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WO2025047925A1 - Récepteur hybride de lymphocytes t - Google Patents

Récepteur hybride de lymphocytes t Download PDF

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Publication number
WO2025047925A1
WO2025047925A1 PCT/JP2024/031141 JP2024031141W WO2025047925A1 WO 2025047925 A1 WO2025047925 A1 WO 2025047925A1 JP 2024031141 W JP2024031141 W JP 2024031141W WO 2025047925 A1 WO2025047925 A1 WO 2025047925A1
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Prior art keywords
tcr
cells
hybrid
amino acid
seq
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Japanese (ja)
Inventor
泰 赤堀
奈緒子 今井
和宏 吉川
慶裕 宮原
美欧 劉
航平 根岸
立楠 王
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T Cell Nouveau Inc
Mie University NUC
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T Cell Nouveau Inc
Mie University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to modified T cell receptors, etc.
  • T cell infusion therapy has been developed as a minimally invasive and highly effective cancer treatment.
  • CD19 CAR T cell infusion therapy developed as a treatment for B cell lymphoma, is technological and has attracted attention for its high therapeutic effectiveness.
  • This CAR T cell therapy is a treatment that suppresses tumors by recognizing cancer cell antigens on the cell surface, and the receptor used for genetic modification is a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • Cancer is formed by cancer cells. Cancer cells are formed by the accumulation of genetic mutations in normal somatic cells, which results in cancer-specific gene expression. 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 major histocompatibility complexes. It is known that these are expressed on cancer cells and recognized by T cell receptors on T cells, resulting in tumor suppression. Therefore, genetically modified T cell therapy using TCRs with high cancer therapeutic effects is being developed. However, most of the TCRs isolated so far have low affinity, and it is not easy to isolate such TCRs with high therapeutic effects. Although it is possible to increase affinity using genetic modification technology, the cross-recognition effect is also enhanced at the same time, making it difficult to develop a safe and effective TCR therapy.
  • the present inventor attempted to develop a CAR that recognizes and treats cancer-specific pMHC complexes.
  • the present inventor developed a unique screening method using a human antibody library, isolated antibodies that recognize HLA-A2402 WT1 p235-243, HLA-A2402 PRAMEp301-308, and HLA-A0201 MAGE-A4 p230-239, and used them to create a CAR to advance the development of a cancer treatment method.
  • the tumor-suppressing effect of the developed CAR was insufficient, and the inhibition of the growth of the engrafted cancer was limited.
  • Patent Document 1 describes a fusion receptor of an antibody (including a constant region) that recognizes pMHC and a TCR. However, no sufficient antitumor effects have been reported for this immune receptor.
  • the objective of the present invention is to provide an immune receptor with a stronger tumor-suppressing effect.
  • the present inventors have conducted intensive research and have found that the above problems can be solved by a hybrid TCR comprising polypeptide A including 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 including 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. That is, the present invention encompasses the following aspects.
  • Item 1 A polypeptide A comprising a light chain variable region of an antibody having an affinity for an antigen peptide-MHC complex and a first subunit constant region of a TCR, and a polypeptide B comprising a heavy chain variable region of the antibody and a second subunit constant region of a TCR, Hybrid TCR.
  • Item 2 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.
  • Item 2. A hybrid TCR according to item 1.
  • Item 3 The hybrid TCR according to Item 1, wherein the first subunit is an ⁇ chain and the second subunit is a ⁇ chain, or the first subunit is a ⁇ chain and the second subunit is an ⁇ chain.
  • Item 4 The hybrid TCR according to Item 1, wherein the antigen peptide is a peptide derived from MAGE-A4 or PRAME.
  • Item 5 The hybrid TCR according to Item 1, wherein the dissociation constant of the antibody with respect to an antigen peptide-MHC complex is 100 nM or less.
  • the antigen 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 the antigen 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
  • the polypeptide A comprises a leader sequence of a light chain variable region
  • the polypeptide B comprises a leader sequence of a heavy chain variable region.
  • Item 2. A hybrid TCR according to item 1.
  • Item 8 A polynucleotide encoding a hybrid TCR according to any one of items 1 to 7.
  • Item 9 A cell containing the polynucleotide described in Item 8.
  • Item 10 The cell according to Item 9, wherein the hybrid TCR is expressed on the cell membrane.
  • Item 11 The cell according to Item 10, which is a lymphocyte.
  • Item 12 A pharmaceutical composition containing the cells described in Item 10.
