Improved Anti-FAP alpha-chimeric antigen receptor

Abstract

The present invention identifies a novel single-stranded anti-FAP alpha antibody, and provides a novel CAR-T containing the scFV, the CAR-T hardly reducing FAP alpha expression in cancer cells, and a novel cancer therapeutic agent containing the CAR-T.　The anti-FAP alpha antibody or an antigen-binding fragment thereof according to the present disclosure includes, for example, the following (L) and (H): (L) Light chain variable region containing light chains CDR1-CDR3 having the amino acid sequences of SEQ ID NO: 5-7, respectively; and (H) Heavy chain variable region containing heavy chains CDR1-CDR3 having the amino acid sequences of SEQ ID NO: 78-80, respectively.

Description

Improved anti-FAPα chimeric antigen receptor

[0001] The present disclosure relates to an improved anti-FAPα antibody or an antigen-binding fragment thereof, a chimeric antigen receptor (CAR) comprising an scFV of the antibody, a T cell (CAR-T) comprising the CAR, and a cancer therapeutic agent comprising the CAR-T. The present disclosure also relates to a method for producing the CAR-T.

[0002] In recent years, attempts have been made to treat cancer (CAR-T therapy) using T cells (chimeric antigen receptor T cells, CAR-T) that express single-chain antibodies (scFv) that bind to cancer-specific antigens or target antigens. To date, the only CAR-Ts approved in Japan are Carvykti (Janssen) and Abecma (Bristol-Myers Squibb), which target the B-cell maturation antigen (BCMA), and Breyanzi (Bristol-Myers Squibb), Yescarta (Kite Pharma), and Kymliah (Novartis), which target CD19.

[0003] One of the challenges of CAR-T therapy is that many patients who initially respond to treatment experience relapse. For example, cancers that relapse after administration of CD19 CAR-T or BCMA CAR-T show loss of expression of the cancer antigens CD19 and BCMA. Furthermore, some relapsed patients have been reported to exhibit low antigen expression rather than loss of antigen expression, and a mechanism for the low antigen expression of cancer cells in relapsed patients has recently been reported (Non-Patent Document 1). This report showed that CAR-T binds to antigens on target cells, causing a process of antigen transfer into the CAR-T (trogocytosis), which leads to antigen loss on the target cells. This suggests that in patients who relapse after CAR-T therapy, CAR-T removes antigens from cancer cells, resulting in a decrease in antigen density on the cancer cells, resulting in cancer cell escape from CAR-T.

[0004] The stroma surrounding cancer cells (cancer stroma) plays an important role in cancer invasion and metastasis. Furthermore, the cancer stroma acts as a barrier, preventing adequate delivery of anticancer drugs to cancer cells. FAPα (fibroblast activation protein α) is highly expressed in cancer-associated fibroblasts (CAFs), a major component of the cancer stroma, while its expression is low in normal tissues. Therefore, FAPα-targeted therapy is expected to have fewer side effects. One known antibody against FAPα is sibrotuzumab, a humanized version of the murine anti-FAPα monoclonal antibody (F19). Clinical applications of this antibody have been investigated, but it failed to meet the criteria in a phase II clinical trial for metastatic colorectal cancer (Non-Patent Document 2). To date, antibody therapy using an antibody against FAPα, or CAR-T therapy using CAR-T cells engineered based on this antibody, have not been approved.

[0005] Nature volume 568, 112-116 (2019)Onkologie volume 26 (1): 44-48 (2003)

[0006] Therefore, the object of the present disclosure is to discover a new single-chain antibody against FAPα, and to provide a new CAR-T containing the scFV thereof, which is less likely to reduce FAPα expression in cancer cells, and a new cancer therapeutic agent containing the CAR-T.

[0007] To achieve the above objective, the present inventors conducted extensive research and found that the nucleic acid encoding the scFv (scFv) linking the light and heavy chain variable regions of the anti-FAPα humanized monoclonal antibody sibrotuzumab (F19) was replaced with a library containing nucleic acids encoding the light chain variable region from human peripheral blood B cells to prepare an F19 scFv library with a common heavy chain variable region. Using this library, they screened for new F19 mutant scFvs that bind to FAPα-expressing cells and generated T cell clones expressing CARs carrying these scFvs (F19 mutant CAR-T). Similar to T cells expressing a CAR carrying the sibrotuzumab scFV (F19 CAR-T), the F19 mutant CAR-T was found to be activated by FAPα antigen on antigen-expressing cells. However, unlike F19 CAR-T, it did not reduce the intensity of FAPα expression in antigen-expressing cells. Furthermore, the F19 mutant CAR-T was found to have affinity for FAPα, but to a lesser extent, compared to F19 CAR-T. Furthermore, the F19 mutant CAR-T was found to be less exhausted by FAPα than F19 CAR-T. Furthermore, the F19 mutant CAR-T exhibited a longer-term tumor regression effect in vivo compared to F19 CAR-T.

[0008] According to the present disclosure, administering F19 mutant CAR-T can attack CAFs that express FAPα and reduce the cancer stroma, thereby assisting the approach of anticancer drugs to cancer or directly attacking cancers that express FAPα. Furthermore, because F19 mutant CAR-T does not reduce FAPα expression, it can suppress cancer escape from CAR-T and is expected to have a stable tumor reduction effect.

[0009] Figure 1a is a histogram plot showing the relationship between the CD69 expression level and cell number in each CAR-T stimulated with K562 / FAPα cells in Example 2. The horizontal axis indicates the CD69 expression intensity in the cells, and the vertical axis indicates the number of CD69-expressing cells. Figure 1b is a graph showing the geometric mean of CD69 expression in each CAR-T stimulated with K562 / FAPα cells in Example 2. Stimulation: left bar. Unstimulation: right bar. The vertical axis indicates the geometric mean of CD69 expression. Figure 1c is a graph showing the percentage of CD69-positive cells in each CAR-T stimulated with K562 / FAPα cells in Example 2. The vertical axis indicates the percentage (%) of CD69-positive cells. Figure 2a is a histogram plot showing the relationship between the FAPα expression level and cell number in K562 / FAPα cells treated with each F19 mutant CAR-T in Example 3. The horizontal axis shows the expression intensity of FAPα in K562 / FAPα cells, and the vertical axis shows the number of K562 / FAPα cells expressing FAPα. Figure 2b is a graph showing the geometric mean of FAPα staining in surviving target cells treated with each F19 mutant CAR-T in Example 3. The vertical axis shows the geometric mean of FAPα staining. Figure 2c is a graph showing the proportion of FAPα expression-negative target cells treated with each F19 mutant CAR-T in Example 3. The vertical axis shows the proportion (%) of FAPα-negative target cells. Figure 3a is a graph showing the relationship between the proportion of stained F19 mutant CAR-T and recombinant FAPα concentration in Example 4. The horizontal axis shows recombinant FAPα concentration (nM), and the vertical axis shows the percentage (%) of maximum staining value. Figure 3b is a graph showing the EC50 of FAPα for each F19 mutant CAR-T in Example 4. The vertical axis shows EC50 (nM). Figure 4 is a graph showing the relationship between tumor volume and the number of days elapsed in cancer-bearing mice administered with each F19 mutant CAR-T in Example 5. The horizontal axis shows the number of days elapsed after U87 cell transplantation (days), and the vertical axis shows tumor volume (mm 3) in Figure 5. Figure 5 is a graph showing the relationship between tumor volume and the number of days elapsed in tumor-bearing mice administered with each F19 mutant CAR-T in Example 5. The horizontal axis shows the number of days elapsed after U87 cell transplantation, and the vertical axis shows tumor volume (mm 3 ) are shown. Figure 6 is a histogram plot showing the relationship between the expression levels of exhaustion markers (PD-1, LAG-3, and TIGIT) and the cell counts in each F19 mutant CAR-T stimulated with FAPα in Example 6. The horizontal axis shows the expression intensity of exhaustion markers (PD-1, LAG-3, and TIGIT) in the cells, and the vertical axis shows the number of cells expressing the exhaustion markers. Figure 7 is a graph showing the proportion of surviving target cells over time relative to the initial number of target cells treated with each F19 mutant CAR-T and cultured in 2D in Example 8. The vertical axis shows the proportion of surviving target cells relative to the initial number of target cells. The horizontal axis shows time. Figure 8 is a graph showing the proportion of surviving target cells over time relative to the initial number of target cells treated with each F19 mutant CAR-T and cultured in 3D in Example 8. The vertical axis shows the proportion of surviving target cells relative to the initial number of target cells. The horizontal axis shows time. Figure 9(A) is a line graph showing the average body weight (g) over time for each administration group in Example 9. The horizontal axis shows the number of days (days) since U87MG cell transplantation, and the vertical axis shows body weight (g). Figure 9(B) shows the tumor volume (mm ) for each treatment group (GFP, 7L1, 7L8, 7L12) in Example 9. 3 ) over time. The horizontal axis represents the number of days after U87MG cell transplantation, and the vertical axis represents tumor volume (mm 3 ) is shown. Figure 9(C) shows the appearance of mice 30 days after administration of each treatment group (GFP, 7L1, 7L8, 7L12) in Example 9. Figure 9(D) is a graph showing the tumor inhibition rate (%) 30 days after administration of each treatment group (GFP, 7L1, 7L8, 7L12) in Example 9. The horizontal axis shows the growth inhibition rate (%), and the vertical axis shows the type of CAR-T. Figure 9(E) is a line graph showing the survival rate (%) over time for each treatment group (GFP, 7L1, 7L8, 7L12) in Example 9. The horizontal axis shows the number of days (days) after U87MG cell transplantation, and the vertical axis shows the survival rate (%).

[0010] (Antibodies, etc. of the present disclosure) The present disclosure provides antibodies against FAPα (antibodies of the present disclosure) or antigen-binding fragments thereof (antigen-binding fragments of the present disclosure), which comprise a light chain variable region and a heavy chain variable region. Note that the antibodies of the present disclosure and the antigen-binding fragments of the present disclosure may also be collectively referred to as the antibodies, etc. of the present disclosure.

[0011] The antibodies and the like of the present disclosure are antibodies and the like against fibroblast activation protein alpha (FAPα). FAPα may be, for example, a protein isolated and purified from mammalian cells (e.g., human, mouse, rat, rabbit, sheep, pig, cow, horse, cat, dog, monkey, chimpanzee, etc.). Furthermore, FAPα may be, for example, a protein chemically synthesized or biochemically synthesized using a cell-free translation system, or a recombinant protein produced from a transformant into which a nucleic acid containing a nucleotide sequence encoding the amino acid sequence shown below has been introduced. When FAPα in the present disclosure is human FAPα, it may be, for example, a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence registered under NCBI accession number AAB49652.1 (SEQ ID NO: 1). The NCBI accession number is the number assigned to the full-length amino acid sequence of the protein (the same applies hereinafter).

[0012] Human FAPα (SEQ ID NO: 1)

[0013] Examples of amino acid sequences substantially identical to the amino acid sequence represented by SEQ ID NO: 1 include amino acid sequences having, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence represented by SEQ ID NO: 1. Here, "identity" refers to, for example, the degree of identity when the sequences to be compared are appropriately aligned, and means the occurrence rate (%) of exact amino acid matches between the sequences. The identity can be calculated, for example, using analysis software such as BLAST or FASTA with default parameters.

[0014] The antibody etc. of the present disclosure may be, for example, a so-called antibody having an immunoglobulin molecular structure, or an antigen-binding fragment thereof. In the case of an antibody, for example, its isotype is not particularly limited. Examples of the isotype include IgG, IgM, IgA, IgD, IgE, etc., and preferably IgG. In addition, examples of the IgG subclass include IgG1, IgG2, IgG3, IgG4, etc.

[0015] The antigen-binding fragment in the present disclosure refers to a portion of the antibody, e.g., a partial fragment, that recognizes (binds to) FAPα. Examples of the antigen-binding fragment include Fab, Fab', F(ab')2, variable region fragment (Fv), disulfide-linked Fv, single-chain Fv (scFv), bispecific antibody, and polymers thereof.

[0016] The antibodies and the like of the present disclosure comprise a light chain variable region and a heavy chain variable region. The light chain variable region and the heavy chain variable region comprised in the antibodies and the like of the present disclosure each comprise three complementarity determining regions (CDRs). The CDRs are regions in the light chain variable region and heavy chain variable region that are particularly highly variable in primary structure and are located close to each other in the three-dimensional structure, determining specificity for the antigen to which they bind. When the light chain variable region comprises three CDRs, they typically comprise, from the N-terminus, a light chain CDR1 (CDRL1), a light chain CDR2 (CDRL2), and a light chain CDR3 (CDRL3), in this order. Similarly, when the heavy chain variable region comprises three CDRs, they typically comprise, from the N-terminus, a heavy chain CDR1 (CDRH1), a heavy chain CDR2 (CDRH2), and a heavy chain CDR3 (CDRH3), in this order.

[0017] The light chain variable region and heavy chain variable region contained in the antibodies and the like of the present disclosure may each further comprise four framework regions (FRs) adjacent to each CDR. When the light chain variable region comprises three CDRs and four FRs, it typically comprises, from the N-terminus, light chain FR1 (FRL1), light chain CDR1 (CDRL1), light chain FR2 (FRL2), light chain CDR2 (CDRL2), light chain FR3 (FRL3), light chain CDR3 (CDRL3), and light chain FR4 (FRL4), in this order. Similarly, when the heavy chain variable region comprises three CDRs and four FRs, it typically comprises, from the N-terminus, heavy chain FR1 (FRH1), heavy chain CDR1 (CDRH1), heavy chain FR2 (FRH2), heavy chain CDR2 (CDRH2), heavy chain FR3 (FRH3), heavy chain CDR3 (CDRH3), and heavy chain FR4 (FRH4), in this order.

[0018] When the antigen-binding fragment of the present disclosure is an scFv, the scFv may have, for example, the heavy chain variable region and the light chain variable region linked via a linker. The order of the heavy chain variable region, the linker, and the light chain variable region may be, from the N-terminus, the heavy chain variable region, the linker, and the light chain variable region, or the order of the light chain variable region, the linker, and the heavy chain variable region may be, from the N-terminus. In the Examples described below, the scFv of the present disclosure has the light chain variable region, the linker, and the heavy chain variable region linked in this order.

[0019] The antibodies, etc. of the present disclosure may have, for example, a constant region in addition to the heavy chain variable region and the light chain variable region, and the constant region may be, for example, a human constant region or a mouse constant region. In the case of an antibody (immunoglobulin), the heavy chain constant region includes, for example, regions called CH1, CH2, and CH3, and the light chain constant region includes, for example, a region called CL. When the antibodies, etc. of the present disclosure have the constant regions, for example, the heavy chain variable region binds to at least one of CH1, CH2, and CH3, the light chain variable region binds to the CL, and the heavy chain variable region binds directly to, for example, CH1.

