example, cytokines or chemokines include IL2, IL7, IL12, IL15, IL18, IL21, IL23, GM-CSF, CCL19, and CCL21. The cytokine receptor or chemokine receptor may be a receptor for any of these secreted factors. These factors may be included in the CAR expression vector or expressed by an independent expression vector. For example, CAR can be linked to a self-cleaving peptide such as P2A in the CAR expression vector. Alternatively, genes encoding cytokines, chemokines, cytokine receptors, chemokine receptors, transcription factors, epigenetic factors, and / or immune checkpoint molecules can be deleted by genetic modification.

[0081] 3-3. Effects The host cells of this embodiment express CARs that can bind to FAP. Therefore, for example, if the host cells of this embodiment are T cells, antigen-specific cytotoxic activity against CAFs that express FAP can be induced. Alternatively, antigen-specific cytotoxic activity against activated synovial fibroblasts or activated cardiac fibroblasts can be induced. Thus, the host cells of this embodiment can be used as CAR-T cells in CAR-T cell therapy.

[0082] 4. Compositions and Cell Preparations 4-1. Overview The fourth aspect of the present invention is a composition and a cell preparation. The composition of this aspect contains the CAR of the first aspect and / or the nucleic acid and / or vector of the second aspect as active ingredients and can be used as a pharmaceutical composition for a specific disease or for the production of cells of the third aspect. The cell preparation of this aspect contains host cells as an active ingredient and can be used in adoptive immunotherapy such as CAR-T cell therapy.

[0083] 4-2. Definitions In this specification, “subject” refers to the subject to which the pharmaceutical composition and cell preparation of this embodiment are applied, and is, for example, a tissue, organ, or individual. In the case of an individual, it is, for example, a mammal, and preferably a human individual. The human individual may be a patient, such as a cancer patient.

[0084] In this specification, "subject information" refers to various information about the characteristics and condition of the subject. For example, if the subject is a human individual, this may include age, weight, sex, overall health status, presence or absence of disease, progression and severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment.

[0085] In this specification, “treatment” means the alleviation or elimination of symptoms associated with a disease, and / or the prevention or suppression of the progression of the disease, as well as the cure of the disease.

[0086] The diseases described herein are not limited to any diseases involving fibroblasts. Examples of diseases include fibrosis, immune system disorders, and cancer. Cancer is preferred as the disease.

[0087] In this specification, "fibrosis" refers to the rigidity caused by the migration and proliferation of excessive fibroblasts and subsequent deposition of extracellular matrix, which occur in the process of compensating for the loss or dysfunction of parenchymal cells in tissues. The causes of parenchymal cell loss or dysfunction and the sites of onset are not particularly limited. Specific causes include, for example, chemical stimuli such as drugs (e.g., antitumor agents, antibiotics, antibacterial agents, antiarrhythmic agents, anti-inflammatory agents, antirheumatic agents), physical stimuli such as excessive pressure, physiological stimuli such as inflammatory responses, and abnormalities in the body such as diseases or disorders (e.g., nephritis, myocarditis, enteritis, pneumonia), or combinations thereof. Sites of onset include, for example, the respiratory system such as the trachea and lungs, the digestive system such as the esophagus, stomach and intestines, the urinary system such as the kidneys, the circulatory system such as blood vessels, heart, and liver, or combinations thereof. Specific examples of fibrosis include sclerosing peritonitis, scleroderma, uterine leiomyoma, cardiac fibrosis, retroperitoneal fibrosis, and myelofibrosis.

[0088] In this specification, "immune system disease" refers to a disease characterized by an abnormality of the immune system. Specifically, examples include autoimmune diseases and inflammatory diseases.

[0089] An "autoimmune disease" refers to a disease that produces an immune response to an autoantigen. In this specification, autoimmune diseases may be either organ-specific autoimmune diseases or systemic autoimmune diseases. Specific examples of autoimmune diseases include Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, type 1 diabetes, scleroderma, and psoriasis.

[0090] "Inflammatory disease" refers to a disease characterized by a high level of inflammation or degeneration in tissues. In this specification, inflammatory diseases include both chronic and acute inflammatory diseases. Specifically, examples include celiac disease, vasculitis, lupus, chronic obstructive pulmonary disease (COPD), irritable bowel syndrome, atherosclerosis, arthritis, ankylosing spondylitis, Crohn's disease, colitis, chronic active hepatitis, dermatitis, and psoriasis.

