Hybrid t-cell receptor

Abstract

Provided is an immune receptor having a higher tumor-inhibitory action. This hybrid TCR comprises a polypeptide A that includes a light chain variable region of an antibody having binding ability with respect to an antigen peptide-MHC complex and a first subunit constant region of a TCR, and a polypeptide B that includes a heavy chain variable region of the aforementioned antibody and a second subunit constant region of a TCR.

Description

Hybrid T-cell receptor

[0001] The present invention relates to modified T cell receptors and the like.

[0002] Cancer is the leading cause of death among Japanese people, and the development of new treatments is essential. In recent years, T-cell infusion therapy has been developed as a minimally invasive and highly effective cancer treatment. In particular, CD19 CAR T-cell infusion therapy, developed as a treatment for B-cell lymphoma, is groundbreaking and has attracted attention for its high therapeutic effectiveness. However, the development of treatments for other types of cancer, especially solid tumors, has not progressed.

[0003] CAR T cell therapy is a treatment that suppresses tumors by recognizing cancer cell antigens on the cell surface. The receptor used for genetic modification is a chimeric antigen receptor (CAR), which combines the antigen recognition site of an antibody with the signal transduction site of a TCR. Recent advances in antibody isolation technology have made it possible to isolate antibodies with high recognition specificity, but the problem remains that there are only a few cancer-specific antigens on the cell surface.

[0004] Cancer is formed by cancer cells. Cancer cells are formed through the accumulation of genetic mutations in normal somatic cells, resulting in cancer-specific gene expression. Most of the proteins produced by cancer-specific gene expression are expressed intracellularly, degraded into peptides by the proteasome, and presented on major histocompatibility complexes (MHCs). These proteins are expressed on cancer cells, recognized by T cell receptors on T cells, and tumor suppression is known to occur. Therefore, genetically modified T cell therapies using TCRs with high cancer therapeutic efficacy are currently being developed. However, most TCRs isolated to date have low affinity, making it difficult to isolate such therapeutically effective TCRs. While it is possible to increase affinity through genetic modification, cross-recognition is also enhanced, making the development of safe and effective TCR therapy difficult.

[0005] International Publication No. 2017 / 070608

[0006] To address these issues, the present inventors attempted to develop CARs that recognize cancer-specific pMHC complexes for therapeutic use. Using a unique screening method using a human antibody library, the inventors isolated antibodies that recognize HLA-A2402 WT1 p235-243, HLA-A2402 PRAMEp301-308, and HLA-A0201 MAGE-A4 p230-239. These antibodies were then used to create CARs and advance the development of cancer treatments. However, the tumor-suppressing activity of the CARs developed was insufficient, and the inhibition of tumor growth in engrafted tumors was limited.

[0007] Patent Document 1 describes a fusion receptor of an antibody (including a constant region) that recognizes pMHC and a TCR. However, no sufficient antitumor effect has been reported for this immunoreceptor.

[0008] An objective of the present invention is to provide an immunoreceptor with a stronger tumor-suppressing effect.

[0009] In view of the above-mentioned problems, the present inventors have conducted extensive research and found that the above-mentioned problems can be solved by a hybrid TCR comprising polypeptide A comprising a light chain variable region of an antibody having binding ability to an antigen peptide-MHC complex and a first subunit constant region of a TCR, and polypeptide B comprising a heavy chain variable region of the antibody and a second subunit constant region of a TCR. Based on this finding, the present inventors have conducted further research and have completed the present invention. Specifically, the present invention encompasses the following aspects.

[0010] Item 1. A hybrid TCR comprising: polypeptide A comprising a light chain variable region of an antibody having binding ability to an antigen peptide-MHC complex and a first subunit constant region of a TCR; and polypeptide B comprising a heavy chain variable region of the antibody and a second subunit constant region of a TCR.

[0011] Item 2. The hybrid TCR according to Item 1, wherein the light chain variable region and the first subunit constant region are linked directly or via a linker, and the heavy chain variable region and the second subunit constant region are linked directly or via a linker.

[0012] Item 3. The hybrid TCR according to Item 1, wherein the first subunit is an α chain and the second subunit is a β chain, or the first subunit is a β chain and the second subunit is an α chain.

[0013] Item 4. The hybrid TCR according to Item 1, wherein the antigen peptide is a peptide derived from MAGE-A4 or PRAME.

[0014] Item 5. The hybrid TCR according to Item 1, wherein the dissociation constant of the antibody for an antigen peptide-MHC complex is 100 nM or less.

[0015] Item 6. The hybrid TCR of Item 1, wherein the antigenic peptide is MAGE-A4, and the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, and the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6, or wherein the antigenic peptide is PRAME, and the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a light chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a light chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9, and the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a heavy chain CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a heavy chain CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0016] Item 7. The hybrid TCR according to Item 1, wherein the polypeptide A comprises a leader sequence of a light chain variable region, and the polypeptide B comprises a leader sequence of a heavy chain variable region.

[0017] Item 8. A polynucleotide encoding the hybrid TCR according to any one of Items 1 to 7.

[0018] Item 9. A cell containing the polynucleotide according to Item 8.

[0019] Item 10. The cell according to Item 9, wherein the hybrid TCR is expressed on the cell membrane.

[0020] Item 11. The cell according to Item 10, which is a lymphocyte.

[0021] Item 12. A pharmaceutical composition comprising the cells according to Item 10.

[0022] Item 13. The pharmaceutical composition according to Item 12, which is used to treat or prevent cancer.

[0023] According to the present invention, an immunoreceptor with a stronger tumor-suppressing effect can be provided.