[0020] In the antibodies etc. of the present disclosure, the light chain variable region was prepared using a human B cell light chain variable region library. Specifically, the light chain variable region is a light chain variable region selected from the group consisting of (L1) to (L18) below. (L1) A light chain variable region comprising light chain complementarity determining regions (CDRL) 1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7; (L2) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10; (L3) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13; (L4) A light chain variable region comprising light chain complementarity determining regions (CDRL) 1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13; (L5) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16; (L6) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 19;(L6) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 21, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22; (L7) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 23, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 24, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 25; (L8) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 26, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 27, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 28; (L9) (L10) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 29, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31; (L11) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 37;(L12) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 39, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40; (L13) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 43; (L14) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 44, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 45, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 46; (L15) (L16) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 49; (L17) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 55;(L18) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:56, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:57, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:58.

[0021] The amino acid sequences of CDRL1, CDRL2, and CDRL3 contained in the light chain variable region of the antibody sibrotuzumab (clone: ​​F19), and CDRL1, CDRL2, and CDRL3 contained in the light chain variable region of the antibodies of the present disclosure, are shown in Table 1.

[0022]

[0023] Furthermore, in the antibodies etc. of the present disclosure, the light chain variable region may be a light chain variable region selected from the group consisting of (L1v) to (L18v) below. (L1v) The light chain variable region of (L1), which is a polypeptide selected from the group consisting of (L1v1) to (L1v3) below: (L1v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 60, (L1v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 60, (L1v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 60; (L2v) The light chain variable region of (L2), which is a polypeptide selected from the group consisting of (L2v1) to (L2v3) below: (L2v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 61, (L2v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 61, (L2v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 61; (L3v) The light chain variable region of (L3), which is a polypeptide selected from the group consisting of (L3v1) to (L3v3) below: (L3v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 62, (L3v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 62, (L3v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 62; (L4v) The light chain variable region of (L4), which is a polypeptide selected from the group consisting of (L4v1) to (L4v3) below: (L4v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 63, (L4v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 63, (L4v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 63;(L5v) The light chain variable region of (L5), which is a polypeptide selected from the group consisting of (L5v1) to (L5v3) below: (L5v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 64, (L5v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 64, (L5v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 64; (L6v) The light chain variable region of (L6), which is a polypeptide selected from the group consisting of (L6v1) to (L6v3) below: (L6v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 65, (L6v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 65, (L6v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 65; (L7v) The light chain variable region of (L7), which is a polypeptide selected from the group consisting of (L7v1) to (L7v3) below: (L7v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 66, (L7v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 66, (L7v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 66; (L8v) The light chain variable region of (L8), which is a polypeptide selected from the group consisting of (L8v1) to (L8v3) below: (L8v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 67, (L8v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 67, (L8v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 67;(L9v) The light chain variable region of (L9), which is a polypeptide selected from the group consisting of (L9v1) to (L9v3) below: (L9v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:68, (L9v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO:68, (L9v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO:68; (L10v) The light chain variable region of (L10), which is a polypeptide selected from the group consisting of (L10v1) to (L10v3) below: (L10v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:69, (L10v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO:69, (L10v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO:69; (L11v) The light chain variable region of (L11), which is a polypeptide selected from the group consisting of (L11v1) to (L11v3) below: (L11v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 70, (L11v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 70, (L11v3) a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO: 70; (L12v) The light chain variable region of (L12), which is a polypeptide selected from the group consisting of (L12v1) to (L12v3) below: (L12v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 71, (L12v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 71, (L12v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 71;(L13v) The light chain variable region of (L13), which is a polypeptide selected from the group consisting of (L13v1) to (L13v3) below: (L13v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 72, (L13v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 72, (L13v3) a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO: 72; (L14v) The light chain variable region of (L14), which is a polypeptide selected from the group consisting of (L14v1) to (L14v3) below: (L14v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 73, (L14v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 73, (L14v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 73; (L15v) A light chain variable region of (L15), which is a polypeptide selected from the group consisting of (L15v1) to (L15v3) below: (L15v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 74, (L15v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 74, (L15v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 74; (L16v) A light chain variable region of (L16), which is a polypeptide selected from the group consisting of (L16v1) to (L16v3) below: (L16v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 75, (L16v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 75; (L16v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 75;(L17v) The light chain variable region of (L17), which is a polypeptide selected from the group consisting of (L17v1) to (L17v3) below: (L17v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 76, (L17v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 76, (L17v3) a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO: 76; (L18v) The light chain variable region of (L18), which is a polypeptide selected from the group consisting of (L18v1) to (L18v3) below: (L18v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 77, (L18v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 77, (L18v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 77;

[0024] The amino acid sequences of the light chain variable regions of the antibody sibrotuzumab (clone: ​​F19) and the antibodies of the present disclosure are shown in Table 2.

[0025]

[0026] In the antibodies etc. of the present disclosure, the heavy chain variable region was prepared using the heavy chain variable region of the anti-FAPα humanized monoclonal antibody sibrotuzumab (clone: ​​F19). Specifically, in the antibodies etc. of the present disclosure, the heavy chain variable region is a heavy chain variable region comprising heavy chain complementarity-determining regions (CDRH) 1, CDRH2, and CDRH3, wherein CDRH1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 78, CDRH2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 79, and CDRH3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 80. The heavy chain variable region is common to the light chain variable region.

[0027] The amino acid sequences of CDRH1, CDRH2, and CDRH3 contained in the heavy chain variable region of the antibodies, etc. of the present disclosure are shown in Table 3. The heavy chain variable region is common to the antibody of sibrotuzumab (clone: ​​F19) and the antibodies, etc. of the present disclosure.

[0028]

[0029] Furthermore, in the antibodies etc. of the present disclosure, the heavy chain variable region may be a heavy chain variable region that is a polypeptide selected from the group consisting of the following (Hv1) to (Hv3): (Hv1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 81. (Hv2) A polypeptide comprising an amino acid sequence that is 90% or more identical to the amino acid sequence set forth in SEQ ID NO: 81. (Hv3) A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 81.

[0030] The amino acid sequences of the heavy chain variable regions of the antibodies of the present disclosure are shown in Table 4.

[0031]

[0032] When the light chain variable region of an antibody or the like of the present disclosure is a polypeptide selected from the group consisting of (L1v1) to (L18v1), the light chain variable region is a light chain variable region comprising, in this order, CDRL1, CDRL2, and CDRL3 contained in a light chain variable region selected from the group consisting of (L1) to (L18). Therefore, the amino acid sequences of CDRL1, CDRL2, and CDRL3 of a polypeptide selected from the group consisting of (L1v1) to (L18v1) can refer to the amino acid sequences of CDRL1, CDRL2, and CDRL3 contained in a light chain variable region selected from the group consisting of (L1) to (L18).

[0033] When the heavy chain variable region of an antibody or the like of the present disclosure is the (Hv1) polypeptide, the heavy chain variable region is a heavy chain variable region comprising the CDRH1, CDRH2, and CDRH3 in this order. Therefore, the amino acid sequences of CDRH1, CDRH2, and CDRH3 of the (Hv1) polypeptide can refer to the amino acid sequences of CDRH1, CDRH2, and CDRH3.

[0034] In (L1v2) to (L18v2) and (Hv2), "identity" refers to the degree of identity when the sequences being compared are appropriately aligned, and means the percentage of exact amino acid matches between the sequences. The degree of "identity" is not limited as long as the binding activity and specificity of the antibody or the like of the present disclosure to FAPα are maintained, and may be, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, respectively. The identity can be calculated using, for example, analysis software such as BLAST or FASTA, with default parameters.

[0035] When the light chain variable region of an antibody or the like of the present disclosure is a polypeptide selected from the group consisting of (L1v2) to (L18v2), the amino acid sequences of at least one (preferably all) of the CDRs contained in the polypeptide may be 100% conserved. In the case of (L1v2), an example of such a light chain variable region is an amino acid sequence that contains CDRL1 (the amino acid sequence set forth in SEQ ID NO:4), CDRL2 (the amino acid sequence set forth in SEQ ID NO:5), and CDRL3 (the amino acid sequence set forth in SEQ ID NO:6), and that has 90% or greater identity to the amino acid sequence set forth in SEQ ID NO:60. In the case of light chain variable regions other than (L1v2) (i.e., the light chain variable regions of (L2v2) to (L18v2)), an example of such a light chain variable region is an amino acid sequence that contains the amino acid sequences of CDRL1, CDRL2, and CDRL3 contained in the corresponding light chain variable region, and that has 90% or greater identity to the amino acid sequence of each corresponding light chain variable region.

[0036] When the heavy chain variable region of an antibody or the like of the present disclosure is the (Hv2) polypeptide, the amino acid sequences of at least one (preferably all) of the CDRs contained in the polypeptide may be 100% conserved. Examples of such heavy chain variable regions include CDRH1 (the amino acid sequence shown in SEQ ID NO:78), CDRH2 (the amino acid sequence shown in SEQ ID NO:79), and CDRH3 (the amino acid sequence shown in SEQ ID NO:80), and include amino acid sequences that share 90% or more identity with the amino acid sequence shown in SEQ ID NO:81.

[0037] In the light chain variable region or heavy chain variable region of an antibody, etc., of the present disclosure, the range of "one or several" regarding substitutions, etc. is not limited as long as the binding activity and specificity of the antibody, etc., of the present disclosure to FAPα are maintained, and may be, for example, 1 to 12, 1 to 11, 1 to 10, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1, respectively.

[0038] The amino acid substitution may be, for example, a conservative substitution. The conservative substitution refers to substituting one or several amino acids with other amino acids and / or amino acid derivatives so as not to substantially alter the function of the antibody or the like of the present disclosure. The "substituting amino acid" and the "substituted amino acid" preferably have similar properties and / or functions. Specifically, they preferably have similar chemical properties, such as hydrophobicity and hydrophilicity index (hydropathy), polarity, and charge, or physical properties, such as secondary structure. Amino acids or amino acid derivatives having similar properties and / or functions are known in the art, for example. Specific examples of nonpolar amino acids (hydrophobic amino acids) include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine; polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine; positively charged amino acids (basic amino acids) include arginine, histidine, and lysine; and negatively charged amino acids (acidic amino acids) include aspartic acid and glutamic acid.

[0039] When the light chain variable region of an antibody or the like of the present disclosure is a polypeptide selected from the group consisting of (L1v3) to (L18v3), the amino acid sequences of at least one (preferably all) of the CDRs contained in the polypeptide may be 100% conserved. In the case of (L1v3), examples of such a light chain variable region include an amino acid sequence comprising CDRL1 (the amino acid sequence of SEQ ID NO:4), CDRL2 (the amino acid sequence of SEQ ID NO:5), and CDRL3 (the amino acid sequence of SEQ ID NO:6), in which one or more amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO:60. In the case of light chain variable regions other than (L1v3) (i.e., the light chain variable regions of (L2v3) to (L18v3)), examples of such a light chain variable region include an amino acid sequence comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 contained in the corresponding light chain variable region, in which one or more amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence of the corresponding light chain variable region.

[0040] When the heavy chain variable region of an antibody or the like of the present disclosure is the (Hv3) polypeptide, the amino acid sequences of at least one (preferably all) of the CDRs contained in the polypeptide may be 100% conserved. Examples of such heavy chain variable regions include CDRH1 (the amino acid sequence shown in SEQ ID NO:78), CDRH2 (the amino acid sequence shown in SEQ ID NO:79), and CDRH3 (the amino acid sequence shown in SEQ ID NO:80), and include amino acid sequences in which one or more amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence shown in SEQ ID NO:81.

[0041] The binding of the antibody or the like of the present disclosure to FAPα can be confirmed by a method for detecting antibody-antigen binding, such as surface plasmon resonance (SPR) or flow cytometry.

[0042] The antibody, etc. of the present disclosure may further contain, for example, a labeling substance. The labeling substance is not particularly limited and includes, for example, a fluorescent substance, a dye, an isotope, an enzyme, etc. Examples of the fluorescent substance include fluorophores such as pyrene, TAMRA, fluorescein, Cy3 dye, Cy5 dye, FAM dye, rhodamine dye, Texas Red dye, JOE, MAX, HEX, and TYE, and examples of the dye include Alexa dyes such as Alexa488 and Alexa647. The antibody, etc. of the present disclosure may be modified, for example, directly or indirectly with the small molecular weight compound. In the latter case, the antibody, etc. of the present disclosure may be modified with the small molecular weight compound, for example, via a linker.

[0043] The antibody or the like of the present disclosure may be immobilized, for example, on a carrier, a porous body, etc. The carrier is not particularly limited and examples thereof include a substrate, beads, a container, etc., and examples of the container include a microplate, a tube, etc.

[0044] The method for producing the antibodies and the like of the present disclosure is not particularly limited, and can be, for example, produced by genetic engineering based on the amino acid sequence information described above. Specifically, for example, the method can be performed as follows. However, the present disclosure is not limited to this example.

[0045] First, a vector containing a nucleic acid sequence encoding the amino acid sequences of the CDRLs (CDRL1, CDRL2, CDRL3), the light chain variable region, the light chain comprising the light chain variable region and light chain constant region, the CDRHs (CDRH1, CDRH2, CDRH3), the heavy chain variable region, and / or the heavy chain comprising the heavy chain variable region and heavy chain constant region in the antibodies etc. of the present disclosure is introduced into a host to obtain a transformant. The transformant is then cultured and a fraction containing an antibody that binds to FAPα is recovered, and the antibody is isolated or purified from the recovered fraction.

[0046] Examples of the vector include a vector containing a nucleic acid sequence encoding the light chain variable region, a vector containing a nucleic acid sequence encoding the heavy chain variable region, a vector containing a nucleic acid sequence encoding the heavy chain, and a vector containing a nucleic acid sequence encoding the light chain. The host is not particularly limited as long as it can be introduced with the vector and can express the nucleic acid sequence in the vector. Examples of the host include mammalian cells such as HEK cells, CHO cells, COS cells, NSO cells, and SP2 / 0 cells. The method for introducing the vector into the host is not particularly limited, and known methods can be used.

[0047] The method for culturing the transformant is not particularly limited and can be appropriately determined depending on the type of the host. The antibody-containing fraction can be recovered, for example, by disrupting the cultured transformant and collecting the fraction as a liquid fraction. The method for isolating or purifying the antibody is not particularly limited and known methods can be used.