[0091] In this specification, the types of cancer are not limited, but examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Specific types of cancer include, for example, malignant melanoma, oral cancer, laryngeal cancer, pharyngeal cancer, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, small intestine cancer, colorectal cancer (including colon and rectal cancer), liver cancer, pancreatic cancer, biliary tract cancer (including gallbladder and bile duct cancer), kidney cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, cervical cancer, endometrial cancer, ovarian cancer, skin cancer, brain tumor, head and neck cancer, glioma, neuroblastoma, mesothelioma, osteosarcoma, soft tissue sarcoma, hematological cancer, lymphoma, and myeloma. Examples of hematological cancers include leukemia (e.g., B-cell leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., non-Hodgkin lymphoma), and myeloma (e.g., multiple myeloma). The cancers to which the cell preparations of this embodiment are suitable are those involving cancer-associated fibroblasts (CAFs). For example, invasive and / or metastatic cancers can be suitable targets for the cell preparations and pharmaceutical compositions of this embodiment.

[0092] 4-3. Composition 4-3-1. Components The components of the composition and cell preparation of this embodiment will be described below. The composition and cell preparation of this embodiment include one or more active ingredients and a solvent and / or carrier as essential components. Each component will be described in detail below.

[0093] (Active ingredient) The cell preparation of this embodiment includes the host cell described in the third embodiment as an essential active ingredient. The cell preparation of this embodiment may contain one or more host cells.

[0094] Furthermore, the composition of this embodiment may include the CAR described in the first embodiment and / or the nucleic acid and / or vector described in the second embodiment as essential active ingredients.

[0095] The amount of active ingredient contained in the cell preparation and composition of the present invention is not particularly limited. Generally, the amount varies depending on the type of active ingredient, the dosage form, and the type of solvent and carrier, which are other components described later. Therefore, it should be determined appropriately taking each condition into consideration. It is sufficient that a single dose of the cell preparation contains an effective amount of the active ingredient. However, if it is necessary to administer a large amount of the cell preparation and / or pharmaceutical composition to a subject in order to obtain the pharmacological effect of the active ingredient, it may be administered in several divided doses to reduce the burden on the subject. In this case, it is sufficient that the total amount of the active ingredient contains an effective amount.

[0096] Furthermore, for example, the composition of this embodiment, which includes the nucleic acid and / or vector (e.g., an expression vector) described in the second embodiment, can be used as a composition for use in the production of host cells described in the third embodiment.

[0097] "Effective dose" refers to the amount necessary for the active ingredient to exert its function, and which does not cause little to no harmful side effects to the subject to which it is applied. This effective dose can vary depending on various conditions such as the subject's information, the route of application, and the number of applications. Therefore, when the cell preparation and / or pharmaceutical composition of this embodiment is used as a pharmaceutical, the amount of active ingredient will ultimately be determined by the judgment of a physician or pharmacist.

[0098] The amount of host cells contained in the cell preparation according to this embodiment is, for example, 105 cells ~10 10 Cells or 10 6 cells ~10 9 Cells, preferably 10 7 cells ~10 8 Examples include cells, etc.

[0099] The cell preparations and compositions of this embodiment may contain additional active ingredients. The type of additional active ingredients is not particularly limited. Examples include CARs (such as CARs capable of binding to surface antigens of tumor cells) and CAR-expressing T cells.

[0100] (Solvent) The compositions and cell preparations of this embodiment may contain a pharmaceutically acceptable solvent as necessary. "Pharmaceutically acceptable solvent" means a solvent commonly used in the pharmaceutical technology field. Examples include water or aqueous solutions, or organic solvents. Examples of aqueous solutions include physiological saline, isotonic solutions containing glucose or other adjuvants, phosphate buffers, phosphate-buffered saline, sodium acetate buffers, glycol, or ethanol solutions. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, sodium chloride, and other low concentrations of nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, etc. An example of an organic solvent is ethanol.

[0101] (Carrier) The compositions and cell preparations of this embodiment may contain a pharmaceutically acceptable carrier as needed. "Pharmaceutically acceptable carrier" means an additive commonly used in the pharmaceutical technology field. Examples include excipients and human serum albumin.

[0102] Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides; inorganic salts such as hydrochloride, hydrobromide, phosphate, or sulfate; salts of organic acids such as acetate, propionate, malonate, or benzoate; metal salts; citric acid, tartaric acid, glycine, polyethylene glycol, kaolin, silicic acid, or combinations thereof.

[0103] In addition to the above, if necessary, the composition may also contain solubilizers, suspending agents, diluents, dispersants, surfactants, analgesics, stabilizers, absorption enhancers, bulking agents, preservatives, antiseptics, antioxidants, buffering agents, isotonic agents, etc., which are commonly used in pharmaceutical compositions.

[0104] The carrier is used to avoid or inhibit the degradation of the active ingredient by enzymes, etc., within the subject's body, as well as to facilitate formulation and administration methods, and to maintain the dosage form and efficacy. It should be used as appropriate as needed.

[0105] 4-3-2. Dosage Form The dosage form of the cell preparation of the present invention is not particularly limited. Any form that can deliver the active ingredient to the target site in the subject's body without inactivating it is acceptable.