[0024] This is a conceptual diagram showing how hybrid TCRs recognize cancer-specific pMHC and suppress cancer cells. Cancer-specific gene expression occurs in cancer cells. Most of the proteins produced by cancer-specific gene expression are expressed intracellularly, where they are degraded into peptides by the proteasome and presented on the major histocompatibility complex (MHC). These are expressed on cancer cells and recognized by hybrid TCRs on T cells, resulting in tumor suppression. The structure of the hybrid TCR is shown (Test Example 1). The hybrid TCR constructed here is expressed using retroviruses, but lentivirus and transposon expression are also possible. The antibody recognition sites, VH and VL, are bound to TCR Cb and TCR Ca, respectively, and a heterodimer is produced to form the CD3 complex via the 2A peptide. Cb57 and Ca48 were replaced with cysteine. Ca116, 119, and 120 were replaced with leucine. This figure shows the transfection and expression of MAGE#17 hybrid TCR and PRAME#98 hybrid TCR into human PBMCs (Test Example 1). Human PBMCs were stimulated with retronectin (TAKARA bio) and OKT3 (Invitrogen) and cultured in GT-T551 medium (TAKARA bio) containing 0.6% human serum. The MAGE-A4 #17 hybrid TCR gene was then retrovirally transfected and cultured. Cells were stained with PE-labeled streptavidin-tetramerized HLA-A0201 MAGE-A4 p230-239, APC-labeled anti-human CD4 antibody (Biolegend), and PE-Cy7-labeled anti-human CD8 antibody (Biolegend), and analyzed using a FACS Foltessa (BD). Figure 1 shows staining of MAGE#17 hybrid TCR-transfected CD8 cells, MAGE#17 hybrid TCR-transfected CD4 cells, non-transfected CD8 cells, and non-transfected CD4 cells. e) PRAME#98 hybrid-TCR-transduced CD8 cells, f) PRAME#98 hybrid-TCR-transduced CD4 cells, g) non-transduced CD8 cells, and h) non-transduced CD4 cells. As explained in Figure 3-1.Hybrid-TCRs produce IFNg at higher levels than CARs, and this production is sustained. Hybrid-TCRs also demonstrate reduced recognition of negative target cells (Test Example 2). Effector and target cells were co-cultured, and the time course of IFNg production in the medium was measured by ELISA. a, b, c: 50,000 effector cells were co-cultured with 50,000 target cells. d, e, f: 10,000 effector cells were co-cultured with 50,000 target cells. Effector cells used were non-transfected T cells (NGMC), CAR T cells (CAR), hybrid-TCR-transfected T cells (Hybrid-TCR), and TCR-transfected T cells (TCR). Target cells used were HLA-A0201. + MAGE-A4 + SK-MEL-37 cells (a, d), HLA-A0201 + MAGE-A4 - The ELISA was performed using HCT116 cells (b, e) and without target cells (c, f). MabTec's human IFNg ELISA kit (MABTECH, 3420-1H-6) was used for the ELISA, and the experimental method followed the protocol. Hybrid-TCR has sufficient cytotoxicity against positive target cells, but no cytotoxic activity against negative target cells (Test Example 3). a, b, c Positive target cells SK-MEL-37 (HLA-A0201 + MAGE-A4 +) 12,000 cells were cultured on an E-plate for 17 hours, and then effector cells (60,000 cells (a), 36,000 cells (b), and 12,000 cells (c)) were added and cultured, and the cell index was tracked over time. The cell index reflects the number of SK-MEL-124 cells on the E-plate. The normalized cell index was normalized to the number of SK-MEL-37 cells immediately before co-culture with the effector cells, with 1 as the normalized value. The graph shows the average value (n=3). Cytotoxicity was observed with CAR T cells, hybrid-TCR, and TCR, but no cytotoxicity was observed with non-transfected cells. This shows that the hybrid-TCR has sufficient cytotoxicity against positive target cells and no cytotoxic activity against negative target cells (Test Example 3). d Negative target cells HCT116 (HLA-A0201) + MAGE-A4 -) 7,000 cells were cultured on an E-plate for 12 hours, after which 7,000 cells of each effector cell type were added and cultured, and the cell index was monitored over time. The cell index reflects the number of SK-MEL-124 cells on the E-plate. The normalized cell index was standardized to the number of SK-MEL-37 cells immediately before co-culture with the effector cells, with 1 as the normalized value. The graph shows the average value (n=3). No cytotoxicity was observed with non-transfected T cells, CAR T cells, hybrid-TCR, or TCR. The hybrid-TCR showed less exhaustion (Test Example 4). CAR, hybrid-TCR, and TCR-transduced T cells were cultured and continuously stimulated with antigen on days 10, 12, 14, 16, and 18 in 12-well plates coated with 0.2 μg of HLA-A0201 MAGE-A4 p230-239 tetramer. Effector cells were stained with PE-Cy7-labeled anti-CD8 antibody (Biolegend), MAGE tetramer and APC-labeled anti-PD-1 antibody (Biolegend), Percp-Cy5.5-labeled anti-TIM-3 antibody (Biolegend), Percp-Cy5.5-labeled anti-LAG-3 antibody (Biolegend), and APC-labeled anti-CTLA-4 antibody (Biolegend), and analyzed using an LSRFortessa (BD). Measurements were performed on days 10, 12, 14, 16, 18, and 20. The percentages of PD-1-, TIM-3-, LAG-3-, and CTLA-4-positive cells are shown in a, e, I, and m, respectively, and the change in mean fluorescence intensity (MFI) for PD-1-positive cells is shown in b, f, j, and n, respectively.CAR, hybrid-TCR, and TCR-transduced T cells were cultured and continuously stimulated with antigen on days 10, 12, and 14 in 12-well plates coated with 0.5 μg of HLA-A0201 MAGE-A4 p230-239 tetramer. From day 16, the cells were cultured in antigen-free plates. Effector cells were stained with PE-Cy7-labeled anti-CD8 antibody, MAGE tetramer, and APC-labeled anti-PD-1 antibody (Biolegend), Percp-Cy5.5-labeled anti-TIM-3 antibody (Biolegend), Percp-Cy5.5-labeled anti-LAG-3 antibody (Biolegend), and APC-labeled anti-CTLA-4 antibody (Biolegend), and analyzed using an LSRFortessa (BD). Measurements were performed on days 10, 12, 14, 16, 18, and 20. The results were analyzed at 20 days, and the percentage of PD-1 positive cells is shown in c, g, k, and o, respectively, and the change in mean fluorescence intensity (MFI) for PD-1 positive cells is shown in d, h, l, and p, respectively. As explained in Figure 6-1. As explained in Figure 6-1. As explained in Figure 6-1. Hybrid-TCR shows a higher percentage of central memory than CAR (Test Example 5). a, b Human PBMCs transfected with CAR, hybrid-TCR, and TCR were cultured and stained with MAGE-A4 tetramer, PECy7-labeled anti-CD8 antibody (Biolegend), APC / Cy7-labeled anti-CD45RO antibody (Biolegend), and FITC-labeled anti-CD62L antibody (eBioscience) on days 10, 12, 14, 16, and 18, and measured using an LSRFortessa. Naive (CD62L+ CD45RO-), Tcm (CD62L+ CD45RO+), The cells were classified into Tem (CD62L- CD45RO+) and Te (CD62- CD45RO-) and their abundance ratios were calculated.a, c Human PBMCs transduced with CAR, hybrid-TCR, or TCR were cultured and stimulated with 0.2 μg of antigen on a 12-well plate at days 10, 12, 14, and 16. Cells were stained with MAGE-A4 tetramer, PECy7-labeled anti-CD8 antibody (Biolegend), APC-labeled anti-CD45RO antibody (Biolegend), and FITC-labeled anti-CD62L antibody (eBioscience) at days 10, 12, 14, 16, and 18, and measured using an LSRFortessa. Cells were classified into naive (CD62L+ CD45RO-), Tcm (CD62L+ CD45RO+), Tem (CD62L- CD45RO+), and Te (CD62- CD45RO-) and their abundance ratios were calculated. As described in Figure 7-1. As described in Figure 7-1. The hybrid TCR exhibited high tumor-suppressive activity. This indicates that no GVHD occurred during this observation period (Test Example 6). HLA-A0201. + MAGE-A4 +NW-MEL-38 cells (2.5x10^6 cells) were administered to NOG mice as tumors on day 0. Four days later, 2.5x10^6 cells of effector cells (PBS, NGMC, CAR, hybrid-TCR, TCR) were infused via the tail vein on day 11. Tumor size and body weight changes were recorded. Infusions were performed with PBS, NGMC, CAR, hybrid-TCR, and TCR, with four mice per group. b. Mean tumor size change for each experimental group. c, d, e, f, g. Individual tumor size changes for each experimental group. h. Mean body weight change for each experimental group. i, j, k, l, m. Individual body weight changes for each experimental group. As described in Figure 8-1. As described in Figure 8-1. Introduction of PRAME#98 hybrid-TCR into human PBMC and confirmation of expression are shown (Test Example 7). Human PBMCs were stimulated with retronectin (TAKARA bio) and OKT3 (Invitrogen) and cultured in GT-T551 medium (TAKARA bio) containing 0.6% human serum. The PRAME#98 hybrid-TCR gene was then retrovirally transduced and cultured. HLA-A2402 PRAME p301-309 was tetramerized with PE-labeled streptavidin, and stained with APC-labeled anti-human CD4 antibody (Biolegend, ), and PE-Cy7-labeled anti-human CD8 antibody (Biolegend, ). Measurements were performed using a FACS Foltessa (BD). Left: PRAME#98 hybrid-TCR-transduced CD8 cells; right: PRAME#98 hybrid-TCR-transduced CD4 cells. This demonstrates that PRAME#98 hybrid-TCR-transduced human PBMCs exhibit enhanced effector function compared to CAR-transduced PBMCs (Test Example 7). Human PBMCs transduced with PRAME#98 hybrid-TCR, human PBMCs transduced with PRAME#98 CAR, and untransduced PBMCs were used as effector cells. K562 A2402 cells (HLA-A2402+ PRAME+) and K562 cells (HLA-A2402- PRAME+) were used as target cells. Effector and target cells were mixed at 5x10^4 and cultured in a CO2 incubator for 20 hours, and the supernatant was collected.The amount of IFNg in the supernatant was measured using 20 microliters of the supernatant with an IFNg ELISA kit (MabTec). The measurement method followed the manufacturer's protocol. After antigen stimulation, the majority of MAGE CAR T cells differentiated into Tem cells, but in hybrid-TCR T cells, Tn and Tscm cells remained in parallel with Tem differentiation, demonstrating that differentiation into Tcm cells was also observed (Test Example 8). (a) shows the experimental schedule. (b) shows the memory phenotype of cells before antigen stimulation. (c) shows the memory phenotype of each cell after antigen stimulation. As explained in Figure 11-1. As explained in Figure 11-1. The oxygen consumption rate (OCR) and spare respiratory capacity level (SRC) of CAR T cells and hybrid-TCR T cells before (a) and after (b) antigen stimulation are shown (Test Example 9). The results of measuring in vivo antitumor activity are shown (Test Example 10). (a) shows the experimental schedule. (b) and (c) show the results of tumor volume measurements. (b) shows the average value for each group, and (c) shows the results of measurements for individual mice. This shows the results of measurements of in vivo antitumor activity (Test Example 11). (a) shows the experimental schedule. (b) and (c) show the results of tumor volume measurements. (b) shows the average value for each group, and (c) shows the results of measurements for individual mice. This shows the results of measurements of in vivo antitumor activity (Test Example 12). (a) shows the experimental schedule. (b) shows the results of measurements of tumor volume. (c) to (e) show the results of cell staining of tumor-infiltrating lymphocytes (TIL). As explained in Figure 15-1. As explained in Figure 15-1. This shows the results of flow cytometry analysis of alpha / beta cells (a) and gamma / delta cells (b) into which hybrid-TCR genes were introduced (Test Example 13). This shows the results of a cytotoxicity test (Test Example 14). (a) shows the results of the cytotoxicity test against the positive target A375, and (b) shows the results of the cytotoxicity test against the negative target HCT116. The results are as explained in Figure 17-1.Figure 1 shows the results of flow cytometry analysis of hybrid-TCR gene-transduced alpha / beta cells (a) and gamma / delta cells (b) (Test Example 15). Figure 2 shows the results of measuring the proliferation rate of hybrid-TCR gene-transduced alpha / beta cells (Test Example 16). HD1 and HD2 represent results using peripheral blood mononuclear cells derived from different healthy individuals. Figure 3 shows the results of measuring the proliferation rate of hybrid-TCR gene-transduced alpha / beta cells (Test Example 17). Figure 4 shows the results of measuring the cytotoxicity of hybrid-TCR gene-transduced alpha / beta cells (Test Example 18). The horizontal axis shows effector cells, and the bottom of each graph shows target cells. The legend indicates the ratio of effector cells to target cells. Figure 5 shows the results of measuring the cytotoxicity of hybrid-TCR-transduced alpha / beta cells (Test Example 19). The legend indicates the effector cells. Figure 6 shows the results of measuring the cytotoxicity of hybrid-TCR-transduced alpha / beta cells (Test Example 20). The legend indicates the effector cells. Figure 7 shows the results of measuring in vivo antitumor activity (Test Example 21). The legend indicates the effector cells administered.

[0025] 1. Definitions In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0026] "Identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF, "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF, "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0027] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having beta-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0028] As used herein, "TCR" refers to a T cell receptor.

[0029] As used herein, "CDR" is an abbreviation for Complementarity Determining Region, also referred to as complementarity-determining region. CDR is a region present in the variable region of an immunoglobulin, and is a region deeply involved in the specific binding of an antibody to an antigen. Furthermore, "light chain CDR" refers to a CDR present in the light chain variable region of an immunoglobulin, and "heavy chain CDR" refers to a CDR present in the heavy chain variable region of an immunoglobulin.

[0030] As used herein, the term "variable region" refers to a region comprising CDR1 to CDR3 (hereinafter simply referred to as "CDRs1-3"). The order in which these CDRs 1-3 are arranged is not particularly limited, but preferably refers to a region in which they are arranged from the N-terminus to the C-terminus in the order of CDR1, CDR2, and CDR3, or in the reverse order, either consecutively or via other amino acid sequences referred to as framework regions (FRs), as described below. The term "heavy chain variable region" refers to a region in which the above-mentioned heavy chain CDRs 1-3 are arranged, and the term "light chain variable region" refers to a region in which the above-mentioned light chain CDRs 1-3 are arranged.