[0048] In the present disclosure, the antibody is a monoclonal antibody. Examples of the monoclonal antibody include monoclonal antibodies obtained by immunizing animals, chimeric antibodies, and humanized antibodies.

[0049] The chimeric antibody is an antibody in which the variable region of an antibody derived from a non-human animal is linked to the constant region of a human antibody. The chimeric antibody can be produced, for example, as follows: First, a gene encoding the light chain variable region contained in the antibody, etc. of the present disclosure is prepared, and this gene is ligated to a gene encoding the light chain constant region of a human antibody, and the resulting product is ligated to an expression vector. Next, a gene encoding the heavy chain variable region contained in the antibody, etc. of the present disclosure is prepared, and this gene is ligated to a gene encoding the heavy chain constant region of a human antibody, and the resulting product is ligated to an expression vector. Cells transfected with the two expression vectors are then cultured, and the desired chimeric antibody secreted into the culture medium is recovered. This allows the chimeric antibody to be prepared. The animal from which the variable region gene is derived is not particularly limited, and examples include rats and mice. The chimeric antibody can be produced by any known method, such as the method described in Japanese Patent Publication No. 3-73280.

[0050] The humanized antibody is an antibody in which only the CDR is derived from a non-human animal and the other regions are derived from humans. The humanized antibody can be produced, for example, as follows: First, a gene encoding the CDR contained in the antibody or the like of the present disclosure is prepared, and then grafted onto a human antibody gene, for example, the variable region (CDR grafting), which is then linked to an expression vector. Cells transfected with the expression vector are then cultured, and a humanized antibody with the desired CDR grafted is secreted into the culture medium. The secreted humanized antibody can be prepared by collecting the antibody. The animal from which the CDR is derived is not particularly limited, and examples include rats and mice. The method for producing a humanized antibody is not limited thereto, and the antibody can be produced by referring to known methods, such as those described in JP-A-4-506458 and JP-A-62-296890.

[0051] (Chimeric Antigen Receptor of the Present Disclosure) The present disclosure provides a chimeric antigen receptor (chimeric antigen receptor (CAR) of the present disclosure) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain comprises an antibody of the present disclosure or an antigen-binding fragment thereof.

[0052] In the present disclosure, the term "domain" refers to a structurally or functionally integrated region of a polypeptide, such as a peptide having a length of 10 amino acids or more.

[0053] The CAR of the present disclosure is characterized in that the antigen-binding domain comprises the antibody or the like of the present disclosure, and other configurations and conditions are not particularly limited. The CAR of the present disclosure can be suitably used, for example, as a CAR expressed by CAR-T cells against FAPα-expressing cancer cells.

[0054] The antigen-binding domain may be, for example, an antibody or an antigen-binding fragment of the present disclosure. The antigen-binding fragment is preferably a single-chain antibody (scFv). When the antigen-binding domain is an scFv, the light chain variable region and the heavy chain variable region are arranged in this order from the N-terminus or C-terminus, preferably from the N-terminus.

[0055] In the scFv, the light chain variable region and the heavy chain variable region are linked by, for example, a linker peptide (Fv linker peptide). Preferably, the Fv linker peptide does not inhibit the binding of the scFv to FAPα. The Fv linker peptide is composed of, for example, 1 to 40, 1 to 18, 1 to 15, 1 to 7, 1 to 3, or 1 or 2 amino acids. The Fv linker peptide is composed of, for example, amino acids such as glycine and serine, and a specific example is (GGGGS (SEQ ID NO: 82)). ｎ The n is, for example, an integer of 1 to 6. The amino acid sequence of the Fv linker peptide is, for example, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 83 or 84 below.

[0056] Fv linker peptide 1 GSTSGSGKPGSGEGSTKG (SEQ ID NO: 83) Fv linker peptide 2 GGGGSGGGGSGGGGS (SEQ ID NO: 84)

[0057] Specific examples of the antigen-binding domain include polypeptides selected from the group consisting of the following (BD1) to (BD3): (BD1) A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 86 to 103. (BD2) A polypeptide that binds to FAPα, comprising an amino acid sequence that is 90% or more identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 86 to 103. (BD3) A polypeptide that binds to FAPα, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 86 to 103, in which one or several amino acids have been deleted, substituted, inserted and / or added.

[0058] The amino acid sequences of the antigen-binding domain of the CAR derived from the antibody sibrotuzumab (clone: ​​F19) and the antigen-binding domain of the CAR of the present disclosure are shown in Table 5.

[0059]

[0060] In the polypeptide (BD1), the amino acid sequences of SEQ ID NOs: 86 to 103 are the amino acid sequences of scFVs obtained by linking the light chain variable region and heavy chain variable region of an antibody, etc. of the present disclosure with an Fv linker peptide (GSTSGSGKPGSGEGSTKG: SEQ ID NO: 83). Therefore, the amino acid sequences of the heavy chain variable region and light chain variable region in the amino acid sequences of SEQ ID NOs: 86 to 103 can refer to the respective amino acid sequences of the light chain variable region and heavy chain variable region of an antibody, etc. of the present disclosure.

[0061] In the (BD1) polypeptide, the order of the amino acid sequences of the light chain variable region and the heavy chain variable region may be reversed. As a specific example, the (BD1) polypeptide may be a polypeptide consisting of an amino acid sequence in which the amino acid sequences of the light chain variable region and the heavy chain variable region in the amino acid sequences of SEQ ID NOs: 86 to 103 are exchanged.

[0062] In the polypeptide (BD2), the "identity" is, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0063] In the polypeptide of (BD2), the amino acid sequence of at least one (preferably all) CDRs contained in the polypeptide may be 100% conserved. In the case of SEQ ID NO: 86, such polypeptides include, for example, amino acid sequences comprising CDRL1 (the amino acid sequence of SEQ ID NO: 4), CDRL2 (the amino acid sequence of SEQ ID NO: 5), CDRL3 (the amino acid sequence of SEQ ID NO: 6), CDRH1 (the amino acid sequence of SEQ ID NO: 78), CDRH2 (the amino acid sequence of SEQ ID NO: 79), and CDRH3 (the amino acid sequence of SEQ ID NO: 80), which have 90% or more identity to the amino acid sequence of SEQ ID NO: 86. In the case of polypeptides other than the polypeptide comprising the amino acid sequence of SEQ ID NO: 86 (polypeptides comprising the amino acid sequences of SEQ ID NOs: 87 to 103), examples of such polypeptides include amino acid sequences comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 contained in the corresponding light chain variable region and the amino acid sequences of CDRH1, CDRH2, and CDRH3 contained in the corresponding heavy chain variable region, which have 90% or more identity to the amino acid sequence of the corresponding scFV.

[0064] In the polypeptide (BD3), "one or several" with regard to substitutions etc. means, for example, 1 to 25, 1 to 24, 1 to 23, 1 to 20, 1 to 15, 1 to 12, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.

[0065] In the (BD3) polypeptide, the amino acid sequence of at least one (preferably all) CDRs contained in the polypeptide may be 100% conserved. In the case of SEQ ID NO: 86, such polypeptides include, for example, CDRL1 (the amino acid sequence of SEQ ID NO: 4), CDRL2 (the amino acid sequence of SEQ ID NO: 5), CDRL3 (the amino acid sequence of SEQ ID NO: 6), CDRH1 (the amino acid sequence of SEQ ID NO: 78), CDRH2 (the amino acid sequence of SEQ ID NO: 79), and CDRH3 (the amino acid sequence of SEQ ID NO: 80), and include amino acid sequences in which one or several amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO: 86. In the case of polypeptides other than the polypeptide comprising the amino acid sequence of SEQ ID NO: 86 (polypeptides comprising the amino acid sequences of SEQ ID NOs: 87 to 103), examples of such polypeptides include amino acid sequences comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 contained in the corresponding light chain variable region and the amino acid sequences of CDRH1, CDRH2, and CDRH3 contained in the corresponding heavy chain variable region, in which one or several amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence of the corresponding scFV.

[0066] The transmembrane domain is, for example, a domain that spans the cell membrane when the CAR of the present disclosure is expressed in a cell. The transmembrane domain may be, for example, the transmembrane domain of a transmembrane protein, or an artificially designed artificial transmembrane domain. In the former case, the transmembrane protein may be, for example, the α or β chain of the T cell receptor, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8 (CD8α or CD8β), CD9, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS, GITR, etc., or a variant thereof having equivalent functions. The variant having equivalent functions is, for example, a polypeptide that consists of an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added, and functions as a transmembrane domain. The "one or several" refers to, for example, 1 to 15, 1 to 10, 1 to 5, or 1 or 2. In the latter case, the artificial transmembrane domain is a polypeptide mainly composed of hydrophobic amino acids such as leucine, isoleucine, and valine. The artificial transmembrane domain may also have, for example, a tripeptide of phenylalanine, tryptophan, and valine at each end. When the transmembrane domain is the transmembrane domain of the transmembrane protein, the transmembrane domain may include, for example, one or several amino acids on the N-terminal or C-terminal side that are continuous with the transmembrane domain of the transmembrane protein. For example, the above-mentioned examples can be used for the "one or several".

[0067] An example of the transmembrane domain is the transmembrane domain of CD28, which is represented by the following amino acid sequence (SEQ ID NO: 104) from positions 153 to 179 in the amino acid sequence registered in NCBI under accession number NP_006130.

[0068] CD28 transmembrane domain FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 104)

[0069] As the transmembrane domain of CD28, for example, a transmembrane unit (SEQ ID NO: 105) including, in addition to the transmembrane domain of CD28, the extracellular region on its N-terminal side and the intracellular region on its C-terminal side may be used.

[0070] CD28 transmembrane unit IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 105)

[0071] The intracellular signaling domain is, for example, a domain capable of transmitting a signal intracellularly when the antigen-binding domain of the CAR of the present disclosure binds to FAPα. Examples of the signaling domain include the intracellular amino acid sequences of the T cell receptor (TCR) complex and costimulatory molecules, as well as variants having equivalent functions to these amino acid sequences. The variants having equivalent functions are, for example, polypeptides consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted, and / or added, and functioning as an intracellular signaling domain. The above explanation can be used for the term "one or several."

[0072] Typically, T cells are activated by antigen-dependent cytoplasmic signals, such as intracellular signals mediated by the TCR complex (primary activation). This activation is then enhanced by antigen-nonspecific cytoplasmic signals, such as intracellular signals mediated by costimulatory molecules (secondary activation). For this reason, T cells have, for example, a domain that activates via the TCR complex (primary activation domain) and a domain that activates nonspecifically (secondary activation domain). Therefore, the intracellular signaling domain has, for example, at least one of the primary activation domain and the secondary activation domain, and preferably both.

[0073] The primary activation domain can regulate primary activation by a TCR complex, for example. Examples of primary activation domains that induce primary activation include intracellular signaling domains containing an immunoreceptor tyrosine-based activation motif (ITAM). On the other hand, examples of primary activation domains that inhibit primary activation include intracellular signaling domains containing an immunoreceptor tyrosine-based inhibition motif (ITIM).

[0074] Examples of the ITAM-containing intracellular signaling domain include intracellular signaling domains such as CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, or variants thereof with equivalent functions. The variants with equivalent functions are, for example, polypeptides consisting of an amino acid sequence with one or more amino acids deleted, substituted, inserted, and / or added, and functioning as an ITAM-containing intracellular signaling domain. The above explanation for "one or more" can be used for reference. Examples of the ITAM-containing intracellular signaling domain of CD3ζ include the amino acid sequence from positions 52 to 164 in the amino acid sequence registered under NCBI accession number NP_932170.1 or the amino acid sequence set forth in SEQ ID NO: 106. Examples of the ITAM-containing intracellular signaling domain of FcεRIγ include the amino acid sequence from positions 45 to 86 in the amino acid sequence registered under NCBI accession number NP_004097.1. An example of an ITAM-containing intracellular signaling domain of FcεRIβ is the amino acid sequence from 201 to 244 in the amino acid sequence registered under NCBI accession number NP_000130.1. An example of an ITAM-containing intracellular signaling domain of CD3γ is the amino acid sequence from 139 to 182 in the amino acid sequence registered under NCBI accession number NP_000064.1. An example of an ITAM-containing intracellular signaling domain of CD3δ is the amino acid sequence from 128 to 171 in the amino acid sequence registered under NCBI accession number NP_000723.1. An example of an ITAM-containing intracellular signaling domain of CD3ε is the amino acid sequence from 153 to 207 in the amino acid sequence registered under NCBI accession number NP_000724.1. An example of the ITAM-containing intracellular signaling domain of CD5 is the amino acid sequence from positions 402 to 495 in the amino acid sequence registered under NCBI accession number NP_055022.2.An example of an ITAM-containing intracellular signaling domain of CD22 is the amino acid sequence from 707 to 847 in the amino acid sequence registered under NCBI accession number NP_001762.2. An example of an ITAM-containing intracellular signaling domain of CD79a is the amino acid sequence from 166 to 226 in the amino acid sequence registered under NCBI accession number NP_001774.1. An example of an ITAM-containing intracellular signaling domain of CD79b is the amino acid sequence from 182 to 229 in the amino acid sequence registered under NCBI accession number NP_000617.1. An example of an ITAM-containing intracellular signaling domain of CD66d is the amino acid sequence from 177 to 252 in the amino acid sequence registered under NCBI accession number NP_001806.2.

[0075] ITAM-containing intracellular signaling domain of CD3ζ (SEQ ID NO: 106) RVKSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0076] Examples of the secondary activation domain include intracellular signaling domains of CD2, CD4, CD5, CD8α, CD8β, CD27, CD28, CD134 (OX40), CD137 (4-1BB), CD154, GITR, ICOS, etc., or variants with equivalent functions. The variants with equivalent functions are, for example, polypeptides that function as intracellular signaling domains and comprise an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, and / or added. The above explanation for the "one or more" can be applied, for example. Specifically, an example of the CD2 intracellular signaling domain is the amino acid sequence from positions 236 to 351 in the amino acid sequence registered under NCBI accession number NP_001758.2. An example of the CD4 intracellular signaling domain is the amino acid sequence from positions 421 to 458 in the amino acid sequence registered under NCBI accession number NP_000607.1. An example of the intracellular signaling domain of CD5 is the amino acid sequence from 402 to 495 in the amino acid sequence registered under NCBI accession number NP_055022.2. An example of the intracellular signaling domain of CD8α is the amino acid sequence from 207 to 235 in the amino acid sequence registered under NCBI accession number NP_001759.3. An example of the intracellular signaling domain of CD8β is the amino acid sequence from 196 to 210 in the amino acid sequence registered under NCBI accession number AAA35664.1. An example of the intracellular signaling domain of CD27 is the amino acid sequence from 213 to 260 in the amino acid sequence registered under NCBI accession number M63928.1. An example of the intracellular signaling domain of CD28 is the amino acid sequence from 181 to 220 (SEQ ID NO: 107) in the amino acid sequence registered under NCBI accession number NP_006130.1. An example of the intracellular signaling domain of CD134 (OX40) is the amino acid sequence from amino acids 241 to 277 (SEQ ID NO: 108) in the amino acid sequence registered under NCBI accession number NP_003318.1.An example of the intracellular signaling domain of CD137 (4-1BB) is the amino acid sequence from positions 214 to 255 (SEQ ID NO: 109) in the amino acid sequence registered under NCBI accession number NP_001552.2. An example of the intracellular signaling domain of GITR is the amino acid sequence from positions 193 to 241 in the amino acid sequence registered under NCBI accession number NP_004186.1. An example of the intracellular signaling domain of ICOS is the amino acid sequence from positions 166 to 199 in the amino acid sequence registered under NCBI accession number NP_036224.1.