[0106] The specific dosage form will vary depending on the method of application, which will be described later. The methods of application can be broadly classified into parenteral administration and oral administration, but parenteral administration is preferred.

[0107] If the method of administration is parenteral, the preferred dosage form is a liquid formulation that can be administered directly to the target site or systemically via the circulatory system. A good example of a liquid formulation is an injectable formulation. Injectable formulations can be formulated by mixing a solvent with excipients, suspensions, surfactants, stabilizers, pH adjusters, etc., in a unit dose form generally accepted for pharmaceutical production.

[0108] 4-3-3. Method of Application The method of application of the cell preparation of the present invention is not particularly limited, and the route of administration is not limited, but parenteral administration can be preferably used. Parenteral administration can be further subdivided into systemic administration and local administration. Local administration includes, for example, intradermal administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intranasal administration, intratumoral administration, tissue administration, and organ administration. Systemic administration by parenteral administration includes intracirculatory administration, such as intravenous administration (intravenous injection), intraarterial administration, and intralymphatic administration. The preferred route of administration is intravenous administration, which may be infusion by drip infusion (for example, a single intravenous infusion).

[0109] 4-4. Other Embodiments The cell preparation of this embodiment can be used for the treatment and / or prevention of diseases. Accordingly, a method for the treatment and / or prevention of diseases is provided, comprising the step of administering the cell preparation of this embodiment to a subject. In this case, the target disease is one or more diseases selected from the group consisting of, for example, fibrosis, immune system diseases and cancer. Cancer patients may be used as subjects. This treatment / prevention method may be adoptive immunotherapy such as CAR-T cell therapy.

[0110] The use of the CAR, nucleic acids and / or vectors encoding the CAR, host cells, or cell preparations of the present invention in the manufacture of pharmaceuticals for treating and / or preventing diseases such as cancer is also provided.

[0111] 5. Method for Producing Host Cells 5-1. Overview The fifth aspect of the present invention is a method for producing host cells. The method of this aspect includes a vector introduction step as an essential step. According to the method of this aspect, the host cells of the third aspect can be produced.

[0112] 5-2. Process The method for producing CAR-introduced cells according to this embodiment includes an introduction step as an essential step, and a cell isolation step and a cell expansion culture step as optional steps. Each step will be described in detail below.

[0113] 5-2-1. Cell Isolation Step The "cell isolation step" is an optional step of the method according to this embodiment, and is a step of isolating host cells. For example, if the cells are peripheral blood mononuclear cells, this can be a peripheral blood mononuclear cell isolation step in which peripheral blood mononuclear cells are isolated from peripheral blood derived from the subject. In this case, the purpose of this step is to isolate peripheral blood mononuclear cells that contain immune cells.

[0114] In this specification, “Peripheral Blood Mononuclear Cells (PBMCs)” means cells or cell populations, including monocytes and / or lymphocytes such as T cells, isolated from the peripheral blood of a human or animal.

[0115] The isolation method can be appropriately selected depending on the target cell type. The following explanation uses peripheral blood mononuclear cells as an example, but is not limited to this; any cell type as exemplified in the third aspect can be isolated in this process.

[0116] When used as cells, peripheral blood mononuclear cells are not particularly limited in their separation method. For example, peripheral blood mononuclear cells can be separated by density gradient centrifugation or hemolysis. Density gradient centrifugation can be performed by layering a diluted whole blood sample on top of a solvent and centrifuging it. The solvent is Ficoll-hypaque (R) Various solvents can be used, with an example being one adjusted to a density of approximately 1.077 g / mL. As a result of density gradient centrifugation, mononuclear cell components can be separated from red blood cells, granulocytes, and plasma as an intermediate layer. Hemolysis is a method of removing red blood cells with a hypotonic solution, but this method does not remove granulocyte components. Therefore, this process can usually be performed by density gradient centrifugation.

[0117] 5-2-2. Expansion Culture Step The "expansion culture step" is an optional step of the method according to this embodiment, and is a step of increasing the number of cells by expanding the culture of isolated cells. This step can be performed simultaneously with or after the cell isolation step.

[0118] The method used in this process can be appropriately selected depending on the type of host cell used, and is not particularly limited.

[0119] For example, if the host cells are peripheral blood mononuclear cells, this can be done by stimulating them with an anti-CD3 antibody. The anti-CD3 antibody used in this process is not particularly limited as long as it can stimulate peripheral blood mononuclear cells; for example, soluble anti-CD3 antibodies or cells expressing membrane-bound anti-CD3 antibodies may be used. Furthermore, the proliferation of peripheral blood mononuclear cells stimulated with the anti-CD3 antibody may be maintained by culturing them in the presence of cytokines such as IL-2.

[0120] This process can be performed to increase the proliferation of specific cells in isolated cells or to improve the efficiency of CAR introduction in the subsequent introduction process.