[0031] The regions of each variable region other than CDR1-3 are referred to as framework regions (FRs) as described above. In particular, the region between the N-terminus of the variable region and CDR1 is defined as FR1, the region between CDR1 and CDR2 as FR2, the region between CDR2 and CDR3 as FR3, and the region between CDR3 and the C-terminus of the variable region as FR4.

[0032] 2. Hybrid TCR In one aspect, the present invention relates to a hybrid TCR (sometimes referred to herein as the "hybrid TCR of the present invention") comprising polypeptide A comprising the light chain variable region of an antibody having binding ability to an antigen peptide-MHC complex and the first subunit constant region of a TCR, and polypeptide B comprising the heavy chain variable region of the antibody and the second subunit constant region of a TCR. This is described below.

[0033] An "antigen" refers to a protein that induces immunity. For example, it is a substance that can induce the activation of lymphocytes such as T cells and B cells by being presented by antigen-presenting cells in lymph nodes or the spleen. An "antigen peptide" refers to an antigen that is a peptide. An antigen peptide is a part of the amino acid sequence contained in an antigen, and includes a T cell-recognized epitope. It may also be a combination of multiple T cell-recognized epitopes.

[0034] Antigens include, but are not limited to, CD19, GD2, GD3, CD20, CD37, CEA, HER2, EGFR, type III mutant EGFR, CD38, BCMA, MUC-1, PSMA, WT1, cancer testis antigens (e.g., NY-ESO-1, MAGE-A4, etc.), mutation peptides (e.g., k-ras, h-ras, p53, etc.), hTERT, PRAME, TYRP1, mesothelin, PMEL, mucin, etc.

[0035] Particularly preferred antigens include MAGE-A4 and PRAME. Particularly preferred antigen peptides include peptides derived from MAGE-A4 or PRAME. Among these peptides, particularly preferred are the MAGE-A4p230-239 peptide (amino acid sequence: GVYDGREHTV (SEQ ID NO: 31)) and the PRAMEp301-309 peptide (amino acid sequence: LYVDSLFFL (SEQ ID NO: 32)).

[0036] The antigen peptide-MHC complex is a complex of an antigen peptide and an MHC (major histocompatibility complex), and is not particularly limited as long as it is in a form that allows the antigen peptide to be presented on immune cells. As MHC, preferably HLA-A*02:01, HLA-A*02:06, HLA-A*02:07, HLA-A*24:02, HLA-A*26:0 1, HLA-A*11:01, HLA-A*39:01, HLA-A*40:02, HLA-A*40:06, HLA-B*15:01, HLA-B*15:1 8, HLA-DRB1*15:02, HLA-DRB1*15:01, HLA-DRB1*04:05, HLA-DRB1*04:06, HLA-DRB1*0 8:03, HLA-DRB1*08:02, HLA-DRB1*12:01, HLA-DRB1*13:02, HLA-DRB1*14:54, etc.

[0037] Particularly preferred examples of antigen peptide-MHC complexes include HLA-A0201 MAGE-A4p230-239 peptide-MHC complexes and HLA-A24 PRAMEp301-309 peptide-MHC complexes.

[0038] The antibodies used in the hybrid TCRs of the present invention have binding ability to antigenic peptide-MHC complexes. That is, the antibodies recognize both antigenic peptides and MHC. The presence or absence of such binding ability can be measured according to or in accordance with the method of Test Example 2 in WO2022 / 124282. For example, when the ratio of the fluorescence intensity in the peptide-pulsed state to the fluorescence intensity in the non-peptide-pulsed state (negative control) is, for example, 2 or more, preferably 3 or more, more preferably 4 or more, even more preferably 5 or more, even more preferably 6 or more, particularly preferably 7 or more, and especially preferably 8 or more, the peptide can be determined to have binding ability to the MHC complex.

[0039] The dissociation constant of an antibody used in the hybrid TCR of the present invention with any antigen peptide-MHC complex is preferably 1 μM or less, more preferably 500 nM or less, even more preferably 200 nM or less, still more preferably 100 nM or less, particularly preferably 50 nM or less, especially more preferably 20 nM or less, and especially more preferably 10 nM or less. The dissociation constant can be measured according to the method of Test Example 3 in WO2022 / 124282.

[0040] The light chain variable region is the light chain variable region of the antibody and includes light chain CDR1, light chain CDR2, and light chain CDR3, and the heavy chain variable region is the heavy chain variable region of the antibody and includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. Parts of the light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 recognize antigen peptides, and another part recognizes MHC.

[0041] In a particularly preferred embodiment (embodiment 1) of the present invention, the antigenic peptide is MAGE-A4, the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1 (HIGSKS), a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2 (DDS), and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 (LAWDSSTAI), and the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4 (GGTFSSYA), a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5 (IIPIFGTA), and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6 (ARSPRRAYHDAFDI).

[0042] In Aspect 1, the light chain variable region is preferably a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having 90% or more (preferably 95% or more, preferably 98% or more, preferably 99% or more) identity to the amino acid sequence set forth in SEQ ID NO: 13, and the heavy chain variable region is preferably a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14 or an amino acid sequence having 90% or more (preferably 95% or more, preferably 98% or more, preferably 99% or more) identity to the amino acid sequence set forth in SEQ ID NO: 14. When there is an amino acid mutation from SEQ ID NO: 13 or 14, the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.

[0043] In a particularly preferred embodiment (embodiment 2) of the present invention, the antigenic peptide is PRAME, the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7 (NIGSKN), a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8 (RDS), and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9 (QVWDSSHV), and the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10 (GGTFSSYA), a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11 (IIPIFGTA), and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12 (ARHHSNYYYYGMDV).

[0044] In aspect 2, the light chain variable region is preferably a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 15 or an amino acid sequence having 90% or more (preferably 95% or more, preferably 98% or more, preferably 99% or more) identity to the amino acid sequence set forth in SEQ ID NO: 15, and the heavy chain variable region is preferably a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 16 or an amino acid sequence having 90% or more (preferably 95% or more, preferably 98% or more, preferably 99% or more) identity to the amino acid sequence set forth in SEQ ID NO: 16. When there is an amino acid mutation from SEQ ID NO: 15 or 16, the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.

[0045] Polypeptide A preferably contains a leader sequence of the light chain variable region, and polypeptide B preferably contains a leader sequence of the heavy chain variable region. The leader sequence can facilitate the placement of the hybrid TCR of the present invention on the cell membrane.

[0046] The leader sequence of the light chain variable region is not particularly limited, but is preferably the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having 90% or more identity (preferably 95% or more, preferably 98% or more, preferably 99% or more) to the amino acid sequence shown in SEQ ID NO: 19. When there is an amino acid mutation, the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.

[0047] The leader sequence of the heavy chain variable region is not particularly limited, but is preferably the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having 90% or more identity (preferably 95% or more, preferably 98% or more, preferably 99% or more) to the amino acid sequence shown in SEQ ID NO: 20. When there is an amino acid mutation, the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.

[0048] The position of the leader sequence is not particularly limited, but is preferably located at the N-terminus of the variable region. That is, the leader sequence of the light chain variable region is preferably located at the N-terminus of the light chain variable region, and the leader sequence of the heavy chain variable region is preferably located at the N-terminus of the heavy chain variable region. In this case, the leader sequence and the variable region may be linked directly or indirectly (for example, via another sequence such as a linker). The linker is not particularly limited, but examples include a linker containing glycine alone or glycine and serine. The number of amino acid residues in the linker is, for example, 1 to 30, preferably 1 to 10, and more preferably 1 to 5. In a particularly preferred embodiment of the present invention, the leader sequence and the variable region are directly linked.

[0049] TCR is a heterodimer consisting of a first subunit and a second subunit. Examples of subunits that constitute TCR include an α chain, a β chain, a γ chain, and a δ chain. The first and second subunits in the hybrid TCR of the present invention are not particularly limited as long as they constitute a TCR as a heterodimer. Examples include a combination in which the first subunit is an α chain and the second subunit is a β chain, a combination in which the first subunit is a β chain and the second subunit is an α chain, a combination in which the first subunit is a γ chain and the second subunit is a δ chain, and a combination in which the first subunit is a γ chain and the second subunit is a δ chain. Among these, particularly preferred are a combination in which the first subunit is an α chain and the second subunit is a β chain, and a combination in which the first subunit is a β chain and the second subunit is an α chain.

[0050] The constant regions of the first / second subunits are parts other than the variable regions of the first / second subunits (regions that contain CDRs and whose amino acid sequences vary depending on the type of antigen), and are not particularly limited as long as they have the function of the constant regions of the first / second subunits (for example, the function of penetrating the cell membrane and positioning the variable regions of the first / second subunits in the appropriate orientation on the cell surface).

[0051] The α-chain constant region is preferably the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having 90% or more identity (preferably 95% or more, preferably 98% or more, preferably 99% or more) to the amino acid sequence shown in SEQ ID NO: 17. When there is an amino acid mutation, the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.

[0052] The β-chain constant region is preferably the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having 90% or more identity (preferably 95% or more, preferably 98% or more, preferably 99% or more) to the amino acid sequence shown in SEQ ID NO: 18. When there is an amino acid mutation, the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.

[0053] The light chain variable region is usually located N-terminal to the first subunit constant region, and the heavy chain variable region is usually located N-terminal to the second subunit constant region.

[0054] The light chain variable region and the first subunit constant region may be linked directly or indirectly (e.g., via another sequence such as a linker). In a particularly preferred embodiment of the present invention, the light chain variable region and the first subunit constant region are linked directly. The heavy chain variable region and the second subunit constant region may be linked directly or indirectly (e.g., via another sequence such as a linker). In a particularly preferred embodiment of the present invention, the heavy chain variable region and the second subunit constant region are linked directly. The linker is the same as the linker that can link the leader sequence and variable region described above.