[0077] CD28 intracellular signaling domain RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 107)

[0078] CD134 (OX40) intracellular signaling domain RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (SEQ ID NO: 108)

[0079] CD137 (4-1BB) intracellular signaling domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 109)

[0080] The CAR of the present disclosure includes, for example, one or more intracellular signaling domains. When including multiple intracellular signaling domains, the intracellular signaling domains may be the same or different. The intracellular signaling domain preferably includes, for example, an intracellular signaling domain containing an ITAM of CD3ζ as the primary activation domain. Furthermore, the intracellular signaling domain preferably includes, for example, at least one intracellular signaling domain selected from the group consisting of CD28, CD134 (OX40), and CD137 (4-1BB) as the secondary activation domain. Specific examples of the intracellular signaling domain include, for example, an intracellular signaling domain containing an ITAM of CD3ζ and at least one intracellular signaling domain selected from the group consisting of CD28, CD134 (OX40), and CD137 (4-1BB). When the intracellular signaling domain comprises an ITAM-containing intracellular signaling domain of CD3ζ and at least one intracellular signaling domain selected from the group consisting of CD28, CD134 (OX40), and CD137 (4-1BB), the ITAM-containing intracellular signaling domain of CD3ζ is preferably located C-terminal to the at least one intracellular signaling domain selected from the group consisting of CD28, CD134 (OX40), and CD137 (4-1BB).When the intracellular signaling domain comprises an ITAM-containing intracellular signaling domain of CD3ζ and an intracellular signaling domain of CD28, the intracellular signaling domain may further comprise at least one intracellular signaling domain of CD134 (OX40) and CD137 (4-1BB).

[0081] The antigen-binding domain and the transmembrane domain are linked directly or indirectly. In the case of the indirect link, the antigen-binding domain and the transmembrane domain are preferably linked via a spacer peptide. The spacer peptide is composed of, for example, 1 to 300, 1 to 100, 10 to 100, or 25 to 50 amino acids. The spacer peptide preferably contains, for example, an amino acid (e.g., cysteine, serine, threonine, etc.) that promotes binding between the CAR of the present disclosure and FAPα and induces and / or enhances signal transduction by the intracellular signaling domain upon binding between the CAR of the present disclosure and FAPα. Specific examples of the spacer peptide include immunoglobulin constant regions such as CH1 regions and L regions, and partial regions of CD4, CD8α, CD8β, or CD28. The spacer peptide may also be an artificially designed amino acid sequence. An example of a partial region of CD8α is the hinge region of CD8α, specifically the amino acid sequence from positions 118 to 178 in the amino acid sequence registered under NCBI accession number NP_001759.3. An example of a partial region of CD8β is the amino acid sequence from positions 135 to 195 in the amino acid sequence registered under NCBI accession number AAA35664.1. An example of a partial region of CD4 is the amino acid sequence from positions 315 to 396 in the amino acid sequence registered under NCBI accession number NP_000607.1. An example of a partial region of CD28 is the amino acid sequence from positions 137 to 152 (SEQ ID NO: 110) in the amino acid sequence registered under NCBI accession number NP_006130.1. The spacer peptide may be a portion of each of the exemplary amino acid sequences.

[0082] Partial region of CD28 (extracellular region) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 110)

[0083] In the CAR of the present disclosure, the domains may be linked together by, for example, a linker peptide (domain linker peptide). The domain linker peptide is composed of, for example, 1 to 40, 1 to 18, 1 to 15, 1 to 7, 1 to 3, or 1 or 2 amino acids. The domain linker peptide is composed of, for example, amino acids such as glycine and serine, and a specific example is (GGGGS (SEQ ID NO: 82)). ｎ The n is, for example, an integer of 1 to 6.

[0084] An example of a CAR of the present disclosure is a polypeptide consisting of the amino acid sequence represented by [light chain variable region]-[Fv linker peptide]-[heavy chain variable region]-[CD28 transmembrane unit]-[CD3ζ ITAM-containing intracellular signaling domain]. The [light chain variable region], [Fv linker peptide], [heavy chain variable region], [CD28 transmembrane unit], and [CD3ζ ITAM-containing intracellular signaling domain] are the amino acid sequences of the light chain variable region of an antibody of the present disclosure, the Fv linker peptide 1 of a CAR of the present disclosure, the heavy chain variable region of an antibody of the present disclosure, the CD28 transmembrane unit of a CAR of the present disclosure, and the CD3ζ ITAM-containing intracellular signaling domain of a CAR of the present disclosure, respectively. Fv linker peptide 1 may also be Fv linker peptide 2 of a CAR of the present disclosure.

[0085] Specific examples of the CAR of the present disclosure include polypeptides selected from the group consisting of the following (CAR1) to (CAR 3): (CAR1) A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 112 to 129; (CAR2) A polypeptide comprising an amino acid sequence having 90% or more identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 112 to 129, and binding to FAPα; (CAR3) A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 112 to 129, in which one or several amino acids have been deleted, substituted, inserted and / or added, and binding to FAPα.

[0086] The amino acid sequences of the CAR derived from the antibody sibrotuzumab (clone: ​​F19) and the CAR of the present disclosure are shown in Table 6.

[0087]

[0088] In the (CAR1) polypeptide, the amino acid sequences of SEQ ID NOs: 112 to 129 are amino acid sequences obtained by linking an scFV obtained by linking the light chain variable region and heavy chain variable region of an antibody, etc. of the present disclosure with an Fv linker peptide (GSTSGSGKPGSGEGSTKG: SEQ ID NO: 83), a transmembrane unit of CD28, and an intracellular signaling domain containing an ITAM of CD3ζ. Therefore, the amino acid sequences of the heavy chain variable region and light chain variable region in the amino acid sequences of SEQ ID NOs: 112 to 129 can be referenced to the respective amino acid sequences of the light chain variable region and heavy chain variable region of an antibody, etc. of the present disclosure.

[0089] In the (CAR1) polypeptide, the order of the amino acid sequences of the light chain variable region and the heavy chain variable region may be reversed. As a specific example, the (CAR1) polypeptide may be a polypeptide consisting of an amino acid sequence in which the amino acid sequences of the light chain variable region and the heavy chain variable region in the amino acid sequences of SEQ ID NOs: 112 to 129 are exchanged.

[0090] In the (CAR2) polypeptide, the "identity" is, for example, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, respectively.

[0091] In the (CAR2) polypeptide, the amino acid sequence of at least one (preferably all) CDRs contained in the polypeptide may be 100% conserved. In the case of SEQ ID NO: 112, such polypeptides include, for example, CDRL1 (the amino acid sequence shown in SEQ ID NO: 4), CDRL2 (the amino acid sequence shown in SEQ ID NO: 5), CDRL3 (the amino acid sequence shown in SEQ ID NO: 6), CDRH1 (the amino acid sequence shown in SEQ ID NO: 78), CDRH2 (the amino acid sequence shown in SEQ ID NO: 79), and CDRH3 (the amino acid sequence shown in SEQ ID NO: 80), and have an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO: 112. In the case of polypeptides other than the polypeptide containing the amino acid sequence shown in SEQ ID NO: 112 (polypeptides containing the amino acid sequences shown in SEQ ID NOs: 113 to 129), examples of such polypeptides include amino acid sequences that contain the amino acid sequences of CDRL1, CDRL2, and CDRL3 contained in the corresponding light chain variable region and the amino acid sequences of CDRH1, CDRH2, and CDRH3 contained in the corresponding heavy chain variable region, and have an amino acid sequence that is 90% or more identical to the amino acid sequence of the corresponding CAR.

[0092] In the (CAR3) polypeptide, "one or several" with regard to substitutions etc. means, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2, or 1.

[0093] In the polypeptide of (BD3), the amino acid sequence of at least one (preferably all) CDRs contained in the polypeptide may be 100% conserved. In the case of SEQ ID NO: 112, such polypeptides include, for example, CDRL1 (the amino acid sequence of SEQ ID NO: 4), CDRL2 (the amino acid sequence of SEQ ID NO: 5), CDRL3 (the amino acid sequence of SEQ ID NO: 5), CDRH1 (the amino acid sequence of SEQ ID NO: 78), CDRH2 (the amino acid sequence of SEQ ID NO: 79), and CDRH3 (the amino acid sequence of SEQ ID NO: 80), and include amino acid sequences in which one or several amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence of SEQ ID NO: 112. In the case of polypeptides other than the polypeptide comprising the amino acid sequence of SEQ ID NO: 112 (polypeptides comprising the amino acid sequences of SEQ ID NOs: 113 to 129), examples of such polypeptides include amino acid sequences comprising the amino acid sequences of CDRL1, CDRL2, and CDRL3 contained in the corresponding light chain variable regions and the amino acid sequences of CDRH1, CDRH2, and CDRH3 contained in the corresponding heavy chain variable regions, in which one or several amino acids have been deleted, substituted, inserted, and / or added relative to the amino acid sequence of the corresponding CAR.

[0094] The CAR of the present disclosure may have, for example, a signal peptide at the N-terminus. Examples of the signal peptide include signal peptides that signal transport to the endoplasmic reticulum. The CAR of the present disclosure may have, for example, a tag. Examples of the tag include peptide tags, protein tags, etc. Examples of the tag include FLAG (registered trademark) tags, HA tags, His tags, Myc tags, V5 tags, truncated NGFR (nerve growth factor receptor), etc. The tag peptide is, for example, added to at least one of the N-terminus and C-terminus of the CAR of the present disclosure. When the tag is truncated NGFR, the tag is positioned, for example, on the C-terminus side of the CAR of the present disclosure. The CAR of the present disclosure and the tag may be linked by a linker peptide.

[0095] (Nucleic Acids Encoding Chimeric Antigen Receptors of the Present Disclosure) The present disclosure provides nucleic acids encoding the chimeric antigen receptors of the present disclosure (nucleic acids of the present disclosure).

[0096] The nucleic acid of the present disclosure may be composed of DNA, RNA, or both. The nucleic acid may be composed of natural nucleic acids, modified nucleic acids obtained by modifying natural nucleic acids, artificial nucleic acids such as ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acids), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), and morpholino, or two or more of these.

[0097] The nucleic acid of the present disclosure can be prepared, for example, by a conventional method based on the amino acid sequence of the CAR of the present disclosure. Specifically, the nucleic acid encoding the CAR of the present disclosure can be prepared by, for example, obtaining a base sequence encoding the amino acid sequence from a database in which the amino acid sequences of each of the aforementioned domains are registered, and then using molecular biological techniques and / or chemical synthesis methods based on the base sequence. The base sequence of the nucleic acid of the present disclosure may be codon-optimized, for example, depending on the origin of the cells expressing the CAR of the present disclosure.

[0098] (Vector Comprising a Nucleic Acid of the Present Disclosure) The present disclosure provides a vector (the vector of the present disclosure) comprising a nucleic acid of the present disclosure.

[0099] The vector of the present disclosure can be prepared, for example, by ligating the nucleic acid of the present disclosure to a gene transfer vector. The type of gene transfer vector is not particularly limited, and examples include retroviral vectors such as oncoretroviral vectors, lentiviral vectors, and pseudotype vectors; and viral vectors such as adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Specific examples of the gene transfer vector include pUC, pCMV, pMX, and pELP. The gene transfer vector can also be appropriately designed depending on, for example, the cells into which the vector of the present disclosure is to be introduced.

[0100] The vector of the present disclosure preferably has a regulatory sequence that regulates the expression of the nucleic acid of the present disclosure. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori). The location of the regulatory sequence in the vector of the present disclosure is not particularly limited. In the vector of the present disclosure, the regulatory sequence may be located, for example, so long as it is capable of functionally regulating the expression of the nucleic acid of the present disclosure, and may be located based on known methods. For example, the regulatory sequence may utilize a sequence already present in the gene transfer vector, or the regulatory sequence may be further inserted into the gene transfer vector, or the regulatory sequence present in the gene transfer vector may be replaced with another regulatory sequence.

[0101] The vector of the present disclosure may further comprise, for example, a coding sequence for a selectable marker. Examples of the selectable marker include a drug resistance marker, a fluorescent protein marker, an enzyme marker, and a cell surface receptor marker.

[0102] (Cells Comprising a Vector of the Present Disclosure) The present disclosure provides cells (cells of the present disclosure) comprising a vector of the present disclosure.

[0103] The host cells into which the vectors of the present disclosure are introduced are not particularly limited, and examples thereof include pluripotent stem cells, immune cells, etc. Furthermore, when the cells of the present disclosure are administered, the host cells are preferably cells derived from the subject of administration.

[0104] Pluripotent stem cells used as host cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic tumor cells (EC cells), and embryonic germ stem cells (EG cells), with ES cells or iPS cells (more preferably human iPS cells) being preferred.

[0105] When the pluripotent stem cells are ES cells, they can be prepared by a method known per se. Methods for producing ES cells include, for example, a method for culturing the inner cell mass of mammalian blastocyst stage embryos (see, for example, Manipulating the Mouse Embryo: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)), a method for culturing early embryos produced by somatic cell nuclear transfer (Wilmut et al., Nature, 385, 810 (1997); Cibelli et al., Science, 280, 1256 (1998); Iritani A. et al., Proteins, Nucleic Acids, and Enzymes, 44, 892 (1999); Baguisi et al., Nature Biotechnology, 17, 456 (1999); Wakayama et al., Nature, 394, 369 (1998); Wakayama et al., Nature Genetics, 22, 127 (1999); Wakayama et al., Proc. Natl. Acad. Sci. USA, 96, 14984 (1999); Rideout III et al., Nature Genetics, 24,109 (2000)). ES cells can be obtained from designated institutions or purchased commercially. For example, human ES cell lines H1, H7, H9, H13, and H14 are available from the WiCell Research Institute in the United States; HES1-6 are available from ES Cell International in Australia; SA002, SA181, and SA611 are available from Cellartis AB in Sweden; HUES1-17 are available from the HUES Cell Facility in the United States; KhES-1 to KhES-5 are available from the Institute of Medical Biology, Kyoto University; and SEES1-SEES7 and SEES-x are available from the National Center for Child Health and Development. When ES cells are produced by somatic cell nuclear transfer, the type and source of somatic cells are the same as those for iPS cell production, described below.