[0121] 5-2-3. Introduction Step The "introduction step" is an essential step of the method of this embodiment, and is a step of introducing a vector containing the nucleic acid encoding the CAR described in the second embodiment into isolated cells. This step can be performed after the cell isolation step if one is performing it, or simultaneously with or after the expansion culture step if one is performing it.

[0122] Cells into which the vector has been introduced in this process, for example, if the cells are peripheral blood mononuclear cells, can be used as CAR-T cells in adoptive immunotherapy such as CAR-T cell therapy.

[0123] There are no particular limitations on the method for introducing the vector into peripheral blood mononuclear cells. The method can be appropriately selected depending on the type of vector, the type of host cell, etc.

[0124] If the vector is a viral vector, the method of viral infection of cells is known in the art. For the introduction of the viral vector, a functional substance that improves the efficiency of viral infection, such as fibronectin or fibronectin fragment (for example, retronectin, which is a fibronectin fragment having a heparin binding site) is used. (R) Or Vecofusin-1 (R) Functional substances such as ) may also be used.

[0125] The following is an example of a viral infection method using retronectin: After treating a cell culture plate with retronectin, the bottom of the plate is blocked with a 2% BSA / PBS solution for 30 minutes. Then, after washing with PBS, retrovirus solution derived from PG13 packaging cells is loaded onto the culture plate, and the plate is centrifuged at 32°C and 2000g for 2 hours. After centrifugation, the virus solution is removed, and cells are seeded onto the plate.

[0126] If the vector is a non-viral vector such as a plasmid, a gene transfer method (transformation method) known in the field, as described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, etc., may be used. Examples include lipofection, electroporation, microinjection, calcium phosphate, DEAE-Dextran, and particle impact.

[0127] Cells into which the vector has been introduced in this process can usually be used as is, but if necessary, only the cells into which the vector has been introduced may be isolated. The method of isolation in this case is not particularly limited, but for example, isolation and / or enrichment may be performed based on the labeled gene in the vector.

[0128] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0129] <Example 1. Relationship between CAR design and localization to the cell membrane> (Objective) To investigate the effect of humanization of the scFV portion and the design of other portions on the localization to the cell membrane.

[0130] (Methods) 1. Design of CAR Expression Vectors A lentiviral vector was used as the CAR expression vector. To prepare the lentiviral vector, a plasmid vector was first designed by inserting the nucleotide sequence encoding the CAR downstream of the SFFV promoter of pHR-SFFV (adgene accession number: 79121; as of the filing date). The design details of each encoded CAR are shown in Table 1 below. The correspondence between each component and the sequence number is shown in Table 2 below. As a specific example of the plasmid vector structure, Figure 9 shows the structure of the plasmid vector "#34_WL_28z". Each CAR was designed as a fusion protein in which a Myc tag (in Figure 9, "myc tag") is included between its scFV (in Figure 9, "anti-FAP_scFV(#34)") and the hinge and transmembrane region (in Figure 9, "CD28 EC"), and mCherry (in Figure 9, "mCherry") is fused to the C-terminus.

[0131]

[0132]

[0133] Next, each plasmid vector and packaging mix (pCMVR8.74 (adgene accession number: 22036), pMD2.G (adgene accession number: 12259), pAdVAntage TMA lentiviral vector capable of inducing CAR expression upon infection was created by co-transfecting packaging cells (Thermo Fisher: 293FT cells) with Promega (Promega). The lentiviral vector was prepared according to the protocol provided by the packaging mix manufacturer.

[0134] 2. Introduction into cells and culture host cells: Human primary cultured CD8 ＋ T cells (STEMCELL technologies, ST-200-0164) were used. Each lentiviral vector was used to infect 1 × 10^6 cells.

[0135] 48 hours after infection, the cells were cultured for 5–7 days in culture medium (X-VIVO15 medium (Lonza: 04-418Q) supplemented with 5% human serum and 100 μM 2-mercaptoethanol, supplemented with human IL-2 at a final concentration of 25 U / mL (Peprotech) and human IL-7 and human IL-15 at a final concentration of 100 ng / mL (both Miltenyi Biotec)).

[0136] Infected T cells were subcultured in culture medium to a cell density of 50 × 10^4 cells / mL and cultured for 5 days after infection. As a control, T cells that were not infected with lentiviral vectors and cultured in culture medium for 5 days were used.

[0137] 3. Expression Level Measurement: The obtained cells were stained with APC-labeled anti-Myc antibody (Cell Signaling Technology: #47029) to identify cells with Myc tags exposed extracellularly. Staining for flow cytometry was performed according to the protocol provided by the antibody manufacturer.

[0138] After staining, mCherry expression and the presence or absence of APC signaling were detected in the cells using a flow cytometer (Beckman Coulter: CytoFLEX S). Flow cytometry analysis was performed according to the manufacturer's protocol.