[0055] Polypeptide A / B may contain, or in one embodiment, may not contain, sequences / regions other than the above-mentioned regions / sequences (e.g., the intracellular domain of CD3, the intracellular domain of a costimulatory factor (e.g., OX40, 4-1BB, GITR, CD27, CD278, CD28, etc.), or a ligand domain (e.g., the GITRL domain, the 4-1BBL domain, the ICOSL domain, etc.)). In one embodiment, the number of amino acid residues in the amino acid sequence present on the N-terminal side of the light / heavy chain variable region of polypeptide A / B (on the N-terminal side of the leader sequence when a leader sequence is positioned on the N-terminal side of the light / heavy chain variable region) is, for example, 0 to 50, preferably 0 to 40, more preferably 0 to 30, even more preferably 0 to 20, still more preferably 0 to 10, particularly preferably 0 to 5, and particularly preferably 0 to 2. From a similar perspective, the number of amino acid residues in the amino acid sequence present on the C-terminal side of the 1st / 2nd subunit constant region of polypeptide A / B is, for example, 0 to 80, preferably 0 to 70, more preferably 0 to 60, even more preferably 0 to 50, still more preferably 0 to 40, particularly preferably 0 to 30, and particularly preferably 0 to 25.

[0056] The hybrid TCR of the present invention may be chemically modified. The polypeptide constituting the hybrid TCR of the present invention may have a C-terminus that is a carboxyl group (-COOH), a carboxylate group (-COO － The ester may be any of -CONH, -CONH, -COOR ... 1－6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3－8 Cycloalkyl groups such as phenyl and α-naphthyl 6－12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1－2 Alkyl group: α-naphthyl-C such as α-naphthylmethyl 1－2 C such as alkyl group 7－14An aralkyl group; a pivaloyloxymethyl group, etc., are used. In the polypeptide constituting the hybrid TCR of the present invention, a carboxyl group (or carboxylate) other than the C-terminus may be amidated or esterified. In this case, for example, the C-terminal esters described above are used as the esters. Furthermore, in the polypeptide constituting the hybrid TCR of the present invention, the amino group of the N-terminal amino acid residue may be protected by a protecting group (e.g., a C-protecting group such as a formyl group or an acetyl group). 1－6 C such as alkanoyl 1－6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1－6 C such as alkanoyl group 1－6 Also included are those protected with an alkyl group such as an acyl group.

[0057] The hybrid TCR of the present invention may be one to which a known protein or peptide such as a protein tag or signal sequence has been added. Examples of protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, and fluorescent protein tag.

[0058] The hybrid TCR of the present invention may be in the form of a pharmaceutically acceptable salt with an acid or base. The salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and both acidic and basic salts can be used. Examples of acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0059] The hybrid TCRs of the present invention may be in the form of a solvate. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, and acetic acid.

[0060] 3. Polynucleotide In one aspect, the present invention relates to a polynucleotide encoding the hybrid TCR of the present invention (sometimes referred to herein as the "polynucleotide of the present invention"). This is described below.

[0061] The polynucleotide of the present invention may contain other sequences in addition to the coding sequence of the hybrid TCR of the present invention. Preferably, the polynucleotide of the present invention contains the hybrid TCR of the present invention in an expressible state. Examples of such other sequences include promoter sequences, enhancer sequences, repressor sequences, insulator sequences, origins of replication, coding sequences for reporter proteins (e.g., fluorescent proteins), and coding sequences for drug resistance genes. The polynucleotide of the present invention may be a linear polynucleotide or a circular polynucleotide (e.g., a vector). The vector may be a plasmid vector or a viral vector (e.g., an adenovirus or a retrovirus). The vector may be, for example, a cloning vector or an expression vector. Examples of expression vectors include vectors for prokaryotic cells such as Escherichia coli or actinomycetes, and vectors for eukaryotic cells such as yeast cells, insect cells, or mammalian cells.

[0062] The polynucleotides of the present invention include not only DNA and RNA, but also those that have been chemically modified by known methods, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residues of each nucleotide can be substituted with chemically modified phosphate residues, such as phosphorothioate (PS), methylphosphonate, and phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide can also be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) can also be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Furthermore, the term "polynucleotide" encompasses not only natural nucleic acids but also bridged nucleic acids (BNAs), locked nucleic acids (LNAs), peptide nucleic acids (PNAs), etc.

[0063] The polynucleotide of the present invention is not particularly limited as long as it encodes polypeptide A and polypeptide B, and may be one type of polynucleotide or two types of polynucleotides (for example, a polynucleotide encoding polypeptide A and a polynucleotide encoding polypeptide B). From the viewpoint of the expression efficiency of a hybrid TCR, one type of polynucleotide encoding polypeptide A and polypeptide B is preferred. In this preferred embodiment, the coding sequence for polypeptide A and the coding sequence for polypeptide B are preferably linked via a coding sequence for a self-cleaving peptide.

[0064] As used herein, the term "self-cleaving peptide" refers to a peptide sequence with cleavage activity occurring between two amino acid residues within the peptide sequence itself. Examples of self-cleaving peptides include 2A peptides or 2A-like peptides. For example, in 2A peptides or 2A-like peptides, cleavage occurs between glycine and proline residues on these peptides. This occurs via a "ribosomal skipping mechanism" in which normal peptide bond formation between glycine and proline residues does not occur during translation, and downstream translation is not affected. The ribosomal skipping mechanism is known in the art and is used for the expression of multiple proteins encoded by a single messenger RNA (mRNA). The self-cleaving peptide used in the present invention can be derived from a viral 2A peptide or a 2A-like peptide with equivalent function. For example, the self-cleaving peptide domain may be selected from the group consisting of 2A peptide (F2A) derived from foot-and-mouth disease virus (FMDV), 2A peptide (E2A) derived from equine rhinitis A virus (ERAV), 2A peptide (P2A) derived from porcine teschovirus (PTV-1), and 2A peptide (T2A) derived from Thosea asigna virus (TaV). The self-cleaving peptide domain may be mutated as appropriate, as long as its activity is not significantly impaired.

[0065] 4. Cells In one aspect, the present invention relates to cells (sometimes referred to herein as "cells of the present invention") containing the polynucleotides of the present invention. This is described below.

[0066] The cells of origin of the cells of the present invention are not particularly limited. If the cells of the present invention are intended to be used in producing the hybrid TCRs of the present invention, the cells of origin may be cells that can be used for protein expression (e.g., insect cells, eukaryotic cells, mammalian cells, etc.).

[0067] When the cell of the present invention comprises a polynucleotide encoding a hybrid TCR of the present invention, the cell is preferably a lymphocyte (e.g., a T cell (e.g., a CD4-positive CD8-negative T cell, a CD4-negative CD8-positive T cell, a T cell prepared from an iPS cell, an αβ-T cell, a γδ-T cell, etc.), a NK cell, a NKT cell, etc.). The lymphocyte is preferably a cell that expresses the hybrid TCR of the present invention, and in a more specific embodiment, the lymphocyte expresses the hybrid TCR of the present invention on the cell membrane.

[0068] Lymphocytes and other cells expressing hybrid TCRs recognize antigen peptide-MHC complexes with the antibody variable region, then transmit the recognition signal inside T cells and other cells, activating a signal that induces cytotoxic activity, which in turn allows the cells to attack or exert cytotoxic activity against other cells or tissues that express the antigen.

[0069] Such lymphocytes and the like specifically recognize cancer tissue (tumor tissue) and are therefore useful for the treatment or prevention of cancer, etc. The type of cancer is not particularly limited, and includes hematological cancers and solid cancers. Examples of hematological cancers include various B-cell malignant lymphomas (B-cell acute lymphoblastic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), myeloproliferative disorders, myelodysplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, and multiple myeloma. Examples of solid cancers include lung cancer, colon cancer, ovarian cancer, breast cancer, brain tumors, gastric cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, melanoma, neuroblastoma, and bladder cancer.

[0070] The cells of the present invention can be obtained by introducing the polynucleotide of the present invention into cells. If necessary, cells containing the polynucleotide of the present invention may be enriched, or may be enriched using a specific marker (CD antigen such as CD8) as an indicator.

[0071] Hybrid TCR-expressing cells have enhanced cytokine production and higher cytotoxicity compared to CAR-expressing cells. They also have increased antigen recognition sensitivity and slower progression of exhaustion. In in vivo antitumor testing using NOG mice, tumor elimination occurred rapidly and the effect was sustained.

[0072] 5. Pharmaceutical Composition In one aspect, the present invention relates to a pharmaceutical composition (sometimes referred to herein as the "pharmaceutical composition of the present invention") containing the cell of the present invention, in which the hybrid TCR of the present invention is expressed on the cell membrane. This is described below.

[0073] The content of the above-mentioned cells in the pharmaceutical composition can be appropriately determined taking into consideration the type of target disease (e.g., solid cancer), the desired therapeutic effect, the administration method, the treatment period, the patient's age, and the patient's weight, etc. For example, the content of the antibody in the pharmaceutical composition can be about 0.001 to 10 parts by weight, where the total pharmaceutical composition is 100 parts by weight. The content of the cells in the pharmaceutical composition can be, for example, about 1 cell / mL to 10^4 cells / mL.

[0074] The administration form of the pharmaceutical composition is not particularly limited as long as the desired effect is obtained, and it can be administered to mammals, including humans, by either oral or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, or topical administration). Since the active ingredient is cells, the preferred administration form is parenteral administration, more preferably intravenous injection. Dosage forms for oral and parenteral administration and methods for their preparation are well known to those skilled in the art, and can be prepared according to standard methods by mixing the antibody or cells of the present invention with a pharmaceutically acceptable carrier, etc.

[0075] Dosage forms for parenteral administration include injectable preparations (e.g., drip infusions, intravenous injections, intramuscular injections, subcutaneous injections, and intradermal injections), topical preparations (e.g., ointments, poultices, and lotions), suppositories, inhalants, ophthalmic preparations, eye ointments, nasal drops, ear drops, and liposomes. For example, injectable preparations are prepared by dissolving or suspending antibodies or cells in distilled water for injection, and solubilizers, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, and the like can be added as needed. The pharmaceutical composition can also be a lyophilized preparation for preparation immediately before use.