[0106] When the pluripotent stem cells are iPS cells, they can be generated by introducing a nuclear reprogramming substance into a somatic cell. The somatic cells that can be used as the starting material for generating iPS cells may be any cells derived from mammals (e.g., mice or humans) other than germ cells. Examples include keratinizing epithelial cells (e.g., keratinizing epidermal cells), mucosal epithelial cells (e.g., epithelial cells of the tongue surface), exocrine gland epithelial cells (e.g., mammary gland cells), hormone-secreting cells (e.g., adrenal medullary cells), metabolic / storage cells (e.g., hepatocytes), luminal epithelial cells that form interface surfaces (e.g., type I alveolar cells), luminal epithelial cells of the inner chain ducts (e.g., vascular endothelial cells), ciliated cells with transport capacity (e.g., airway epithelial cells), cells that secrete extracellular matrix (e.g., fibroblasts), contractile cells (e.g., smooth muscle cells), cells of the blood and immune system (e.g., T lymphocytes), sensory cells (e.g., rod cells), autonomic nervous system neurons (e.g., cholinergic neurons), supporting cells of sensory organs and peripheral neurons (e.g., companion cells), neurons and glial cells of the central nervous system (e.g., astrocytes), pigment cells (e.g., retinal pigment epithelial cells), and their precursor cells (tissue precursor cells). There are no particular limitations on the degree of differentiation of cells, and both undifferentiated progenitor cells (including somatic stem cells) and terminally differentiated mature cells can be used as somatic cells for preparing iPS cells. Examples of undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as adipose-derived stromal (stem) cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

[0107] As nuclear reprogramming substances to be introduced into somatic cells to generate iPS cells, various combinations of reprogramming genes have been reported so far (e.g., WO 2007 / 069666, Nature Biotechnology, 26, 101-106 (2008), Cell, 126, 663-676 (2006), Cell, 131, 861-872 (2007), Nat. Cell Biol., 11, 197-203 (2009), Nature, 451, 141-146 (2008), Science, 318, 1917-1920 (2007), Stem Cells, 26, 1998-2005 (2008), Cell Research (2008) 600-603, Nature 454: 646-650). (2008), Cell Stem Cell, 2: 525-528(2008), WO2008 / 118820, Nat. Cell Biol., 11, 197-203(2009), Nat. Cell Biol., 11, 197-203(2009), Science, 324: 797-801(2009)). In addition, proteins encoded by the above-mentioned reprogramming genes can also be introduced into somatic cells as nuclear reprogramming substances (Cell Stem Cell, 4: 381-384(2009), Cell Stem Cell, doi:10.1016 / j.stem.2009.05.005(2009)). In particular, when considering the therapeutic use of the resulting iPS cells, a combination of the three factors Oct3 / 4, Sox2, and Klf4 is preferred.

[0108] iPS cell colonies can be selected using drug resistance and reporter activity as indicators (Cell, 126, 663-676 (2006), Nature, 448, 313-317 (2007)) or by visual morphological observation (Cell, 131, 861-872 (2007)). Identification of iPS cells can be confirmed by the expression of various ES cell-specific genes and teratoma formation.

[0109] Immune cells used as host cells include lymphocytes, granulocytes, macrophages, etc., preferably lymphocytes (more preferably human lymphocytes). Lymphocytes include T cells, NK cells, NKT cells, B cells, etc., preferably T cells (more preferably human T cells). The immune cells may be, for example, immune cells isolated from a living body, or immune cells induced to differentiate from pluripotent stem cells.

[0110] When the immune cells are isolated from a living body, they can be isolated by means known in the art. For example, when the immune cells are T cells, peripheral blood mononuclear cells (PBMCs) can be collected from a living body by apheresis, and then the T cells can be isolated and collected using an anti-CD3 antibody column, density gradient centrifugation, or the like.

[0111] When the immune cells are induced from pluripotent stem cells, they can be differentiated into T cells by any method known in the art. For example, when the immune cells are T cells, they can be differentiated into T cells by the methods described in International Publication Nos. 2016 / 076415 and 2017 / 221975.

[0112] When the cells of the present disclosure are T cells, the cells of the present disclosure may be obtained by introducing a vector of the present disclosure into T cells obtained from a living organism or T cells induced to differentiate from pluripotent stem cells as host cells, or by introducing a vector of the present disclosure into pluripotent stem cells as host cells and inducing the differentiation of the resulting cells into T cells.

[0113] (Method for producing cells that express a chimeric antigen receptor, comprising the step of introducing a vector of the present disclosure into cells) The present disclosure provides a method for producing cells that express a chimeric antigen receptor (the production method of the present disclosure), comprising the step of introducing a vector of the present disclosure into cells.

[0114] In the production method of the present disclosure, the method for introducing the vector of the present disclosure into cells is not particularly limited, and for example, known methods for introducing nucleic acids into cells can be used, including methods using nucleic acid introduction reagents such as liposomes and cationic lipids, methods using viruses such as retroviruses and lentiviruses, etc. As described above, cells into which the vector of the present disclosure is introduced include pluripotent stem cells, immune cells, etc.

[0115] When the cells into which the vector of the present disclosure is introduced are pluripotent stem cells, the production method of the present disclosure may further include a step of differentiating the pluripotent stem cells into which the vector of the present disclosure is introduced into T cells. As described above, the step of differentiating the pluripotent stem cells into T cells can be carried out according to known means in the art (e.g., WO 2016 / 076415 and WO 2017 / 221975).

[0116] The production method of the present disclosure may further include a step of culturing cells into which the vector of the present disclosure has been introduced. The cell culture period is not particularly limited and may be, for example, 6 hours to 30 days, 6 to 96 hours, or 1 to 30 days. The cell culture temperature is, for example, 28 to 37°C.

[0117] (Cancer therapeutic agent comprising cells of the present disclosure or cells produced by the manufacturing method of the present disclosure) The present disclosure provides a cancer therapeutic agent (cancer therapeutic agent of the present disclosure) comprising cells of the present disclosure or cells produced by the manufacturing method of the present disclosure (hereinafter collectively referred to as cells of the present disclosure).

[0118] The cancer therapeutic agent of the present disclosure is characterized by containing the cells of the present disclosure, and other configurations and conditions are not particularly limited.

[0119] In the cancer therapeutic agent of the present disclosure, the cancer to be treated is not particularly limited as long as it is a cancer in which cancer-associated fibroblasts present in the vicinity thereof highly or specifically express FAPα, or a cancer that highly or specifically expresses FAPα. Examples of cancers in which cancer-associated fibroblasts present in the vicinity thereof highly or specifically express FAPα include head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colon cancer, and esophageal cancer. Examples of cancers that highly or specifically express FAPα include glioblastoma, osteosarcoma, and malignant mesothelioma. Therefore, the cancer to be treated is at least one selected from head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colorectal cancer, esophageal cancer, glioblastoma, osteosarcoma, and malignant mesothelioma.

[0120] The conditions for administering the cancer therapeutic agent of the present disclosure are not particularly limited, and the administration form, administration method, administration timing, dosage, etc. can be appropriately set depending on, for example, the type of cancer to be treated, the stage of cancer progression, the age of the patient, etc. The method for using the cancer therapeutic agent of the present disclosure is not particularly limited, and for example, the cancer therapeutic agent of the present disclosure may be administered to the subject.

[0121] The subjects of administration include, for example, humans and non-human animals, such as mammals including mice, rats, dogs, monkeys, rabbits, sheep, horses, and pigs.

[0122] The administration method is not particularly limited and can be appropriately determined depending on, for example, the subject of administration. Examples of the administration method include parenteral administration, oral administration, etc. Examples of the parenteral administration include topical administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intralymphatic administration, and intratumoral administration.

[0123] The administration form of the cancer therapeutic agent of the present disclosure is not particularly limited, and may be, for example, a liquid such as an injection solution (injection agent) or an intravenous infusion solution.

[0124] In the cancer therapeutic agent of the present disclosure, the amount of the cells of the present disclosure to be compounded is not particularly limited. Furthermore, the administration conditions of the cells of the present disclosure are not particularly limited. When the administration method is intravenous administration, the administration conditions of the cells of the present disclosure are, for example, a single dose (total) for a human adult male of, for example, 1 x 10 ８ ~3 x 10 １０ cells, preferably 1 x 10 ９ ~1 x 10 １０ The cells are administered, for example, once every one to four weeks. In the cancer therapeutic agent of the present disclosure, the cells are preferably contained in an amount that satisfies the administration conditions exemplified above.

[0125] The cancer therapeutic agent of the present disclosure may further contain other additives. The amount of the additive is not particularly limited as long as it does not interfere with the function of the cancer therapeutic agent of the present disclosure. The additive is not particularly limited, and for example, a pharmaceutically acceptable additive is preferred. The type of the additive is not particularly limited, and can be appropriately selected depending on, for example, the type of administration target. Examples of the additive include a base, a stabilizer, a preservative, etc.

[0126] (Cancer treatment method comprising a step of administering to a subject the cells of the present disclosure or cells produced by the production method of the present disclosure) The present disclosure provides a cancer treatment method (cancer treatment method of the present disclosure) comprising a step of administering to a subject the cells of the present disclosure.

[0127] The cancer treatment method of the present disclosure is characterized by including a step of administering the cells of the present disclosure to a subject, and other steps and conditions are not particularly limited. In the administration step of the cancer treatment method of the present disclosure, the cancer to be treated, administration conditions, subjects, administration method, administration form, etc. can be referenced from the description of the cancer treatment agent of the present disclosure.

[0128] (Cells of the Present Disclosure or Cells Produced by the Production Method of the Present Disclosure for Use in Treating Cancer) The present disclosure provides cells of the present disclosure (cells for use of the present disclosure) for use in treating cancer.

[0129] For cells for use in the present disclosure, the cancer to be treated, administration conditions, administration subjects, administration methods, administration forms, etc. can be referenced from the explanation of the cancer therapeutic agent of the present disclosure.

[0130] Next, examples of the present disclosure will be described. However, the present disclosure is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. Note that "mol / L" may also be abbreviated as "M."

[0131] Example 1 Preparation of F19 Mutant CAR-T Cells and FAPα-Expressing Cells (1) Preparation of F19 scFV and Its Mutants F19 scFV was an scFv linking the light and heavy chain variable regions of the anti-FAPα humanized monoclonal antibody sibrotuzumab (F19). Screening of F19 scFV mutants (F19 mutant scFv) was performed according to the method described in WO2020 / 162452. Briefly, among the nucleic acids encoding scFv linking the light and heavy chain variable regions of the anti-FAPα humanized monoclonal antibody sibrotuzumab (F19), the nucleic acid encoding the light chain variable region was replaced with a library containing nucleic acids of the light chain variable region derived from human peripheral blood B cells to prepare an F19 scFv library with a common heavy chain variable region. This library was used to screen for new F19 mutant scFVs that bind to FAPα-expressing cells.As a result, by linking, in this order, a nucleic acid encoding the light chain variable region of F19 svFC (SEQ ID NO:59) or each mutant thereof (7L1 (SEQ ID NO:60), 7L2 (SEQ ID NO:61), 7L3 (SEQ ID NO:62), 7L8 (SEQ ID NO:63), 7L12 (SEQ ID NO:64), 7L20 (SEQ ID NO:65), 7L29 (SEQ ID NO:66), 7L35 (SEQ ID NO:67), 7L36 (SEQ ID NO:68), 7L48 (SEQ ID NO:69), 12L1 (SEQ ID NO:70), 12L3 (SEQ ID NO:71), 12L4 (SEQ ID NO:72), 12L5 (SEQ ID NO:73), 12L18 (SEQ ID NO:74), 12L21 (SEQ ID NO:75), 14L4 (SEQ ID NO:76), or 14L17 (SEQ ID NO:77)), a nucleic acid encoding an Fv linker peptide (GSTSGSGKPGSGEGSTKG (SEQ ID NO:83)), and a nucleic acid encoding the heavy chain variable region of F19 scFV (SEQ ID NO:81), F19 Various nucleic acids encoding scFv (SEQ ID NO: 85) and F19 mutant scFVs (7L1 (SEQ ID NO: 86), 7L2 (SEQ ID NO: 87), 7L3 (SEQ ID NO: 88), 7L8 (SEQ ID NO: 89), 7L12 (SEQ ID NO: 90), 7L20 (SEQ ID NO: 91), 7L29 (SEQ ID NO: 92), 7L35 (SEQ ID NO: 93), 7L36 (SEQ ID NO: 94), 7L48 (SEQ ID NO: 95), 12L1 (SEQ ID NO: 96), 12L3 (SEQ ID NO: 97), 12L4 (SEQ ID NO: 98), 12L5 (SEQ ID NO: 99), 12L18 (SEQ ID NO: 100), 12L21 (SEQ ID NO: 101), 14L4 (SEQ ID NO: 102), or 14L17 (SEQ ID NO: 103)) were prepared.

[0132] (2) Preparation of F19 CAR and Its Mutants Next, nucleic acids encoding the partial region, transmembrane domain, and intracellular signaling domain of human CD28 (SEQ ID NO: 105) and the intracellular signaling domain of human CD3ζ (SEQ ID NO: 106) were linked to the 3' end of the nucleic acids encoding F19 scFv and F19 mutant scFv, respectively, in this order from the 5' end. This resulted in the preparation of nucleic acids encoding F19 CAR containing F19 scFv and nucleic acids encoding F19 mutant CAR containing F19 mutant scFv. The nucleic acids encoding the partial region, transmembrane domain, and intracellular signaling domain of human CD28 and the intracellular signaling domain of human CD3ζ were obtained using the GeneArt™ (Thermo Fisher Scientific) artificial gene synthesis service. Furthermore, the nucleic acids encoding the partial region, transmembrane domain, and intracellular signaling domain of human CD28 had a NotI restriction enzyme site added to the 5' end.