[0139] The determination of whether or not each signal was present was based on a criterion in which all cells of the same type that have not been infected with the lentiviral vector ("WT") are judged as negative.

[0140] For each condition, more than 5,000 cells were measured, and the experiment was repeated twice.

[0141] (Results) The results are shown in Figure 1. Figure 1 is a graph showing the percentage of Myc-tag positive cells among mCherry-expressing cells in cells infected with each CAR-expressing lentiviral vector. This percentage represents the proportion of CAR-expressing cells in which the CAR was actually localized on the cell membrane.

[0142] The CD19-targeting CAR used in this embodiment (in Figure 1, "CD19_BBz") is a CAR targeting cancer cell surface antigens and has a sequence similar to those already on the market. When the mouse anti-FAP antibody F19 was used as is, the cell membrane localization of the introduced CAR was lower than that of the CD19-targeting CAR, regardless of the type of its costimulatory domain.

[0143] On the other hand, when scFV, a humanized version of the anti-FAP antibody, was used as a CAR, the amount of localization to the cell membrane increased significantly, regardless of the type of other regions, and the results far surpassed those of CARs targeting CD19.

[0144] It is not generally known that humanizing scFV used in CAR improves its localization to the cell membrane in this way. Furthermore, US2002 / 0099180, which only lists the provision of antibodies #18 and #34, does not mention that such effects can be obtained by using them in CAR.

[0145] These findings suggest that the results obtained by humanizing scFV in this embodiment were unexpected.

[0146] <Example 2. Relationship between CAR design and proliferation efficiency of expressed cells> (Objective) To investigate the effect of humanization of the scFV portion and the design of other portions on the proliferation efficiency of expressed cells.

[0147] (Methods) The design of the CAR expression vector, preparation of the lentiviral vector, and infection were carried out in the same manner as in Example 1.

[0148] Selected T cells after infection were suspended in culture medium to a cell density of 50 × 10^4 cells / mL, and subcultured by seeding 100 μL into each well of a 96-well plate. The cells were then cultured in culture medium until 6 days after infection. As a control, T cells that had not been infected with a lentiviral vector and cultured in culture medium for 6 days were used.

[0149] 1. Cell Count Measurement: The total number of cells obtained 6, 8, 12, 16, and 19 days after infection was measured using a flow cytometer (Beckman Coulter: CytoFLEX S). Flow cytometry analysis was performed according to the manufacturer's protocol.

[0150] 2. After counting the cells during subculturing, the cells were subculturified in culture medium to a cell density of 50 × 10^4 cells / mL and cultured for 2 to 4 days. Cell counts and subculturing were repeated after each subculturing period, allowing the cells to be cultured up to 19 days after infection.

[0151] 3. Calculation of Growth Rate The growth rate in each subculturing was calculated as the ratio of the number of cells obtained during measurement to the number of cells seeded at the time of measurement. Furthermore, the growth rate 6 days after infection was calculated as the ratio of the number of cells obtained during measurement to the number of cells used for infection. In addition, the cumulative growth rate based on each measurement result was calculated by adding the growth rate calculated from that measurement result to the cumulative growth rate based on the previous measurement result. The experiment was repeated twice for each condition.

[0152] (Results) The results are shown in Figure 2. Figure 2 shows the cumulative growth rate based on the measurement results for each type of CAR introduced.

[0153] CARs containing humanized scFV generally showed a high cumulative replication rate, and this trend was particularly pronounced after 12 days of infection. Furthermore, when comparing humanized scFVs, #34 tended to have a higher replication rate, and among #34s, those with a co-stimulatory domain derived from CD28 showed a particularly high replication rate.

[0154] It was previously unknown that humanization of scFV and other structural modifications could have such an effect on the proliferation rate of CAR-infected cells.

[0155] <Example 3. Relationship between CAR design and cytotoxic activity by expressing cells (1)> (Objective) To investigate the effects of humanization of the scFV portion and the design of other portions on cytotoxic activity by expressing cells.

[0156] (Methods) The design of the CAR expression vector, preparation of the lentiviral vector, and infection were carried out in the same manner as in Example 1.

[0157] Selected T cells after infection were suspended in culture medium to a cell density of 50 × 10⁴ cells / mL, and 2 mL was seeded into each well of a 6-well plate for subculturing. Subsequently, the cells were cultured in culture medium until 12 days after infection, while subculturing to maintain a concentration of 1 × 10⁶ cells / mL to 4 × 10⁶ cells / mL. As a control, T cells that were not infected with lentiviral vectors and cultured in culture medium for 12 days were used in the same manner.

[0158] 1. T cells and target cells obtained 12 days after co-culture infection with target cells were seeded in 384-well plates at a cell count of 1 × 10^4 cells / well and co-cultured for 48 hours in co-culture medium (X-VIVO15 medium supplemented with 5% human serum and 100 μM 2-mercaptoethanol).