[0076] The pharmaceutical composition may further contain other drugs that are effective in diagnosing, treating, or preventing diseases. In addition, the pharmaceutical composition may also contain ingredients such as bactericides, anti-inflammatory agents, cell activators, vitamins, and amino acids, as needed.

[0077] Carriers used in formulating pharmaceutical compositions include excipients, binders, disintegrants, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, and the like that are commonly used in the art.

[0078] The type of disease to be diagnosed, treated, or prevented using the pharmaceutical composition is not particularly limited as long as the diagnosis, treatment, or prevention can be achieved. Specific target diseases include, for example, cancer. The type of cancer is not particularly limited and includes blood cancer and solid cancer. Examples of blood cancer include various B-cell malignant lymphomas (B-cell acute lymphoblastic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B-cell lymphoma, CD20-positive Hodgkin's lymphoma, etc.), myeloproliferative disorders, myelodysplastic / myeloproliferative neoplasms (CMML, JMML, CML, MDS / MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, multiple myeloma, etc. Examples of solid cancers include lung cancer, colon cancer, ovarian cancer, breast cancer, brain tumor, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, melanoma, neuroblastoma, and bladder cancer.

[0079] The subject (subject) to which the pharmaceutical composition is administered is, for example, an animal that is afflicted with or may be afflicted with the above-mentioned disease. "Possibly afflicted" can be determined by known diagnostic methods. The animal is, for example, a mammal, preferably a human.

[0080] The dosage of a pharmaceutical composition can be determined by a clinician based on various factors, such as the route of administration, the type of disease, the severity of symptoms, the patient's age, sex, and body weight, the severity of the disease, pharmacological knowledge such as pharmacokinetic and toxicological characteristics, the use of a drug delivery system, and whether the composition is administered as part of a combination of other drugs. For example, if the active ingredient is an antibody, the dosage of the pharmaceutical composition can be approximately 1 microgram / kg (body weight) to 10 g / kg (body weight) per day. If the active ingredient is cells, the dosage can be approximately 10^4 cells / kg (body weight) to 10^9 cells / kg (body weight). The administration schedule of the pharmaceutical composition can also be determined taking into account factors similar to those for the dosage. For example, the above daily dosage can be administered once a day to once a month.

[0081] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0082] Test Example 1. Hybrid-TCR structure and PBMC expression As shown in Figure 1, hybrid-TCR is a gene-constructed receptor that is introduced into T cells and expressed. It recognizes the cancer-specific peptide-MHC complex (pMHC complex) on cancer cells and exerts cytotoxic effects on cancer cells. This allows for cancer suppression in the body and makes cancer treatment possible.

[0083] The hybrid TCR constructed in this study is a retroviral expression system, but lentiviral and transposon expression are also possible (Figure 2). The antibody recognition sites, VH and VL, are bound to TCR Cb and TCR Ca, respectively, and the 2A peptide generates heterodimers for CD3 complex formation. Cb57 and Ca48 were replaced with cysteine. This is thought to form disulfide bonds between the introduced heterodimer molecules, stabilizing the structure. In addition, Ca116, 119, and 120 were replaced with leucine. This is thought to increase the frequency of intermolecular binding of the introduced heterodimer molecules.

[0084] In this study, we constructed hybrid TCRs by combining the VH and VL domains of the MAGE#17 antibody (WO / 2018 / 225732), which specifically recognizes the HLA-A0201 MAGE-A4p230-239 peptide-MHC complex, and the PRAME#98 antibody (WO2022 / 124282), which specifically recognizes the HLA-A24 PRAMEp301-309 peptide-MHC complex, with TCR Cb and TCR Ca. The nucleotide and amino acid sequences of the MAGE#17 and PRAME#98 hybrid TCRs are shown below.

[0085] <MAGE#17 Hybrid TCR Amino Acid Sequence><VH leader> MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 20) <vh>QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSPRRAYHDAFDIWGQGTMVTVSS (SEQ ID NO: 14)<TCR Cb> EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 18) <p2a>GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 21)<VL leader> MNFGLRLIFLVLTLKGVKC (SEQ ID NO: 19) <vl>SYELTQPPSMSVAPGKTASITCGGDHIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCLAWDSSTAIFGGGTQLTVL (SEQ ID NO: 13)<TCR Ca> YIQNPDPAVYQLRRSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVILLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 17) The amino acid sequence of MAGE-A4 #17 hybrid-TCR is shown above. The sequences are linked in the order of VH leader / VH / TCR / P2A / VL leader / VL / TCR. Cb57 and Ca48 were substituted with cysteine. Ca116, 119, and 120 were substituted with leucine. The CDR sequence is underlined.

[0086] <MAGE#17 Hybrid TCR Base Sequence><VH leader> ATG AAA CAC CTG TGG TTC TTC CTC CTG CTG GTG GCA GCT CCC AGA TGG GTC CTG TCC (SEQ ID NO: 29) <vh>CAG GTC CAG CTG GTA CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG TCC TCG GTG AAG GTC TCC TGC AAG GCT TCT GGA GGC ACC TTC AGC AGC TAT GCT ATC AGC TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT GAG TGG ATG GGA GGG ATC ATC CCT ATC TTT GGT ACA GCA AAC TAC GCA CAG AAG TTC CAG GGC AGA GTC ACG ATT ACC GCT GAC AAA TCC ACG AGC ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG TAT TAC TGT GCG AGA TCC CCC CGG CGG GCA TAT CAT GAT GCT TTT GAT ATC TGG GGC CAA GGG ACA ATG GTC ACC GTC TCT TCA (SEQ ID NO: 23) <TCR Cb> GAA GAT CTG AAG AAC GTG TTC CCC CCA GAG GTG GCC GTG TTC GAG CCC AGC GAG GCC GAG ATC AGC CAC ACC CAG AAA GCC ACC CTG GTC TGC CTG GCC ACC GGC TTC TAC CCC GAC CAC GTG GAA CTG TCT TGG TGG GTG AAC GGC AAA GAG GTG CAC AGC GGC GTC tGC ACC GAC CCC CAG CCC CTG AAA GAG CAG CCC GCC CTG AAT GAT TCT CGA TAT TGT TTA AGC AGC AGA CTG AGA GTG TCC GCC ACC TTC TGG CAG GAC CCC CGG AAC CAC TTC AGA TGC CAG GTG CAG TTC TAC GGC CTG AGC GAG AAC GAC GAG TGG ACC CAG GAC CGG GCC AAG CCT GTG ACC CAG ATC GTG TCT GCC GAG GCCTGG GGC AGA GCC GAC TGC GGC TTT ACC AGC GAG AGC TAC CAG CAG GGC GTG CTG AGC GCC ACC ATC CTG TAC GAG ATC CTG CTG GGC AAG GCC ACC CTG TAC GCC GTG CTG GTG TCC GCT CTG GTG CTG ATG GCC ATG GTG AAA CGG AAG GAC AGC AGA GGC (sequence number 27) <p2a>GGA AGC GGA GCC ACG AAC TTC TCT CTG TTA AAG CAA GCA GGA GAT GTT GAA GAA AAC CCC GGG CCT (SEQ ID NO: 30.) <VL leader> ATG AAC TTT GGG CTC AGA TTG ATT TTC CTT GTC CTT ACT TTA AAA GGT GTG AAG TGT (SEQ ID NO: 28) <vl>TCC TAT GAG CTG ACT CAG CCA CCC TCG ATG TCA GTG GCC CCA GGA AAG ACG GCC AGC ATT ACC TGT GGC GGA GAC CAT ATT GGA AGT AAA AGT GTT CAC TGG TAC CAG CAG AAG CCA GGC CAG GCC CCT GTA CTG GTC GTC TAT GAT GAT AGC GAC CGG CCC TCA GGA ATC CCT GAG CGA TTC TCT GGC TCC AAC TCT GGG AAC ACA GCC ACT CTG ACC ATC AGC GGG ACC CAG GCT ATG GAT GAG GCT GAC TAT TAC TGT CTG GCG TGG GAC AGC AGC ACT GCG ATC TTC GGC GGA GGC ACC CAG CTG ACC GTC CTC (SEQ ID NO: 22) <TCR Ca> TAC ATC CAG AAC CCC GAC CCC GCC GTG TAC CAG CTG CGG AGA TCT AAA TCT TCT GAT AAA TCT GTC TGC CTG TTC ACC GAC TTC GAC AGC CAG ACC AAC GTG TCC CAG AGC AAG GAC AGC GAC GTG TAC ATC ACC GAT AAG tgC GTG CTG GAC ATG CGG AGC ATG GAC TTC AAG AGC AAC AGC GCC GTG GCC TGG TCC AAC AAG AGC GAC TTC GCC TGC GCC AAC GCC TTC AAC AAC AGC ATC ATC CCC GAG GAC ACA TTC TTC CCA AGC CCC GAG AGC AGC TGC GAC GTG AAG CTG GTG GAA AAG AGC TTC GAG ACA GAC ACC AAC CTG AAC TTC CAG AAC CTG ctC GTG ATC ctC cTC CGG ATT CTG CTG CTG AAG GTG GCC GGC TTC AAC CTG CTG ATG ACC CTG CGG CTG TGG TCCAGC TAA (SEQ ID NO: 26).

[0087] <PRAME#98 Hybrid TCR Amino Acid Sequence><VL leader> MNFGLRLIFLVLTLKGVKC (SEQ ID NO: 19) <vl>SYELTQPLSVSVALGQTARITCGGNNIGSKNVHWYQQKPGQAPVLVIYRDSNRPSGIPERFSGSNSGNTATLTISRAQAGDEADYYCQVWDSSHVFGTGTKVTVL (SEQ ID NO: 15)<TCR Cb> EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 18) <p2a>GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 21)<VH leader> MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 20) <vh>QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTGTAYMELSSLRSEDTAVYYCARHHSNYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 16)<TCR Ca> YIQNPDPAVYQLRRSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVILLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 17) The amino acid sequence of PRAME #98 hybrid-TCR is shown above. The sequences are linked in the order of VH leader / VH / TCR / P2A / VL leader / VL / TCR. Cb57 and Ca48 were substituted with cysteine. Ca116, 119, and 120 were substituted with leucine. The CDR sequence is underlined.