[0133] Furthermore, a nucleic acid encoding a truncated NGFR gene (ΔNGFR) was linked to the 3' end of each nucleic acid encoding F19 CAR and F19 mutant CAR via a nucleic acid encoding a furin cleavage site (RAKR: SEQ ID NO: 130), a spacer sequence (SGSG: SEQ ID NO: 131), and a codon-optimized P2A sequence (ATNFSLLKQAGDVEENPGP: SEQ ID NO: 132). This resulted in the preparation of a nucleic acid encoding a tagged F19 CAR and a nucleic acid encoding a tagged F19 mutant CAR. Each nucleic acid was introduced into a pMXs expression vector.

[0134] (3) Preparation of T cells expressing F19 CAR and its mutants. Plat-A cells were transfected with an expression vector for F19 CAR or an expression vector for F19 mutant CARs using a transfection reagent (TransIT293, Takara Bio). Culture supernatant containing ecotropic retrovirus was prepared. Plat-A cells were maintained in DMEM medium containing 10% fetal calf serum (FCS), 1 μg / mL puromycin, and 10 μg / mL blasticidin. Next, the ecotropic virus-containing culture supernatant was added to PG13 cells, which were used as packaging cells, to transduce F19 CAR or each F19 mutant CAR. GaLV-pseudotyped retroviruses containing F19 CAR or each F19 mutant CAR were prepared. PG13 cells were cultured in DMEM medium containing 10% FCS at 37°C in a humid atmosphere with 5% CO2. Unless otherwise specified, the cell culture conditions were the same below.

[0135] Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy individuals and cultured for two days in the presence of 50 ng / mL anti-CD3 antibody (clone: ​​OKT3) and 100 IU / mL human IL-2. The culture medium for the PBMCs was RPMI 1640 medium containing 50 μg / mL gentamicin and 10% human AB serum (Sigma). After the culture, T cells were infected with the GaLV-pseudotyped retrovirus by centrifugation at 1000 × g and 32°C for 1 hour, and then transduced with the F19 CAR or each F19 mutant CAR. The resulting T cells were stained with PE-labeled anti-human NGFR mAb (clone: ​​ME20.4) and then analyzed using a flow cytometer (Gallios flow cytometer, Beckman Coulter) to confirm the expression of the tag on the T cells. Data obtained from the flow cytometer were analyzed using FlowJo Version 7.6.5 software (TreeStar).

[0136] T cells after transfection with the F19 CAR or each F19 mutant CAR were reacted with the FITC-labeled anti-human NGFR mAb. After the reaction, anti-FITC microbeads (Miltenyi Biotec) were used to detect T cells (F19 CAR-T) into which F19 CAR (SEQ ID NO: 111) had been introduced or T cells (F19 mutants) into which each F19 mutant CAR (7L1 (SEQ ID NO: 112), 7L2 (SEQ ID NO: 113), 7L3 (SEQ ID NO: 114), 7L8 (SEQ ID NO: 115), 7L12 (SEQ ID NO: 116), 7L20 (SEQ ID NO: 117), 7L29 (SEQ ID NO: 118), 7L35 (SEQ ID NO: 119), 7L36 (SEQ ID NO: 120), 7L48 (SEQ ID NO: 121), 12L1 (SEQ ID NO: 122), 12L3 (SEQ ID NO: 123), 12L4 (SEQ ID NO: 124), 12L5 (SEQ ID NO: 125), 12L18 (SEQ ID NO: 126), 12L21 (SEQ ID NO: 127), 14L4 (SEQ ID NO: 128), or 14L17 (SEQ ID NO: 129)) had been introduced. Unless otherwise stated, in Examples 2 to 6 below, F19 CAR-T or F19 mutant CAR-T refers to T cells generated by a retrovirus containing nucleic acid encoding the F19 CAR or each F19 mutant CAR.

[0137] (3) Preparation of target antigen-expressing cells Using a retrovirus, a nucleic acid encoding a truncated NGFR gene (ΔNGFR) was linked to the 3' end of a nucleic acid encoding FAPα via nucleic acids encoding a furin cleavage site (RAKR: SEQ ID NO: 130), a spacer sequence (SGSG: SEQ ID NO: 131), and a codon-optimized P2A sequence (ATNFSLLKQAGDVEENPGP: SEQ ID NO: 132). This was then introduced into K562 cells (obtained from ATCC, without expression of HLA or FAPα) to produce antigen-expressing cells (K562 / FAPα) that forcibly express FAPα.

[0138] Example 2 CD69 Assay We investigated whether each F19 mutant CAR-T (7L1, 7L2, 7L3, 7L8, 7L12, 7L20, 7L29, 7L35, 7L36, 7L48, 12L1, 12L3, 12L4, 12L5, 12L18, 12L21, 14L4, and 14L17) is activated by binding to FAPα expressed by K562 / FAPα cells. First, 1.0x10 K562 / FAPα cells were cultured in a 5% COOH / Cell culture medium. 6 Adjust the concentration to 1.0x10 cells / mL and plate at 1.0x10 cells / mL per well in a 96-well U-bottom plate. 5 Next, each F19 mutant CAR-T, positive control (F19 CAR-T), and negative control (dNGFR-T) were seeded at 1.0x10 cells / 100μL. 5 The cells were adjusted to a concentration of 100 cells / 100 μL and seeded into each well of the 96-well U-bottom plate. After 5 hours of incubation at 37°C, the cells were transferred to a V-plate and washed once with 2% FCS / PBS at 1000 x g for 1 minute at 4°C. The cells were then blocked with 20% mouse serum / PBS for 10 minutes at room temperature, and 100 μL of 2% FCS / PBS was added and centrifuged at 1000 x g for 1 minute at 4°C. 10 μL of antibodies (FITC-labeled anti-CD69 antibody, BV421-labeled anti-NGFR antibody, and PE-labeled anti-CD8 antibody) were added to the cells and incubated for 15 minutes at 4°C. 100 μL of 2% FCS / PBS was added and centrifuged at 1000 x g for 1 minute at 4°C. The cell pellet was suspended in 200 μL of 2% FCS / PBS and analyzed by flow cytometry. The results are shown in Figure 1a-c.

[0139] Figure 1a is a histogram plot showing the CD69 expression levels and cell counts for each F19 mutant CAR-T, positive control, and negative control, with or without stimulation with FAPα expressed by K562 / FAPα cells. Figure 1b is a graph showing the geometric mean of CD69 expression for each CAR-T stimulated with K562 / FAPα cells. Figure 1c is a graph showing the percentage of CD69 expression for each CAR-T stimulated with K562 / FAPα cells. As shown in Figures 1a-c, CD69 expression remained unchanged in the negative control, regardless of stimulation. On the other hand, stimulation with FAPα expressed by K562 / FAPα cells for the positive control increased the percentage of cells expressing high levels of CD69 compared to unstimulated control, confirming that the positive control bound to FAPα on target cells and activated them. Furthermore, when each F19 mutant CAR-T was stimulated with FAPα, the percentage of cells highly expressing CD69 increased compared to the unstimulated control, similar to the positive control. Therefore, each F19 mutant CAR-T bound to and activated FAPα on target cells, similar to the positive control.

[0140] Example 3 Antigen Reduction Assay We investigated whether the FAPα expression level of K562 / FAPα cells was reduced by binding to each F19 mutant CAR-T (7L1, 7L2, 7L3, 7L8, 7L12, 7L20, 7L29, 7L35, 7L36, 7L48, 12L1, 12L3, 12L4, 12L5, 12L18, 12L21, 14L4, and 14L17). First, K562 / FAPα cells were cultured at 1.0 x 10 6 Adjust the concentration to 1.0x10 cells / mL and plate at 1.0x10 cells / mL per well in a 96-well U-bottom plate. 5 Next, each F19 mutant CAR-T, positive control (F19 CAR-T), and negative control (dNGFR-T) were seeded at 1.0x10 cells / 100μL. 5The solution was adjusted to 100 cells / 100 μL and seeded into each well of the 96-well U-bottom plate. After 2 hours of incubation at 37°C, the cells were transferred to a V-plate and washed once with 2% FCS / 2 mM EDTA / PBS at 1000 × g for 1 minute. The cells were then blocked with 20% mouse serum / PBS for 10 minutes at room temperature and centrifuged at 1000 × g for 1 minute. 20 μL of mAbs (PE-labeled anti-Linker antibody, APC-labeled anti-FAP antibody, FITC-labeled anti-CD3e antibody, and BV421-labeled anti-CD271 antibody) diluted 50-fold with 2% FCS / PBS were added to the cells and incubated for 30 minutes at 4°C. The cells were then washed with 2% FCS / PBS at 1000 × g for 1 minute. The cells were then suspended in 7AAD diluted 500-fold with 2% FCS / PBS and analyzed by flow cytometry. 7AAD-negative and CD3e-negative cells were used as viable target cells, and the FAPα expression level on these target cells was examined. The results are shown in Figures 2a to 2c.

[0141] Figure 2a is a histogram plot showing the relationship between FAPα expression and cell number in surviving target K562 / FAPα cells treated with each F19 mutant CAR-T. Figure 2b is a graph showing the geometric mean of FAPα staining in surviving target cells treated with each F19 mutant CAR-T. Figure 2c is a graph showing the percentage of surviving target cells treated with each F19 mutant CAR-T with attenuated FAPα expression. As shown in Figures 2a-c, unlike K562 / FAPα cells treated with medium or the negative control, K562 / FAPα cells treated with the positive control showed an increased number of cells with attenuated FAPα expression. On the other hand, the percentage of cells with attenuated FAPα expression decreased in K562 / FAPα cells treated with each F19 mutant CAR-T. This suggests that the positive control CAR had a high binding affinity for FAPα and partially shed FAPα from K562 / FAPα cells. Furthermore, each F19 mutant CAR-T, unlike the positive control, was less likely to shed FAPα from K562 / FAPα cells.

[0142] Example 4 Dimer Staining The binding affinity of each F19 mutant CAR-T (7L1, 7L2, 7L3, 7L8, 7L12, 7L20, 7L29, 7L35, 7L36, 7L48, 12L1, 12L3, 12L4, 12L5, 12L18, 12L21, 14L4, and 14L17) to FAPα was examined. First, 6 μL of 6xHis-human FAPα recombinant protein (molecular weight 88 kDa) (50 μg / mL) and 2.4 μL of PE-labeled anti-His mAb (molecular weight 150 kDa) (100 μg / mL) (Abcam) were mixed prior to use, and a total volume of 20 μL of dimeric protein (6xHis-human FAPα recombinant protein adjusted to approximately 170 nM) was prepared with 11.6 μL of 2% FCS / PBS. A 10 μL aliquot of the prepared dimeric protein was serially diluted 2-fold with an equal volume of 2% FCS / PBS to obtain 11 different concentrations of dimeric protein (approximately 170 nM, 85 nM, 42 nM, 21 nM, 10 nM, 5 nM, 2 nM, 1.33 nM, 0.66 nM, 0.33 nM, and 0.16 nM). The dimeric protein was incubated in the dark at 4°C for 1 hour before use. A negative control without 6xHis-human FAPα recombinant protein was prepared using the same composition as above, but with 2% FCS / PBS instead of 6xHis-human FAPα recombinant protein. Each F19 mutant CAR-T and the positive control (F19 CAR-T) were then diluted at 2.0x10 5The cells were adjusted to 1000xg per well and seeded into each well. The cells were centrifuged at 1000xg for 1 minute and the supernatant was discarded. The cells were vortexed, and 10-20μL of 20% mouse serum / PBS was added and incubated at 4°C for 10 minutes. The cells were again centrifuged at 1000xg for 1 minute and the supernatant was discarded. After vortexing, the cells were added with 10μL of the dimeric protein at each of the above concentrations and incubated at 4°C for 25-30 minutes. The cells were then washed once with 100μL / well of 2% FCS / PBS, centrifuged at 1000xg for 1 minute, and the supernatant was discarded. The cells were vortexed and treated with BV421-labeled anti-NGFR antibody diluted in 2% FCS / PBS and incubated at 4°C for 15 minutes. The cells were then centrifuged at 1000xg for 1 minute and the supernatant was discarded. The cells were vortexed and suspended in 7ADD diluted 500-fold with 2% FCS / PBS, and subjected to flow cytometry analysis. The results are shown in Figure 3a and b.

[0143] Figure 3a is a graph showing the relationship between the percentage of FAPα-positive cells for each F19 mutant CAR-T and the recombinant FAPα concentration. Figure 3b is a graph showing the EC50 of FAPα for each F19 mutant CAR-T. It was found that the FAPα concentration required for staining each F19 mutant CAR-T tended to be higher than that for the positive control. This indicates that the affinity of each F19 mutant CAR-T with FAPα is lower than that for the positive control. This difference in affinity may be related to the difference in the expression level of FAPα on K562 / FAPα cells in Example 3.

[0144] Example 5 In vivo antitumor assay 2.0x10 7 Prepare a U87 cell suspension at 2.0x10 cells / mL. 6 100 μL of cell suspension was added to 7-week-old female mice (NOD.Cg-Prkdc scid Il2rg tm1Wjl The tumors were subcutaneously transplanted into the right flank of 16 mice (n = 16) (generic name: NSG) (supplied by Jackson Laboratory Japan, Inc.) on day 0. The transplanted mice showed no problems with their general condition, weight, or tumor shape, and the tumor volume reached 50 mm.3 ~150 mm 3 Mice of approximately 3.0x10 were selected, and based on the data of the selected mice, a program from the SAS preclinical package was applied to divide the selected mice into groups. The selected mice were divided into four groups, each with five mice, by multivariate block allocation (tumor volume and body weight). 7 F19 mutant CAR-T (7L1, 7L12), positive control (F19 CAR-T), or negative control (dNGFR-T) was administered at 0.2 mL / body (6.0x10 6 The tumor volume was measured and recorded. The results are shown in Figure 4. The tumor volume was calculated by visually and palpably observing the implanted site, measuring the long diameter (mm) and short diameter (mm) of the tumor with a vernier caliper, and using the following formula: Tumor volume (mm 3 ) = major axis (mm) x minor axis (mm) x minor axis (mm) x 0.5

[0145] Figure 4 shows the relationship between tumor volume and days after administration in mice administered with the F19 mutant CAR-T. Tumors in mice administered the negative control continued to grow. Tumors in mice administered the positive control shrank initially but then resumed growth. On the other hand, tumors in mice administered with the F19 mutant CAR-T (7L1, 7L12) continued to shrink throughout the observation period.

[0146] Also, 3.0x10 7 Prepare a U87 cell suspension at 3.0x10 cells / mL. 6 Mice were subcutaneously implanted with a cell suspension at 100μL / 100μL. The mice were divided into groups as described above. On day 7 after U87 cell implantation, 6.0x10 F19 mutant CAR-T (7L12) or positive control (F19 CAR-T) was injected. 6 cells / body, 2.0x10 6 cells / body or 6.0x10 5 The tumor volume was measured and recorded after administration. The results are shown in Figure 5.