[0159] As target cells, we used Nalm-6 cells (RIKEN BRC) into which FAP overexpression vectors and luciferase gene expression vectors had been introduced.

[0160] 2. Measurement of cytotoxic activity: After co-culture, each well contains a cell lysate (Promega, ONE-Glo) containing Luciferin substrate. TM The color reaction was carried out by adding ( ). The color reaction was performed according to the protocol provided by the manufacturer.

[0161] The luciferase signal was measured using a microplate reader (Hitachi High-Tech Corporation: SH-9000Lab).

[0162] Cytotoxic activity was calculated as the percentage decrease in the measured value under each condition compared to the measured value in the absence of T cells. This reflects the percentage decrease in target cells, with 0% cytotoxic activity corresponding to no change from the measured value in the absence of T cells, and 100% cytotoxic activity corresponding to a measured value of 0. The experiment was repeated three times for each condition.

[0163] (Results) The results are shown in Figure 3. Figure 3 shows the cytotoxic activity against FAP-expressing Nalm-6 cells for each type of CAR introduced.

[0164] In particular, higher cytotoxic activity was obtained when the co-stimulatory domain was derived from CD28, and even higher cytotoxic activity was obtained when #34 was used as the scFV.

[0165] <Example 4. Relationship between CAR design and cytotoxic activity by expressing cells (2)> (Objective) To investigate the effect of humanization of the scFV portion and the design of other portions on the cytotoxicity efficiency of expressing cells.

[0166] (Methods) The design of the CAR expression vector, preparation of the lentiviral vector, infection, culture of infected cells, and measurement of cytotoxic activity were carried out in the same manner as in Example 3.

[0167] 1. Co-culture with target cells: Target cells were seeded in 384-well plates at a cell count of 1.6 × 10⁴ cells / well. The following day, T cells obtained 9 days after infection were added and co-cultured in co-culture medium for 72 hours. T cells (effector cells) were added at cell counts of 3.2 × 10⁴ cells / well, 1.6 × 10⁴ cells / well, 0.8 × 10⁴ cells / well, 0.4 × 10⁴ cells / well, 0.2 × 10⁴ cells / well, 0.1 × 10⁴ cells / well, and 0.05 × 10⁴ cells / well. The ratio of effector cells to target cells (E / T ratio) under each condition was 200%, 100%, 50%, 25%, 12.5%, 6.25%, and 3.125%, respectively.

[0168] As target cells, we used A375 cells (ECACC) into which FAP overexpression vectors and luciferase gene expression vectors had been introduced. Each experiment was repeated three times under each condition.

[0169] (Results) The results are shown in Figure 4. Figure 4 shows the cytotoxic activity against FAP-expressing A375 cells for each type of CAR introduced.

[0170] All CAR-transformed T cells showed higher cytotoxic activity compared to non-CAR-transformed T cells (indicated as "WT" in Figure 4). When #34 was used as the scFV, extremely high cytotoxic activity was observed, particularly under harsh conditions for T cells (low E / T ratio), even when the number of T cells was low relative to the number of target cells. This level of activity was significantly higher than when F19 was used as the scFV. No significant difference in cytotoxic activity was observed depending on the type of linker.

[0171] <Example 5. Relationship between CAR design and cytotoxic activity by expressing cells (3)> (Objective) To investigate the effects of humanization of the scFV portion and the design of other portions on the long-term cytotoxic activity of expressing cells.

[0172] (Methods) The design of the CAR expression vector, preparation of the lentiviral vector, infection, culture of infected cells, and measurement of cytotoxic activity were carried out in the same manner as in Example 3.

[0173] 1. Co-culture with target cells (1) Target cells at 5 × 10^4 cells / well and T cells obtained 6 days after infection at 1.25 × 10^4 cells / well were seeded in a 96-well plate (E / T ratio: 25%) and co-cultured in co-culture medium for 48 hours. Nalm-6 cells, the same as in Example 3, were used as the target cells.

[0174] Half of the cell sample after co-culture was used for measurement of cytotoxic activity (labeled "1st" in Figure 5), and the other half was used for further co-culture. For the measurement of cytotoxic activity, target cells of 5 × 10^4 cells / well were seeded into a new 96-well plate at the start of co-culture, and the measurement results of the target cells cultured in the same manner were used as the value for 0% cytotoxic activity to calculate cytotoxic activity.

[0175] 2. Co-culture with target cells (2) After 48 hours of co-culture, the cell sample was again seeded with target cells at 5 × 10^4 cells / well into a new 96-well plate and co-cultured in co-culture medium for 72 hours.