[0088] <PRAME#98 Hybrid TCR base sequence><VL leader> ATG AAC TTT GGG CTC AGA TTG ATT TTC CTT GTC CTT ACT TTA AAA GGT GTG AAG TGT (SEQ ID NO: 28) <vl>TCC TAT GAG CTG ACT CAG CCA CTC TCA GTG TCA GTG GCC CTG GGA CAG ACG GCC AGG ATT ACC TGT GGC GGA AAC AAC ATT GGA AGT AAA AAT GTG CAC TGG TAC CAG CAG AAG CCA GGC CAG GCC CCT GTG CTG GTC ATC TAT AGG GAT AGC AAC CGG CCC TCT GGa ATt CCT GAG CGA TTC TCT GGC TCC AAC TCG GGG AAC ACG GCC ACC CTG ACC ATC AGC AGA GCC CAA GCC GGG GAT GAG GCT GAC TAT TAC TGT CAG GTG TGG GAC AGC AGC CAT GTC TTC GGA ACT GGG ACC AAG GTC ACC GTC CTA (SEQ ID NO: 24) <TCR Cb> GAA GAT CTG AAG AAC GTG TTC CCC CCA GAG GTG GCC GTG TTC GAG CCC AGC GAG GCC GAG ATC AGC CAC ACC CAG AAA GCC ACC CTG GTC TGC CTG GCC ACC GGC TTC TAC CCC GAC CAC GTG GAA CTG TCT TGG TGG GTG AAC GGC AAA GAG GTG CAC AGC GGC GTC tGC ACC GAC CCC CAG CCC CTG AAA GAG CAG CCC GCC CTG AAT GAT TCT CGA TAT TGT TTA AGC AGC AGA CTG AGA GTG TCC GCC ACC TTC TGG CAG GAC CCC CGG AAC CAC TTC AGA TGC CAG GTG CAG TTC TAC GGC CTG AGC GAG AAC GAC GAG TGG ACC CAG GAC CGG GCC AAG CCT GTG ACC CAG ATC GTG TCT GCC GAG GCC TGG GGC AGA GCC GAC TGC GGC TTT ACC AGC GAG AGC TAC CAG CAG GGCGTG CTG AGC GCC ACC ATC CTG TAC GAG ATC CTG CTG GGC AAG GCC ACC CTG TAC GCC GTG CTG GTG TCC GCT CTG GTG CTG ATG GCC ATG GTG AAA CGG AAG GAC AGC AGA GGC (sequence number 27) <p2a>GGA AGC GGA GCC ACG AAC TTC TCT CTG TTA AAG CAA GCA GGA GAT GTT GAA GAA AAC CCC GGG CCT (sequence number 30)<VH leader> ATG AAA CAC CTG TGG TTC TTC CTC CTG CTG GTG GCA GCT CCC AGA TGG GTC CTG TCC (sequence number 29) <vh>CAG GTG CAG CTG GTG CAG TCT GGG GCT GAG GTG AAG AAG CCT GGG TCC TCG GTG AAG GTC TCC TGC AAG GCT TCT GGA GGC ACC TTC AGC AGC TAT GCT ATC AGC TGG GTG CGA CAG GCC CCT GGA CAA GGG CTT GAG TGG ATG GGA GGG ATC ATC CCT ATC TTT GGT ACA GCA AAC TAC GCA CAG AAG TTC CAG GGC AGA GTC ACG ATT ACC GCG GAC GAA TCC ACG GGC ACA GCC TAC ATG GAG CTG AGC AGC CTG AGA TCT GAG GAC ACG GCC GTG TAT TAC TGT GCG AGA CAC CAC AGT AAC TAC TAC TAC TAC GGT ATG GAC GTC TGG GGC CAA GGG ACC ACG GTC ACC GTC TCt tcA (SEQ ID NO: 25) <TCR Ca> TAC ATC CAG AAC CCC GAC CCC GCC GTG TAC CAG CTG CGG AGA TCT AAA TCT TCT GAT AAA TCT GTC TGC CTG TTC ACC GAC TTC GAC AGC CAG ACC AAC GTG TCC CAG AGC AAG GAC AGC GAC GTG TAC ATC ACC GAT AAG tgC GTG CTG GAC ATG CGG AGC ATG GAC TTC AAG AGC AAC AGC GCC GTG GCC TGG TCC AAC AAG AGC GAC TTC GCC TGC GCC AAC GCC TTC AAC AAC AGC ATC ATC CCC GAG GAC ACA TTC TTC CCA AGC CCC GAG AGC AGC TGC GAC GTG AAG CTG GTG GAA AAG AGC TTC GAG ACA GAC ACC AAC CTG AAC TTC CAG AAC CTG ctC GTG ATC ctC cTC CGG ATT CTG CTG CTGAAG GTG GCC GGC TTC AAC CTG CTG ATG ACC CTG CGG CTG TGG TCC AGC TAA (SEQ ID NO: 26).

[0089] Retroviral vectors for the MAGE#17 and PRAME#98 hybrid TCRs (Fig. 2) were constructed and transfected into human PBMCs for expression (Fig. 3a, b, c, d, e, f, g, h).

[0090] Test Example 2: Hybrid-TCR-introduced T cells enhance cytokine production compared to CAR-introduced T cells. + MAGE-A4 + ) and effector cells were co-cultured, and the time course of the amount of IFNg produced in the medium was measured by ELISA.

[0091] The results are shown in Figure 4. The amount of IFNg produced by co-culture of MAGE#17 Hybrid-TCR with positive target cells was significantly higher than that produced by MAGE#17 CAR, and this was particularly evident in cultures with a reduced number of effectors (Figure 4a, b). + MAGE-A4 - ) co-culture did not result in IFNg production.

[0092] Test Example 3: Hybrid-TCR-introduced T cells have enhanced cytotoxicity compared to CAR-introduced T cells. The positive target SK-MEL-37 cells (HLA-A0201+ MAGE-A4+) were co-cultured with effector cells, and the change over time in current value due to cytotoxic activity was measured using xCelligence.

[0093] The results are shown in Figure 5. The cell index decrease observed in co-culture of MAGE#17 Hybrid-TCR with positive target cells was faster than that observed in MAGE#17 CAR, and this decrease was more pronounced in assays with low E:T ratios (Figure 5a, b, c). Neither effector cell showed cytotoxicity against negative target cells, HCT116 (Figure 5d).

[0094] Test Example 4: Hybrid-TCR-introduced T cells show slower progression of exhaustion than CAR-introduced T cells or TCR-introduced T cells. MAGE#17 CAR, MAGE#17 hybrid-TCR, and HLA-A0201 MAGE-A4p230-239 peptide-MHC complex-specific recognizing TCR (MAGE TCR) were subjected to continuous antigen stimulation using immobilized tetramers, and the expression of exhaustion markers on effector cells was examined.

[0095] The results are shown in Figure 6. Expression of these exhaustion markers peaked on day 12 after stimulation on day 10 and remained constant thereafter. However, the percentage of cells expressing PD-1, LAG-3, and CTLA-4, as well as the MFI (measured factor index), which indicates the intensity of expression, were lowest in the hybrid-TCR group (Figures 6a, b, e, f, I, j, m, n). Furthermore, in the experimental group in which antigen stimulation was discontinued from day 16 onward, the expression of exhaustion markers subsequently decreased, with the hybrid-TCR group showing the lowest expression (Figures 6c, d, g, h, k, l, o, p).

[0096] Test Example 5: Hybrid-TCRs cultured without stimulation have higher ratios of T naive (Tn), T central memory (Tcm), and T effector memory (Tem) compared to CAR and TCR. In effector cells subjected to chronic antigen stimulation, the ratio of memory types is nearly equal between hybrid-TCR and TCR, with a clear difference in ratio compared to CAR, which has fewer Tn and more T effector (Te). When cultured without stimulation, MAGE#17 CAR and MAGE TCR show a significant increase in Te and a significant decrease in Tn. Furthermore, the ratios of Tcm and Tem are low. On the other hand, in the MAGE#17 hybrid-TCR, the Tn ratio remains at approximately 60%, the Te ratio remains at approximately 25%, and approximately 7% of Tcm and Tem are produced (Figure 7a, b). On the other hand, with continuous antigen stimulation, memory differentiation of MAGE#17 hybrid-TCR and MAGE TCR was almost similar, with Tn at approximately 44-47%, Te at approximately 30%, Tcm at 16-18%, and Tem at approximately 8%. In contrast, with MAGE#17 CAR, Tn decreased to approximately 20%, Te increased to 55%, Tcm was slightly lower at approximately 10%, and Tem increased by approximately 15%, showing a tendency toward Te compared to memory formation of MAGE#17 hybrid-TCR and MAGE TCR (Figure 7a, c).

[0097] Experimental Example 6: Tumor Regression and Sustained in the Treatment Group Infused with Hybrid-TCR-Transduced T Cells. NW-MEL-38 cells were loaded onto the tumors. On day 4, five experimental groups (PBMC, NGMC, MAGE#17 CAR, MAGE#17 hybrid-TCR, and MAGE TCR) were infused with effector cells on day 11, and the antitumor effects were examined (Figure 8a). Tumor suppression was not observed in the PBS and NGMC groups (Figure 8b, c, d). In contrast, tumor growth suppression was observed in the MAGE#17 CAR-infused group, but the effect was limited, and tumor suppression was insufficient after day 28 after tumor loading (Figure 8b, e). On the other hand, rapid tumor suppression was observed in the MAGE#17 hybrid-TCR and MAGE TCR groups, with tumors disappearing by day 17 of tumor loading (day 13 after infusion) and remaining in this state (Figure 8b, f, g). Although weight loss was observed in one MAGE#17 CAR-treated mouse during the experimental period, suggesting the occurrence of GVHD, no significant weight loss was observed in the other experimental mice, suggesting the absence of GVHD during the experimental period (Fig. 8h, I, j, k, l, m). Therefore, the tumor suppression observed in this study was suggested to be due to the infused effector cells. The antitumor activity of the MAGE#17 Hybrid-TCR was confirmed to be superior to that of the MAGE#17 CAR-treated mouse and almost equivalent to that of the MAGE TCR (Fig. 8e, f, g).