[0147] Figure 5 is a graph showing the relationship between tumor volume and the number of days elapsed in mice administered with F19 mutant CAR-T. 6 The tumors in mice administered the positive control at 6.0x10 cells / body temporarily shrank, but then began to grow again, and the growth rate increased with the decrease in the number of administered cells (Figure 5). 6 cells / body or 2.0x10 6 The tumors in mice administered with F19 mutant CAR-T (7L12) at 100 cells / body size remained small and did not grow during the observation period, reaching 6.0x10 5 The tumors began to grow only in the mice receiving the F19 mutant CAR-T (7L12) cells / body (Figure 5). Furthermore, the difference in tumor volume between the positive control and F19 mutant CAR-T (7L12) groups increased with the decrease in the number of cells administered.

[0148] These findings suggest that the tumors in mice administered the positive control had a high binding affinity for FAPα, and that the FAPα on some tumor cells was shed, resulting in reduced FAPα expression on tumor cells and their inability to be recognized by the positive control, resulting in a lack of antitumor effect. On the other hand, unlike the positive control, the F19 mutant CAR-T (7L1, 7L12) did not shed FAPα on tumor cells, thereby not reducing FAPα expression on tumor cells and thus maintaining its antitumor effect.

[0149] Example 6: Antigen-Stimulated F19 Mutant CAR-T Cell Exhaustion Assay To assess T cell exhaustion, untreated 96-well plates were coated with PBS containing recombinant human FAPα protein (R&D Systems) at 0, 0.1, 0.3, 1, or 3 μg / mL. After a brief wash with PBS, a positive control (F19-CAR-T), a negative control (mock-transduced T), or F19 mutant CAR-T (7L1, 7L8, or 7L12) was added to the FAPα-coated wells and incubated at 37°C for 72 hours. After incubation, cells were harvested and transferred to a V-bottom 96-well plate for surface staining. Cells were stained with fluorochrome-conjugated antibodies against PD-1, LAG-3, and TIGIT and analyzed by flow cytometry using a CytoFLEX S (Beckman Coulter). The results are shown in Figure 6.

[0150] Figure 6 shows histogram plots showing the relationship between the expression of exhaustion markers (PD-1, LAG-3, and TIGIT) and cell number in each F19 mutant CAR-T stimulated with FAPα. Compared to the positive control (F19-CAR-T), the F19 mutant CAR-Ts (7L1, 7L8, and 7L12) had lower expression levels of exhaustion markers (PD-1, LAG-3, and TIGIT). Furthermore, the expression of exhaustion markers was similar among the F19 mutant CAR-Ts (7L1, 7L8, and 7L12). Of note, the F19 mutant CAR-T (7L12) showed a significantly lower expression of CD8 － TIGIT expression was slightly lower among T cell subsets.

[0151] Example 7: Preparation of F19 Mutant CAR-T Cells. The F19 CAR expression vector or each F19 mutant CAR expression vector was prepared using a lentiviral gene transfer system (pALD-Lenti System, purchased from Aldevron). The F19 CAR expression vector or each F19 mutant CAR expression vector was introduced into HEK293T cells using a transfection reagent (TransIT-Lenti Transfection Reagent, Mirus Bio), to prepare VSV-G-pseudotyped lentiviruses containing F19 CAR or each F19 mutant CAR. The HEK293T cell culture medium consisted of DMEM medium containing 50 μg / mL gentamicin and 10% FCS, and was cultured at 37°C, 5% CO2, and in a humid atmosphere. Unless otherwise noted, the cell culture conditions were the same below. Two days after transfection, the HEK293T cell culture supernatant was collected and concentrated lentivirus was prepared using a concentration reagent (Lenti-X Concentrator, Takara Bio). The virus was stored at -80°C.

[0152] Next, T cells were isolated from healthy human peripheral blood mononuclear cells (Uncharacterized PBMCs, Cellular Technology Limited) using a T cell isolation reagent (Pan T Cell Isolation Kit, Miltenyi Biotec) and cultured for two days in the presence of a T cell stimulating reagent (T Cell TransAct, Miltenyi Biotec) and 100 IU / mL human IL-2. The human peripheral blood mononuclear cells were cultured in AIM-V medium containing 50 μg / mL gentamicin and 5% FCS. After the culture, T cells were seeded on plates coated with concentrated lentivirus and centrifuged at 1000 × g and 32°C for 1 hour to infect the T cells with the lentivirus and transduce the F19 CAR or each F19 mutant CAR. The resulting T cells were stained with PE-labeled anti-Whitlow / 218 Linker (clone: ​​E3U7Q) and then examined using a flow cytometer (CytoFlex, Beckman Coulter) to confirm the expression of Linker on the T cells. Analysis software (CytExpert, Beckman Coulter) was used to analyze the data obtained with the flow cytometer. Unless otherwise specified, in the following Examples 8 and 9, F19 CAR-T or F19 mutant CAR-T refers to T cells generated by lentiviruses containing nucleic acids encoding F19 CAR or each F19 mutant CAR.

[0153] Example 8: In Vitro Cytotoxicity Assay. Target cells (U-87MG, U2OS, IMR32) expressing mCherry-NLS were seeded in 96-well plates and cultured in 2D and 3D culture for 24 hours to allow for adhesion and initial proliferation. After this culture period, effector cells (GFP-expressing T cells (control) or F19 mutant CAR-Ts (7L1, 7L8, 7L12)) were added at effector-to-target (E:T) ratios of 0.03, 0.1, 0.3, 1, or 3. Live cell imaging and analysis were performed over 120 hours using the IncuCyte S3 Live-Cell Analysis System (Sartorius, Germany). The system captured images at regular intervals and automatically quantified the total number of mCherry-labeled nuclei per well. Nuclear mCherry signal was used as a surrogate for viable target cell numbers. Data were normalized to the initial (baseline) target cell number to account for variability in seeding and proliferation rates. The results are shown in Figures 7 and 8.

[0154] Figure 7 shows the percentage of viable target cells over time relative to the initial target cell number when treated with each F19 mutant CAR-T and cultured in 2D. Figure 8 shows the percentage of viable target cells over time relative to the initial target cell number when treated with each F19 mutant CAR-T and cultured in 3D. (1) Cytotoxic Activity of F19 Mutant CAR-T (7L12) F19 mutant CAR-T (7L12) exhibited potent cytotoxicity against highly FAPα-expressing U-87MG target cells (U-87MG cells stably expressing mCherry-NLS (nuclear localization signal)) at all effector-to-target (E:T) ratios tested (0.03, 0.1, 0.3, 1, and 3). A clear dose-dependent effect was observed, with a higher E:T ratio associated with a greater reduction in mCherry-NLS-positive nuclei and increased target cell death. (2) Comparison of Cytotoxicity among F19 Mutant CAR-Ts. F19 mutant CAR-Ts (7L1) and (7L8) also exhibited cytotoxicity against U-87MG cells, although the effect was less than that of F19 mutant CAR-T (7L12). In contrast, GFP-expressing T cells (control) did not exhibit significant cytotoxicity at any E:T ratio, confirming the specificity of the CAR-mediated response. (3) Verification of Selectivity Based on FAPα Expression Levels. All F19 mutant CAR-Ts, including F19 mutant CAR-T (7L12), exhibited minimal cytotoxicity against FAPα-negative IMR32 cells (IMR32 cells stably expressing mCherry-NLS). Notably, only the F19 mutant CAR-T(7L12) showed measurable cytotoxicity against U2OS cells with low FAPα expression (U2OS cells stably expressing mCherry-NLS), whereas the F19 mutant CAR-T(7L1) and F19 mutant CAR-T(7L8) showed no significant activity against these cells. This observation indicates that the F19 mutant CAR-T(7L12) is more sensitive to differences in FAPα expression levels and suggests superior target selectivity compared to the F19 mutant CAR-T(7L1) and F19 mutant CAR-T(7L8).These findings also support the specificity of the FAPα-directed CAR design and its potential efficacy in selectively targeting FAPα-expressing cells. (4) Time-resolved cytotoxicity profile: Time-lapse analysis using the IncuCyte S3 system revealed that the cytotoxic effect of the F19 mutant CAR-T (7L12) began to appear within 24-40 hours after effector cell addition and gradually persisted over 120 hours. In contrast, the onset of cytotoxicity was delayed or negligible for the control and less effective F19 mutant CAR-T (7L1).

[0155] Example 9 In vivo antitumor assay A suspension of U87MG human glioblastoma cells (ATCC) was added to a 1:1 mixture of Matrigel (Corning Matrigel Basement Membrane Matrix (Corning, Cat. No. 354234)) and PBS at a concentration of 2.0 × 10 7 The suspension was prepared at a concentration of 2 × 10 6 100 μL of the cells was added to each ASID mouse (NOD.Cg-Prkdc scid / / 2rg em1Narl Male mice (6-8 weeks old, National Center for Biomodels, Taiwan) were injected subcutaneously into the right flank of each mouse. 3 ~150 mm 3 Once the tumor volume reached 100 mg / kg, animals were assigned to four treatment groups (n = 5 per group) using block randomization based on initial tumor volume and body weight. Tumor size (longest and shortest diameters) was measured with a digital caliper, and tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = major axis (mm) × minor axis (mm) × minor axis (mm) × 0.5 Day 0 was the day of U87MG cell inoculation. On day 7 after inoculation, 2.0 × 10 cells corresponding to each treatment group were 6 Mice received a single intravenous injection of T cells.

[0156] Figure 9 shows the relationship between body weight, tumor volume, tumor inhibition rate, and survival rate and time elapsed in tumor-bearing mice treated with each F19 mutant CAR-T. Treatment with F19 mutant CAR-T (7L12) statistically significantly reduced tumor volume compared with GFP-expressing T cells (control), F19 mutant CAR-T (7L1), and F19 mutant CAR-T (7L8) (p<0.05). No significant changes in body weight were observed in the F19 mutant CAR-T (7L12) treatment group throughout the study period, indicating that this treatment was well tolerated in vivo. (1) Body Weight Monitoring of ASID-Treated Mice. Figure 9(A) shows a line graph showing the mean body weight (g) over time for each treatment group. Body weight remained stable throughout the study period in all control (GFP) and F19 mutant CAR-T treatment groups, suggesting the absence of significant systemic toxicity. (2) Tumor growth curve in tumor-bearing ASID mice subcutaneously implanted with U87MG. Figure 9(B) shows the tumor volume (mm ) of each treatment group (GFP, 7L1, 7L8, 7L12). 3) over time. Tumor volume was measured using calipers and calculated using the method described above. Tumor growth was significantly suppressed in the F19 mutant CAR-T (7L12)-treated group compared to the other groups. (3) Tumor Suppression. Figure 9(C) shows the appearance of mice 30 days after administration of each treatment group (GFP, 7L1, 7L8, 7L12). Figure 9(D) shows the tumor inhibition rate (%) 30 days after administration of each treatment group (GFP, 7L1, 7L8, 7L12). F19 mutant CAR-T (7L12) demonstrated potent antitumor activity in a U87MG subcutaneous tumor model. At the end of the study, mice treated with F19 mutant CAR-T (7L12) showed significantly reduced tumor volume compared to the GFP control group and the other F19 mutant CAR-T groups (7L1 or 7L8). The tumor growth inhibition (TGI) rate was calculated based on final tumor volume, confirming the superior efficacy of F19 mutant CAR-T(7L12) in suppressing tumor progression. (4) Survival Rate Figure 9(E) is a line graph showing the percent survival rate over time for each treatment group (GFP, 7L1, 7L8, and 7L12). Kaplan-Meier survival analysis demonstrated that mice treated with F19 mutant CAR-T(7L12) had prolonged survival. While mice treated with the control, F19 mutant CAR-T(7L1), or F19 mutant CAR-T(7L8) reached the humane endpoint early due to tumor burden, those treated with F19 mutant CAR-T(7L12) maintained prolonged survival. These results suggest that F19 mutant CAR-T(7L12) not only suppresses tumor growth but also improves overall survival in this glioblastoma xenograft model.

[0157] From the above, each of the F19 mutant CAR-Ts confirmed in the examples does not reduce the expression of FAPα, and therefore can be expected to suppress cancer escape from CAR-T and have a stable cancer shrinkage effect.

[0158] Although the present disclosure has been described above with reference to embodiments and examples, the present disclosure is not limited to the above embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0159] The patents, patent applications, and publications cited herein are incorporated by reference into this specification in their entirety as if the contents were specifically set forth herein.