[0176] The cell samples after this co-culture were subjected to measurement of cytotoxic activity (indicated as "2nd" in Figure 5). Here, half of the cells carried over from the wells where only 5 × 10^4 cells / well of target cells were seeded in the first co-culture were used, and the cytotoxic activity was calculated using the measurement results of target cells cultured in the same way after adding 5 × 10^4 cells / well of target cells to a new 96-well plate at the start of the second co-culture, with the value of 0% cytotoxic activity being used as the baseline. The experiment was repeated twice for each condition.

[0177] (Results) The results are shown in Figure 5. Figure 5 shows the cytotoxic activity (1st) in the first co-culture and the cytotoxic activity (2nd) in the second co-culture for each type of introduced CAR against FAP-expressing Nalm-6 cells.

[0178] In the initial co-culture, all CAR-introduced T cells showed higher cytotoxic activity compared to non-CAR-introduced T cells (in Figure 5, labeled "WT"). Furthermore, CARs with scFV #34 showed higher cytotoxic activity compared to those with F19. In particular, CARs composed of scFV #34 and a Whitlow linker almost completely killed FAP-expressing Nalm-6 cells (in Figure 5, labeled "#34_WL_28z").

[0179] In subsequent co-cultures, no cytotoxic activity was observed in T cells without CAR introduction (labeled "WT" in Figure 5). In the subsequent co-cultures, all T cells with CAR introduction showed cytotoxic activity, and the cytotoxic activity was higher when using a CAR with scFV #34 compared to when using F19. In particular, when a CAR composed of scFV #34 and a Whitlow linker was introduced, it showed cytotoxic activity of approximately 50%, which was higher than the results obtained in the initial co-culture for T cells without CAR introduction, despite repeated exposure.

[0180] <Example 6. Relationship between CAR design and cytotoxic activity by expressing cells (4)> (Objective) To investigate the effect of humanization of the scFV portion and the design of other portions on the cytotoxic activity of fibroblasts by expressing cells.

[0181] (Methods) The design of the CAR expression vector, preparation of the lentiviral vector, infection, culture of infected cells, and measurement of cytotoxic activity were carried out in the same manner as in Example 3.

[0182] 1. Co-culture with target cells: Target cells were seeded in a 384-well plate at a cell count of 0.6 × 10^4 cells / well. The following day, T cells obtained 6 days after infection were added, and the cells were co-cultured in culture medium for 72 hours. T cells (effector cells) were added at cell counts of 1.8 × 10^4 cells / well, 0.6 × 10^4 cells / well, and 0.2 × 10^4 cells / well. The ratio of effector cells to target cells (E / T ratio) under each condition was 300%, 100%, and 33%, respectively.

[0183] As target cells, synovial cells derived from rheumatoid arthritis patients (JCRB cell bank) into which a luciferase gene expression vector was introduced were used. Each experiment was repeated twice under each condition.

[0184] (Results) The results are shown in Figures 6-8. Figures 6-8 show the cytotoxic activity of each type of introduced CAR against synovial cells derived from rheumatoid arthritis patients. Synovial cells include fibroblasts that express FAP.

[0185] All CAR-transformed T cells showed higher cytotoxic activity compared to non-CAR-transformed T cells (labeled "WT" in Figures 6-8). When #34 was used as the scFV, extremely high cytotoxic activity was observed, especially under harsh conditions for T cells (low E / T ratio), where the number of T cells was low relative to the number of target cells. This level of activity was significantly higher than when F19 was used as the scFV. Furthermore, there was no significant difference in cytotoxic activity depending on the type of linker, nor was there a significant difference depending on the individual from which the target cells originated.

[0186] Furthermore, it was found that when synovial cells, including cells that naturally express FAP, were used as target cells, higher cytotoxic activity was obtained regardless of the E / T ratio, compared to the experimental system using A375 cells that artificially expressed FAP (Figure 4).

[0187] <Example 7. Relationship between CAR design and cytotoxic activity by expressing cells (5)> (Objective) To investigate the effects of humanization of the scFV portion and the design of other portions on the cytotoxic activity of fibroblasts in vivo by expressing cells.