[0098] Test Example 7. A hybrid TCR with enhanced effector function was also generated for the PRAME#98 antibody that recognizes PRAME. We successfully generated a PRAME#98 hybrid TCR that could be expressed with the combination of VL-Cbeta and VH-Calpha (Figure 9). This PRAME#98 hybrid TCR recognized K562 A2402, which is HLA-A2402+ PRAME+, but did not recognize K562, which is A2402- PRAME+. Furthermore, compared to the PRAME#98 CAR, which recognizes PRAME in the same way, enhanced IFNg production and enhanced effector function were confirmed (Figure 10).

[0099] Test Example 8: After antigen stimulation, the majority of MAGE CAR T cells differentiate into Tem, but in MAGE hybrid-TCR T cells, Tn and Tscm remain in parallel with Tem differentiation, and differentiation into Tcm is also observed (MAGE#17 hybrid-TCR). Antigen stimulation was performed as follows: First, a 24-well plate was reacted with 2 micrograms of neutravidin / 0.2 ml of PBS at 4°C for one day to immobilize the cells, and then washed with PBS. This was then reacted with 2 micrograms of MAGE-A4 / HLA-A*0201 monomer / 0.2 ml of PBS at 4°C overnight to immobilize the cells, and then washed with PBS. This was used as the antigen plate.

[0100] Next, as shown in Figure 11a, 5x10^5 MAGE hybrid-TCR T cells, MAGE CAR T cells, and NGMCs cultured for 10 days were placed on antigen plates and cultured. The culture medium was GT-T551 medium (Takara-Bio) containing 300 IU / ml human recombinant IL-2 (Novartis) and 0.6% autologous plasma. The cells were cultured for 48 hours and then harvested. The cell number was adjusted to 5x10^5 for the next round of antigen stimulation. After a total of four rounds of antigen stimulation, cell staining and seahorse assays were performed.

[0101] The cells prepared by the above method were subjected to antibody staining and memory phenotype analysis using a Fortessa X-20 (BD). The antibodies used were: PE-labeled MAGE-A4 / HLA-A*0201 streptavidin tetramer, PE / Cy7-labeled anti-human CD8a antibody (clone RPA-T8, Biolegend), APC-labeled anti-human CD4 antibody (clone OKT-3, Biolegend), FITC-labeled anti-human CD62L antibody (clone DREG-56, Biolegend), and BV711-labeled anti-human CD45RA antibody (clone HI100, Biolegend).

[0102] The results are shown in Figure 11. (b) shows the memory phenotype of cells before antigen stimulation; no difference was observed between CAR and hybrid-TCR. (c) shows the memory phenotype of each cell after antigen stimulation. After antigen stimulation, the majority of MAGE CAR T cells differentiated into Tem, but in hybrid-TCR T cells, Tn and Tscm remained in parallel with Tem differentiation, and differentiation into Tcm was also observed.

[0103] Test Example 9. After antigen stimulation, spare respiratory capacity level (SRC) is reduced in MAGE CAR T cells, but maintained in MAGE hybrid-TCR (MAGE#17 hybrid-TCR). The oxygen consumption rate (OCR) was measured for effector cells before and after antigen stimulation, as shown in Figure 11. Measurements were performed using the Seahorse XF Cell Mito Stress Test Kit (Agilent) according to the measurement procedure.

[0104] The results are shown in Figure 12. Before antigen stimulation, no difference was observed in OCR measurements between CAR T cells and hybrid-TCR T cells (a). However, after antigen stimulation, the spare respiratory capacity level (SRC) of MAGE CAR T cells decreased, while that of MAGE hybrid-TCR T cells was maintained (b), suggesting that mitochondrial function was maintained.

[0105] Test Example 10. MAGE hybrid-TCR T cells have a stronger antitumor effect than CAR T cells (MAGE#17 hybrid-TCR). The experimental schedule is shown in Figure 13a. 2.5x10^6 NW-MEL-38 cells were inoculated subcutaneously into NOG mice, and 2.5x10^6 effector cells were administered via the tail vein four days later, and tumor volume was measured. Four NOG mice were used in each group.

[0106] The results are shown in Figure 13. CAR T cell administration demonstrated a tumor-suppressing effect compared to PBS(-) and NGMC administration, but tumors began to regrow from day 28 (b, c). In contrast, in the experimental group administered hybrid-TCR T cells, tumors nearly disappeared by day 14, and no tumor regrowth was observed up to day 50 (b, c). These results demonstrate that hybrid-TCR T cells have a stronger antitumor effect than CAR T cells.

[0107] Test Example 11. MAGE hybrid-TCR T cells have sufficient antitumor effect even at low doses (MAGE#17 hybrid-TCR). The experimental schedule is shown in Figure 14a. 3x10^6 NW-MEL-38 cells were inoculated subcutaneously into NOG mice, and 14 days later, 3x10^6, 1x10^6, or 3x10^5 effector cells were administered via the tail vein, and tumor volume was measured. Three NOG mice were used in each group.

[0108] The results are shown in Figure 14. Tumor disappearance was observed when 3x10^6 and 1x10^6 effector cells were administered. When 3x10^5 cells were administered, almost complete tumor suppression was observed in one mouse, and tumor regrowth occurred in the other two mice, but the growth was slow and sufficient antitumor effect was observed (b, c). These results demonstrate that hybrid-TCR T cells have sufficient antitumor effect even when administered at low doses.

[0109] Test Example 12. MAGE hybrid-TCR T cell administration resulted in more active effector cells remaining in tumors than CAR T cell administration, suppressing even large solid tumors (MAGE#17 hybrid-TCR). The experimental schedule is shown in Figure 15a. 5x10^6 NW-MEL-38 cells were inoculated subcutaneously into NOG mice, and 29 days later, 3x10^6 effector cells were administered via the tail vein. Tumor diameter was measured. Cancer tissue was collected on day 37 (8 days after effector administration) and day 43 (14 days after effector administration), and tumor-infiltrating lymphocytes (TILs) were isolated and analyzed by cell staining. Three NOG mice were used in each group.

[0110] The results are shown in Figure 15. In large solid tumors 29 days after subcutaneous inoculation, the CAR T cell-infused group showed little antitumor effect, whereas the hybrid-TCR T cell-infused group showed a significant antitumor effect (b). Furthermore, tumors containing hybrid-TCR T cells contained more CD8 and CD4 effector cells than those containing CAR T cells, and IFN-γ-positive cells were also more abundant in the hybrid-TCR T cell-infused group (c, d). Analysis of TIL memory phenotypes revealed that Tcm and Tem were more abundant in the hybrid-TCR T cell-infused group than in the CAR T cell-infused group (e). These results suggest that hybrid-TCR T cell-infused tumors retain more active effector cells than CAR T cell-infused tumors, suppressing even large solid tumors.

[0111] Test Example 13. Transduction of MAGE hybrid-TCR gene into alpha / beta and gamma / delta cells and expression analysis (MAGE#17 hybrid-TCR) Gene transduction into alpha / beta cells, culture, and antibody staining were performed as follows: 1x10^6 peripheral blood mononuclear cells from healthy individuals were cultured in a 12-well plate coated with 5 micrograms of retronectin (Takara Bio) and 1 microgram of OKT-3 (Invitrogen) in GT-T551 medium (Takara Bio) containing 0.6% autologous plasma and 300 IU / ml of IL-2 (Novartis). Retroviral gene transduction was performed 3 and 4 days later. After 10 days of culture, the cells were stained with PE-labeled MAGE-A4p230-239 / HLA-A*0201 streptavidin, APC-labeled anti-human CD4 antibody (Biolegend), and APC / Cy7-labeled anti-human CD8 antibody (Biolegend), and analyzed by flow cytometry using a Fortessa X-20 (BD). The results are shown in Figure 16a.

[0112] Gene transduction, culture, and antibody staining of gamma / delta cells were performed as follows. 1x10^6 peripheral blood mononuclear cells from healthy donors were cultured in 1.5 ml of YM-AB medium containing 0.67 micrograms / ml of PTA in a 24-well plate. One day later, they were cultured in YM-AB medium containing 25 nanograms / ml of IL-7 and 25 nanograms / ml of IL-15. Retroviral transduction was performed 3 and 4 days later. After 10 days of culture, the cells were stained with PE-labeled MAGE p230-239 / HLA-A*0201 streptavidin, APC-labeled anti-human CD4 antibody (Biolegend), APC / Cy7-labeled anti-human CD8 antibody (Biolegend), PE / Cy7-labeled anti-human Vd2 antibody (Biolegend), and PerCP / Cy5.5-labeled anti-human CD3 antibody (Biolegend). Analysis was performed by flow cytometry using a Fortessa X-20 (BD). The results are shown in Figure 16b. It can be seen that the expression of endogenous Vd2 / Vg9 is suppressed in PBMCs transfected with PRAME hybrid-TCR (b).

[0113] Test Example 14. Cytotoxicity Test of MAGE Hybrid-TCR T Cells and CAR T Cells Transfected into gamma / delta Cells (MAGE#17 Hybrid-TCR) The cytotoxicity test was performed using the N-SPC Non-RI Cytotoxicity Assay Kit (Suzuta Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. Specifically, A375 cells were used as the positive target for MAGE-A4+A2+, and HCT116 cells were used as the negative target for MAGE-A4-A2+. MAGE Hybrid-TCR T cells, CAR T cells, and NGMC were used as effector cells. The target cells were 2x10^4, and effector cells were prepared at 20x10^5, 6x10^4, and 2x10^4. First, target cells were labeled with BM-HT and washed with 10% FCS / RPMI1640. After that, the target cells were mixed with the designated effector cells and co-cultured for 2 hours. After that, EU solution was added and luminescence was measured using a TriStar2S LB942 Multimode Reader (BER THOLD).