[0160] This application claims priority based on Japanese Patent Application No. 2024-083802, filed May 23, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0161] <Supplementary Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. (Supplementary Note 1) An antibody or antigen-binding fragment thereof against FAPα, comprising the light chain variable region of the following (L) and the heavy chain variable region of the following (H): (L) A light chain variable region selected from the group consisting of the following (L1) to (L18): (L1) A light chain variable region comprising light chain complementarity determining regions (CDRL) 1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7; (L2) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 8, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 10; (L3) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein (L4) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13; (L5) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 19; (L6)(L7) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 21, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22; (L8) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 26, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 27, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 28; (L9) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 23, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 24, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 25; (L10) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 29, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31; (L11) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 37; (L12) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 37, and CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38.A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 39, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40; (L13) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 43; (L14) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 44, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 45, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 46; (L15) (L16) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 49; (L17) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 55; (L18)(H) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:56, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:57, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:58; (H) A heavy chain variable region comprising heavy chain complementarity determining region (CDRH)1, CDRH2, and CDRH3, wherein CDRH1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:78, CDRH2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:79, and CDRH3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:80. (Supplementary Note 2) The antibody or antigen-binding fragment thereof according to Supplementary Note 1, wherein the light chain variable region is a light chain variable region selected from the group consisting of the following (L1v) to (L18v): (L1v) the light chain variable region of (L1), which is a polypeptide selected from the group consisting of the following (L1v1) to (L1v3): (L1v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 60, (L1v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 60, (L1v3) a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 60; (L2v) the light chain variable region of (L2), which is a polypeptide selected from the group consisting of the following (L2v1) to (L2v3): (L2v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 61, (L2v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 61, (L2v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 61; (L3v) A light chain variable region of the above (L3), which is a polypeptide selected from the group consisting of the following (L3v1) to (L3v3): (L3v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 62;(L3v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 62, (L3v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 62; (L4v) a light chain variable region of (L4), which is a polypeptide selected from the group consisting of (L4v1) to (L4v3) below: (L4v1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 63, (L4v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 63, (L4v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 63; (L5v) a light chain variable region of (L5), which is a polypeptide selected from the group consisting of (L5v1) to (L5v3) below: (L5v1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 64, (L5v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 64, (L5v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 64; (L6v) A light chain variable region of (L6), which is a polypeptide selected from the group consisting of (L6v1) to (L6v3) below: (L6v1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 65, (L6v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 65, (L6v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 65; (L7v) A light chain variable region of (L7), which is a polypeptide selected from the group consisting of (L7v1) to (L7v3) below: (L7v1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 66, (L7v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 66;(L7v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 66; (L8v) A light chain variable region of (L8), which is a polypeptide selected from the group consisting of (L8v1) to (L8v3) below: (L8v1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 67, (L8v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 67, (L8v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 67; (L9v) A light chain variable region of (L9), which is a polypeptide selected from the group consisting of (L9v1) to (L9v3) below: (L9v1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 68, (L9v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 68, (L9v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 68; (L10v) A light chain variable region of (L10), which is a polypeptide selected from the group consisting of (L10v1) to (L10v3) below: (L10v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 69, (L10v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 69, (L10v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 69; (L11v) A light chain variable region of (L11), which is a polypeptide selected from the group consisting of (L11v1) to (L11v3) below: (L11v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 70, (L11v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 70;(L11v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 70; (L12v) A light chain variable region of (L12), which is a polypeptide selected from the group consisting of the following (L12v1) to (L12v3): (L12v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 71, (L12v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 71, (L12v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 71; (L13v) A light chain variable region of (L13), which is a polypeptide selected from the group consisting of the following (L13v1) to (L13v3): (L13v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 72, (L13v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 72, (L13v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 72; (L14v) A light chain variable region of (L14), which is a polypeptide selected from the group consisting of (L14v1) to (L14v3) below: (L14v1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 73, (L14v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 73, (L14v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 73; (L15v) A light chain variable region of (L15), which is a polypeptide selected from the group consisting of (L15v1) to (L15v3) below: (L15v1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 74; (L15v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 74;(L15v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 74; (L16v) A light chain variable region of (L16), which is a polypeptide selected from the group consisting of the following (L16v1) to (L16v3): (L16v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 75, (L16v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 75, (L16v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 75; (L17v) A light chain variable region of (L17), which is a polypeptide selected from the group consisting of the following (L17v1) to (L17v3): (L17v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 76, (L17v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 76; (L17v3) A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 76; (L18v) A light chain variable region of (L18), which is a polypeptide selected from the group consisting of (L18v1) to (L18v3) below: (L18v1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 77; (L18v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 77; (L18v3) A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 77. (Supplementary Note 3) The antibody or antigen-binding fragment thereof according to Supplementary Note 1 or 2, wherein the heavy chain variable region is a polypeptide selected from the group consisting of the following (Hv1) to (Hv3): (Hv1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 81; (Hv2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 81;(Hv3) A polypeptide comprising the amino acid sequence of SEQ ID NO: 81 with one or several amino acids deleted, substituted, inserted, and / or added. (Appendix 4) The antibody or antigen-binding fragment thereof according to Appendix 2 or 3, wherein the amino acid sequence of at least one CDR contained in the polypeptide selected from the group consisting of (L1v2) to (L18v2) and (Hv2) is 100% conserved. (Appendix 5) The antibody or antigen-binding fragment thereof according to Appendix 2 or 3, wherein the amino acid sequence of at least one CDR contained in the polypeptide selected from the group consisting of (L1v3) to (L18v3) and (Hv3) is 100% conserved. (Appendix 6) A chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to any one of Appendices 1 to 5. (Appendix 7) The chimeric antigen receptor according to Appendix 6, wherein the antigen-binding fragment is an scFv. (Appendix 8) The chimeric antigen receptor according to Appendix 7, wherein the transmembrane domain comprises the transmembrane domain of CD28. (Appendix 9) The chimeric antigen receptor according to Appendix 7 or 8, wherein the intracellular signaling domain comprises at least one intracellular signaling domain selected from the group consisting of CD28, CD134 (OX40), and CD137 (4-1BB), and the intracellular signaling domain of CD3ζ. (Appendix 10) A nucleic acid encoding the chimeric antigen receptor according to any one of Appendixes 6 to 9. (Appendix 11) A vector comprising the nucleic acid of Appendix 10. (Appendix 12) A cell comprising the vector of Appendix 11. (Appendix 13) The cell according to Appendix 12, wherein the cell is a pluripotent stem cell. (Appendix 14) The cell according to Appendix 12, wherein the cell is a T cell. (Appendix 15) The cell according to Appendix 14, wherein the T cell is a T cell obtained by differentiating the pluripotent stem cell of Appendix 13. (Appendix 16) A method for producing cells that express a chimeric antigen receptor, comprising the step of introducing into cells the expression vector according to Appendix 11. (Appendix 17) The method according to Appendix 16, wherein the cells are pluripotent stem cells. (Appendix 18)The production method according to Appendix 17, further comprising the step of differentiating the pluripotent stem cells introduced with the expression vector into T cells. (Appendix 19) The production method according to Appendix 16, wherein the cells are T cells. (Appendix 20) A cancer therapeutic agent comprising the cells according to Appendix 14 or 15, or cells produced by the production method according to Appendix 18 or 19. (Appendix 21) The cancer therapeutic agent according to Appendix 20, wherein the cancer is at least one selected from head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colorectal cancer, esophageal cancer, glioblastoma, osteosarcoma, and malignant mesothelioma. (Appendix 22) A method for treating cancer, comprising the step of administering to a subject the cells according to claim 14 or 15, or cells produced by the production method according to claim 18 or 19. (Supplementary Note 23) The cancer treatment method according to Supplementary Note 22, wherein the cancer is at least one selected from head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colorectal cancer, esophageal cancer, glioblastoma, osteosarcoma, and malignant mesothelioma. (Supplementary Note 24) The cell according to Supplementary Note 14 or 15, or a cell produced by the production method according to Supplementary Note 18 or 19, for use in cancer treatment. (Appendix 25) The cell according to Appendix 14 or 15, or the cell produced by the production method according to Appendix 18 or 19, wherein the cancer is at least one selected from head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colorectal cancer, esophageal cancer, glioblastoma, osteosarcoma, and malignant mesothelioma.

[0162] By administering the cancer therapeutic agent of the present disclosure to cancer patients, it is possible to reduce the cancer stroma, assist approaches to cancer such as anticancer drugs, or directly attack cancers that express FAPα. Furthermore, because the cancer therapeutic agent of the present disclosure does not reduce the expression of FAPα, it is possible to inhibit cancer escape from the cancer therapeutic agent of the present disclosure, and a stable cancer reduction effect can be expected.

Claims

1. An antibody or antigen-binding fragment thereof against FAPα, comprising the light chain variable region of the following (L) and the heavy chain variable region of the following (H): (L) A light chain variable region selected from the group consisting of the following (L1) to (L18): (L5) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 19; (L1) A light chain variable region comprising light chain complementarity determining region (CDRL) 1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 5, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 6, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7; (L2) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein (L3) a light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 11, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13; (L4) a light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 14, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16;(L6) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 21, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22; (L7) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 23, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 24, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 25; (L8) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 26, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 27, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 28; (L9) (L10) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 29, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 30, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 31; (L11) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 35, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 36, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 37;(L12) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 38, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 39, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40; (L13) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 41, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 42, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 43; (L14) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 44, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 45, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 46; (L15) (L16) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 47, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 48, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 49; (L17) A light chain variable region comprising CDRL1, CDRL2 and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 53, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 54, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 55;(L18) A light chain variable region comprising CDRL1, CDRL2, and CDRL3, wherein CDRL1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:56, CDRL2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:57, and CDRL3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:58; (H) A heavy chain variable region comprising heavy chain complementarity determining region (CDRH)1, CDRH2, and CDRH3, wherein CDRH1 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:78, CDRH2 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:79, and CDRH3 is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:

80.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region is a light chain variable region selected from the group consisting of the following (L1v) to (L18v): (L5v) the light chain variable region of (L5), which is a polypeptide selected from the group consisting of the following (L5v1) to (L5v3): (L5v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:64, (L5v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO:64, (L5v3) a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO:64; (L1v) the light chain variable region of (L1), which is a polypeptide selected from the group consisting of the following (L1v1) to (L1v3): (L1v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:60, (L1v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO:60, (L1v3) A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 60; (L2v) A light chain variable region of (L2), which is a polypeptide selected from the group consisting of (L2v1) to (L2v3) below: (L2v1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 61, (L2v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 61, (L2v3) A polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 61; (L3v) A light chain variable region of (L3), which is a polypeptide selected from the group consisting of (L3v1) to (L3v3) below: (L3v1) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 62, (L3v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 62, (L3v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence shown in SEQ ID NO: 62;(L4v) The light chain variable region of (L4), which is a polypeptide selected from the group consisting of (L4v1) to (L4v3) below: (L4v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 63, (L4v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 63, (L4v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 63; (L6v) The light chain variable region of (L6), which is a polypeptide selected from the group consisting of (L6v1) to (L6v3) below: (L6v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 65, (L6v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 65, (L6v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 65; (L7v) The light chain variable region of (L7), which is a polypeptide selected from the group consisting of (L7v1) to (L7v3) below: (L7v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 66, (L7v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 66, (L7v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 66; (L8v) The light chain variable region of (L8), which is a polypeptide selected from the group consisting of (L8v1) to (L8v3) below: (L8v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 67, (L8v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 67, (L8v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 67;(L9v) The light chain variable region of (L9), which is a polypeptide selected from the group consisting of (L9v1) to (L9v3) below: (L9v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:68, (L9v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO:68, (L9v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO:68; (L10v) The light chain variable region of (L10), which is a polypeptide selected from the group consisting of (L10v1) to (L10v3) below: (L10v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:69, (L10v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO:69, (L10v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO:69; (L11v) The light chain variable region of (L11), which is a polypeptide selected from the group consisting of (L11v1) to (L11v3) below: (L11v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 70, (L11v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 70, (L11v3) a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO: 70; (L12v) The light chain variable region of (L12), which is a polypeptide selected from the group consisting of (L12v1) to (L12v3) below: (L12v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 71, (L12v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 71, (L12v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 71;(L13v) The light chain variable region of (L13), which is a polypeptide selected from the group consisting of (L13v1) to (L13v3) below: (L13v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 72, (L13v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 72, (L13v3) a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO: 72; (L14v) The light chain variable region of (L14), which is a polypeptide selected from the group consisting of (L14v1) to (L14v3) below: (L14v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 73, (L14v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 73, (L14v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 73; (L15v) A light chain variable region of (L15), which is a polypeptide selected from the group consisting of (L15v1) to (L15v3) below: (L15v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 74, (L15v2) A polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 74, (L15v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 74; (L16v) A light chain variable region of (L16), which is a polypeptide selected from the group consisting of (L16v1) to (L16v3) below: (L16v1) A polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 75, (L16v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 75; (L16v3) a polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence shown in SEQ ID NO: 75;(L17v) The light chain variable region of (L17), which is a polypeptide selected from the group consisting of (L17v1) to (L17v3) below: (L17v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 76, (L17v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 76, (L17v3) a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO: 76; (L18v) The light chain variable region of (L18), which is a polypeptide selected from the group consisting of (L18v1) to (L18v3) below: (L18v1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 77, (L18v2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 77, (L18v3) A polypeptide comprising an amino acid sequence in which one or several amino acids have been deleted, substituted, inserted and / or added relative to the amino acid sequence set forth in SEQ ID NO: 77; 3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the heavy chain variable region is a polypeptide selected from the group consisting of (Hv1) to (Hv3) below: (Hv1) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 81; (Hv2) a polypeptide comprising an amino acid sequence having 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 81; (Hv3) a polypeptide comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO:

81.

4. The antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the amino acid sequence of at least one CDR contained in a polypeptide selected from the group consisting of (L1v2) to (L18v2) and (Hv2) is 100% conserved.

5. The antibody or antigen-binding fragment thereof according to claim 2 or 3, wherein the amino acid sequence of at least one CDR contained in a polypeptide selected from the group consisting of (L1v3) to (L18v3) and (Hv3) is 100% conserved.

6. A chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain comprises an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The chimeric antigen receptor of claim 6, wherein the antigen-binding fragment is an scFv.

8. The chimeric antigen receptor of claim 7, wherein the transmembrane domain comprises the transmembrane domain of CD28.

9. The chimeric antigen receptor of claim 7 or 8, wherein the intracellular signaling domain comprises at least one intracellular signaling domain selected from the group consisting of CD28, CD134 (OX40), and CD137 (4-1BB), and the intracellular signaling domain of CD3ζ.

10. A nucleic acid encoding the chimeric antigen receptor of any one of claims 6 to 9.

11. A vector comprising the nucleic acid of claim 10.

12. A cell comprising the vector of claim 11.

13. The cell of claim 12, wherein the cell is a pluripotent stem cell.

14. The cell of claim 12, wherein the cell is a T cell.

15. The cell according to claim 14, wherein the T cell is a T cell obtained by differentiating the pluripotent stem cell according to claim 13.

16. A method for producing cells that express a chimeric antigen receptor, comprising the step of introducing the vector according to claim 11 into cells.

17. The method of claim 16, wherein the cells are pluripotent stem cells.

18. The method of claim 17, further comprising the step of differentiating the pluripotent stem cells into which the vector has been introduced into T cells.

19. The method of claim 16, wherein the cells are T cells.

20. A cancer therapeutic agent comprising the cells described in claim 14 or 15, or cells produced by the production method described in claim 18 or 19.

21. The cancer therapeutic agent according to claim 20, wherein the cancer is at least one selected from head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colon cancer, esophageal cancer, glioblastoma, osteosarcoma, and malignant mesothelioma.

22. A method for treating cancer, comprising the step of administering to a subject the cells described in claim 14 or 15, or cells produced by the production method described in claim 18 or 19.

23. The cancer treatment method described in claim 22, wherein the FAPα-expressing cancer is at least one selected from head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colon cancer, esophageal cancer, glioblastoma, osteosarcoma, and malignant mesothelioma.

24. A cell according to claim 14 or 15, or a cell produced by the method according to claim 18 or 19, for use in the treatment of cancer.

25. A cell described in claim 14 or 15, or a cell produced by the production method described in claim 18 or 19, wherein the cancer is at least one selected from head and neck squamous cell carcinoma, hepatocellular carcinoma, pancreatic cancer, cholangiocarcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colon cancer, esophageal cancer, glioblastoma, osteosarcoma, and malignant mesothelioma.

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