[0188] (Methods) The design of the FAP CAR expression vector, preparation of the lentiviral vector, infection, and culture of infected cells were carried out in the same manner as in Example 3. 1. Design of the Mesothelin CAR Expression Vector Similar to the FAP CAR vector, the Mesothelin CAR expression vector was designed as a plasmid vector by inserting the nucleotide sequence encoding Mesothelin CAR (SEQ ID NO: 37) downstream of the SFFV promoter of pHR-SFFV. The P2A peptide sequence and EGFP sequence were placed at the C-terminus of the CAR to ensure simultaneous expression of CAR and EGFP. 2. Transplantation of Target Cells Normal human lung fibroblasts CCD-19Lu (ATCC) were used as FAP-expressing cells. (R) CCL-210 TM ), pancreatic cancer cells AsPC1 (ATCC) as Mesothelin-expressing cells (R) CRL-1682 TM ) was used as the target cell. 1.5 × 10^6 cells of CCD-19Lu and 5.0 × 10^5 cells of AsPC1 were mixed, and immunodeficient mice, NSG mice (Jackson Laboratory; strain name NOD.Cg-Prkdc) were used. scid Il2rg tm1Wjl The cells were transplanted subcutaneously (SzJ). 3. Transplantation of CAR-expressing cells Two weeks after target cell transplantation, the tumor volume was 50 mm 3 ~200mm 3 The tumor-reducing effect of CAR was tested using individuals that met the following criteria. First, these individuals were intravenously injected with 5.0 × 10^6 cells of FAP CAR-expressing T cells or, as a control, wild-type T cells that do not express CAR (day 0, "1st" in Figure 10). Two weeks later (day 14), 5.0 × 10^6 cells of Mesothelin CAR-expressing T cells were intravenously injected ("2nd" in Figure 10). 4. Measurement of Tumor Volume Tumor diameter was measured once or twice a week for four weeks starting from day 0. Tumor diameter was measured using calipers to obtain the longest and shortest diameters, and tumor volume was calculated as longest diameter × longest diameter × shortest diameter ÷ 2.

[0189] On day 28, the tumor volume of each group was statistically compared using the Dunnett method. Each group consisted of 5 or 6 mice.

[0190] (Results) The results are shown in Figure 10. Figure 10 shows the changes in tumor volume derived from AsPC1 and CCD-19Lu transplanted subcutaneously.

[0191] All T cells with FAP CAR introduced tended to reduce tumor size compared to wild-type T cells without CAR introduction (indicated as "WT" in Figure 10), but when F19_GS_28z was used, the final tumor volume on day 28 was comparable to that of WT. On the other hand, when scFV #34 was used, the tumor was significantly smaller on day 28 compared to both the "WT" group (p=0.005) and the "F19_GS_28z" group (p=0.009).

[0192] This trend was observed even before the introduction of Mesothelin CAR-expressing T cells. This indicates that the FAP CAR of the present invention not only enhances the effect of CAR-expressing T cells on cancer cells, but also exerts a significant inhibitory effect on tumor growth on its own.

[0193] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A chimeric antigen receptor (CAR) capable of binding to fibroblast-activating protein α (FAP), comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the antigen-binding domain of the humanized scFV is specifically capable of binding to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 13, and comprising: (a) a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1 or 35, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2 or 36, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 3; and (b) a light chain variable region comprising LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO:

6.

2. The CAR according to claim 1, wherein the antigen-binding domain comprises any of the following: (i) a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 7 or 9, and a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 8 or 10; (ii) a heavy chain variable region consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 7 or 9, excluding the CDR sequence portion, and a light chain variable region consisting of an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 8 or 10, excluding the CDR sequence portion; or (iii) a heavy chain variable region consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in respect to the amino acid sequence shown in SEQ ID NO: 7 or 9, excluding the CDR sequence portion, and a light chain variable region consisting of an amino acid sequence in which one or more amino acids are added, deleted and / or substituted in respect to the amino acid sequence shown in SEQ ID NO: 8 or 10, excluding the CDR sequence portion.

3. The CAR according to claim 1, wherein the linker of the humanized scFV comprises a Whitlow sequence, a Yol sequence, or an L1 sequence.

4. The CAR according to claim 1, wherein the intracellular region includes the CD3ζ signaling domain.

5. The CAR according to claim 4, wherein the intracellular region further comprises at least one co-stimulatory domain.

6. The CAR according to claim 5, wherein the at least one co-stimulatory domain comprises a signaling region of any one protein selected from the group consisting of 4-1BB / CD137, CD28, OX-40, CD27, CD40L, GITR, and ICOS.

7. The nucleic acid encoding CAR as described in claim 1.

8. A vector comprising the nucleic acid described in claim 7.

9. The vector according to claim 8, which is an expression vector.

10. A pharmaceutical composition comprising the CAR described in any one of claims 1 to 6 and / or the nucleic acid described in claim 7.

11. The pharmaceutical composition according to claim 10, for use in the treatment of one or more diseases selected from the group consisting of fibrosis, immune system disorders, and cancer.

12. The pharmaceutical composition according to claim 11, wherein the immune system disease includes autoimmune diseases and / or inflammatory diseases.

13. A host cell containing the nucleic acid described in claim 7.

14. The host cell according to claim 13, which is a peripheral blood mononuclear cell.

15. A cell preparation comprising the host cells described in claim 13.

16. The cell preparation according to claim 15, for use in the treatment of one or more diseases selected from the group consisting of fibrosis, immune system disorders, and cancer.

17. A composition comprising the nucleic acid according to claim 7 for use in the preparation of host cells according to claim 13 or 14.

18. A method for producing host cells according to claim 13 or 14, comprising the step of introducing the vector according to claim 8 or 9 into the cells.

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