[0114] The results are shown in Figure 17. (a) shows the cytotoxicity against the positive target A375, and (b) shows the cytotoxicity against the negative target HCT116. The MAGE hybrid-TCR generated with gamma / delta cells exhibited sufficient cytotoxicity against the positive target cells, but no cytotoxicity was observed against the negative target cells. On the other hand, the CAR T cells exhibited reduced cytotoxic activity against the positive target cells.

[0115] Test Example 15. PRAME hybrid-TCR gene can be successfully transferred into alpha / beta and gamma / delta cells (PRAME#98 hybrid-TCR) Gene transfer into alpha / beta cells, culture, and antibody staining were performed as follows: 1 x 10^6 peripheral blood mononuclear cells from healthy individuals were cultured in a 12-well plate coated with 5 micrograms of retronectin (Takara Bio) and 1 microgram of OKT-3 (Invitrogen) in GT-T551 medium (Takara Bio) containing 0.6% autologous plasma and 300 IU / mL of IL-2 (Novartis). Gene transfer was performed using the retronectin method 3 and 4 days later. After 10 days of culture, the cells were stained with PE-labeled PRAME p301-309 / HLA-A*2402 streptavidin, APC-labeled anti-human CD4 antibody (Biolegend), and APC / Cy7-labeled anti-human CD8 antibody (Biolegend), and analyzed by flow cytometry using a Fortessa X-20 (BD). The results are shown in Figure 18a.

[0116] Gene transfer, culture, and antibody staining of gamma / delta cells were performed as follows: 1x10^6 peripheral blood mononuclear cells from healthy individuals were cultured in 1.5mL of YM-AB medium containing 0.67µg / mL PTA in a 24-well plate. After one day, the cells were cultured in YM-AB medium containing 25µg / mL IL-7 (Biolegend) and 25µg / mL IL-15 (Biolegend). After 3 and 4 days, retroviral gene transfer was performed. After 10 days of culture, the cells were stained with PE-labeled PRAME p301-309 / HLA-A*2402 streptavidin, APC-labeled anti-human CD4 antibody (Biolegend), APC / Cy7-labeled anti-human CD8 antibody (Biolegend), PE / Cy7-labeled anti-human Vd2, and PerCP / Cy5.5-labeled anti-human CD3 antibody (Biolegend), and analyzed by flow cytometry using a Fortessa X-20 (BD). The results are shown in Figure 18b. It can be seen that endogenous Vd2 / Vg9 expression was suppressed in PBMCs transduced with PRAME hybrid-TCR (b).

[0117] Test Example 16. PRAME hybrid-TCR T cells and PRAME CAR T cells transfected into peripheral blood mononuclear alpha / beta cells derived from healthy volunteers exhibit sufficient proliferation (PRAME#98 hybrid-TCR). Peripheral blood mononuclear cells were isolated from two healthy volunteers (HD1, HD2), and alpha / beta cells were cultured using the same procedure as in Test Example 15. On days 4 and 5 of culture, retroviral vectors carrying hybrid-TCR and CAR genes were transfected using the retronectin method to transduce these genes. Cell numbers were counted on days 4, 5, 7, and 9 of culture, and the proliferation rate relative to the start of culture was examined. The results are shown in Figure 19. PRAME hybrid-TCR T cells, CAR T cells, and NGMC cells exhibited similar proliferation.

[0118] Test Example 17. PRAME hybrid-TCR T cells and PRAME CAR T cells transfected into peripheral blood mononuclear gamma / delta cells derived from healthy volunteers exhibit sufficient proliferation (PRAME#98 hybrid-TCR). Peripheral blood mononuclear cells were isolated from healthy volunteers, and gamma / delta cells were cultured using the procedure described in Test Example 15. PRAME hybrid-TCR was retrovirally transfected into 3 x 10^5 gamma / delta cells on Day 3, and the cells were cultured using the same procedure. Cell numbers were counted on Days 4, 6, 7, and 8 to examine the proliferation rate relative to the start of culture. The results are shown in Figure 20. PRAME hybrid-TCR T cells exhibited proliferation similar to that of NGMC cells.

[0119] Test Example 18. PRAME hybrid-TCR T cells and PRAME CAR T cells transfected into alpha / beta cells showed sufficient cytotoxicity (PRAME#98 hybrid-TCR). Cytotoxicity tests were performed using the N-SPC Non-RI Cytotoxicity Assay Kit (Suzuta Pure Chemical Industries, Ltd.) according to the manufacturer's instructions. Specifically, K562-A24 cells, SK-MEL-124 cells, and MKN-45 cells were used as positive targets for PRAME+A24+, while K562 cells and NW-MEL-38 cells were used as negative targets for PRAME+A24-. PRAME hybrid-TCR T cells, CAR T cells, and NGMC were used as effector cells. The target cells were 1 x 10^4, and effector cells were 3 x 10^4 and 1 x 10^4. First, target cells were labeled with BM-HT, washed with 10% FCS / RPMI 1640, and then mixed with the desired effector cells. After co-culture for 4 hours, EU solution was added and luminescence was measured using a TriStar2S LB942 Multimode Reader (BERTHOLD). The results are shown in Figure 21. Cytotoxicity was observed against the positive targets K562-A24 cells, SK-MEL-124 cells, and MKN-45 cells, but not against the negative targets K562 and NW-MEL-38.

[0120] Test Example 19. PRAME hybrid-TCR T cells and PRAME CAR T cells transfected into alpha / beta cells exhibited sufficient time-dependent cytotoxicity (PRAME#98 hybrid-TCR). The time-dependent cytotoxicity test was performed using xCelligence (Agilent) according to the manufacturer's instructions. Specifically, 20,000 888mel cells (A24+PRAME+) were cultured on E-plates for 12 hours, followed by the addition and culture of 20,000 effector cells prepared from alpha / beta cells from healthy donors. The cell index was monitored over time. The cell index reflects the number of 888mel cells on the E-plate. The normalized cell index was calculated by setting the number of 888mel cells immediately before co-culture with effector cells as 1. The results are shown in Figure 22. The graph shows the mean (n = 2). Cytotoxicity was observed in CAR T cells and hybrid-TCR T cells, but not in non-gene-transfected PBMCs (NGMCs).

[0121] Test Example 20. Gamma / delta effector cells demonstrated sufficient time-dependent cytotoxicity (PRAME#98 hybrid-TCR). The time-dependent cytotoxicity test was performed using xCelligence (Agilent) according to the manufacturer's instructions. Specifically, 20,000 888mel cells (A24+PRAME+) were cultured on an E-plate for 12 hours, and then 20,000 cells of each effector cell line prepared from gamma / delta cells from healthy donors were added and cultured. The cell index was monitored over time. The cell index reflects the number of 888mel cells on the E-plate. The normalized cell index was calculated by setting the number of 888mel cells immediately before co-culture with the effector cells as 1. The results are shown in Figure 23. The graph shows the average (n=2). Cytotoxicity was observed with PRAME hybrid-TCR T cells, but not with PRAME CAR T cells or non-gene-transfected PBMCs (NGMCs).

[0122] Test Example 21. PRAME hybrid-TCR T cells have a stronger antitumor effect than PRAME CAR T cells (PRAME#98 hybrid-TCR). 1x10^7 K562-A24 cells were inoculated subcutaneously into NOG mice. Nine days later, 5x10^6 effector cells were administered via the tail vein, and tumor volume was measured. Four NOG mice were used in each group. The results are shown in Figure 24. Administration of PRAME CAR T cells was more effective in suppressing tumor growth than administration of NGMC, but tumors began to regrow on day 26. In contrast, in the experimental group administered PRAME hybrid-TCR T cells, tumors nearly disappeared by day 22, and no tumor regrowth was observed up to day 33.< / vh> < / vl> < / vh> < / vl> < / vl> < / vh> < / vl> < / vh>

Claims

1. A hybrid TCR comprising: polypeptide A comprising a light chain variable region of an antibody having binding affinity to an antigen peptide-MHC complex and a first subunit constant region of a TCR; and polypeptide B comprising a heavy chain variable region of the antibody and a second subunit constant region of a TCR.

2. The hybrid TCR according to claim 1, wherein the light chain variable region and the first subunit constant region are linked directly or via a linker, and the heavy chain variable region and the second subunit constant region are linked directly or via a linker.

3. The hybrid TCR of claim 1, wherein the first subunit is an alpha chain and the second subunit is a beta chain, or the first subunit is a beta chain and the second subunit is an alpha chain.

4. The hybrid TCR of claim 1, wherein the antigenic peptide is a peptide derived from MAGE-A4 or PRAME.

5. The hybrid TCR of claim 1, wherein the dissociation constant of the antibody to an antigen peptide-MHC complex is 100 nM or less.

6. The hybrid TCR of claim 1, wherein the antigenic peptide is MAGE-A4, the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:1, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:2, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:3, and the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:4, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:5, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:6, or wherein the antigenic peptide is PRAME, the light chain variable region comprises a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:7, a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:8, and a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:9, and the heavy chain variable region comprises a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO:10, a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO:11, and a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:

12.

7. The hybrid TCR of claim 1, wherein said polypeptide A comprises a light chain variable region leader sequence, and said polypeptide B comprises a heavy chain variable region leader sequence.

8. A polynucleotide encoding a hybrid TCR according to any one of claims 1 to 7.

9. A cell containing the polynucleotide described in claim 8.

10. The cell described in claim 9, wherein the hybrid TCR is expressed on the cell membrane.

11. The cell of claim 10, which is a lymphocyte.

12. A pharmaceutical composition comprising the cells described in claim 10.

13. The pharmaceutical composition according to claim 12, for use in the treatment or prevention of cancer.

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