  • Item 13 The pharmaceutical composition according to Item 12, for use in the treatment or prevention of cancer.
  • the present invention can provide an immune receptor with a stronger tumor suppression effect.
  • 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 by the 2A peptide.
  • Cb57 and Ca48 have been replaced with cysteine.
  • Ca116, 119, and 120 have been replaced with leucine. This shows the introduction of MAGE#17 hybrid-TCR and PRAME#98 hybrid-TCR into human PBMCs and confirmation of their expression (Test Example 1).
  • Human PBMCs were stimulated with retroectin (TAKARA bio) and OKT3 (Invitrogen) and cultured in GT-T551 medium (TAKARA bio) containing 0.6% human serum, after which the MAGE-A4 #17 hybrid-TCR gene was introduced using a retrovirus and cultured.
  • the cells were then stained with HLA-A0201 MAGE-A4 p230-239 tetramerized with PE-labeled streptavidin, APC-labeled anti-human CD4 antibody (Biolegend,), and PE-Cy7-labeled anti-human CD8 antibody (Biolegend,) and measured using a FACS Foltessa (BD).
  • Hybrid-TCR produces more IFNg than CAR and the production is sustained. Hybrid-TCR also shows low recognition of negative target cells (Test Example 2).
  • Effector cells and target cells were mixed and co-cultured, and the change in the amount of IFNg produced in the medium over time 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.
  • the effector cells used were non-gene-transduced T cells (NGMC), CAR T cells (CAR), hybrid-TCR-transduced T cells (Hybrid-TCR), and TCR-transduced T cells (TCR).
  • the target cells were HLA-A0201 + MAGE-A4 + SK-MEL-37 cells (a, d), HLA-A0201 + MAGE-A4 - HCT116 cells (b, e), and no target cells (c, f).
  • ELISA was performed using MabTec's human IFNg ELISA kit (MABTECH, 3420-1H-6), and the experimental method was according to the protocol.
  • Hybrid-TCR has sufficient cytotoxicity against positive target cells, but no cytotoxicity against negative target cells (Test Example 3).
  • SK-MEL-37 HLA-A0201 + MAGE-A4 + ) were cultured on an E-plate for 17 hours, and then 60,000 (a), 36,000 (b), and 12,000 (c) effector cells 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.
  • Hybrid-TCR shows sufficient cytotoxicity against positive target cells, but no cytotoxic activity against negative target cells (Test Example 3).
  • d 7,000 negative target cells HCT116 HLA-A0201 + MAGE-A4 -
  • the cell index reflects the number of SK-MEL-124 cells on the E-plate.
  • the normalized cell index was standardized by setting the number of SK-MEL-37 cells immediately before co-culture with the effector cells as 1.
  • the 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 measured using an LSRFortessa (BD). Measurements were performed on days 10, 12, 14, 16, 18, and 20.
  • the percentages of cells positive for PD-1, TIM-3, LAG-3, and CTLA-4 are shown in a, e, I, and m, respectively, and the changes in mean fluorescence intensity (MFI) for PD-1 positive cells are shown in b, f, j, and n, respectively.
  • MFI mean fluorescence intensity
  • CAR, hybrid-TCR and TCR-transduced T cells were cultured and cultured on 12-well plates immobilized with 0.5ug of HLA-A0201 MAGE-A4 p230-239 tetramer on days 10, 12 and 14 for continuous antigen stimulation, and then cultured on plates without antigen from day 16.
  • These effector cells were stimulated with PE-Cy7-labeled anti-CD8 antibody, MAGE tetramer and APC-labeled anti-PD-1 antibody (Biolegend), Percp-Cy5.5-labeled anti-PD-1 antibody, and 1.2 ⁇ g/mL of 100% 100% 15% 1 ...
  • 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.
  • the abundance ratios were calculated by classifying the cells into naive (CD62L+ CD45RO-), Tcm (CD62L+ CD45RO+), Tem (CD62L- CD45RO+), and Te (CD62- CD45RO-).
  • PBMCs transduced with CAR, hybrid-TCR or TCR were cultured and stimulated with antigen using 0.2ug solid-phase 12-well plates on days 10, 12, 14 and 16.
  • Cells were stained with MAGE-A4 tetramer, PECy7-labelled anti-CD8 antibody (Biolegend), APC-labelled anti-CD45RO antibody (Biolegend), and FITC-labelled anti-CD62L antibody (eBioscience) on days 10, 12, 14, 16 and 18 and measured using an LSRFortessa.
  • PRAME#98 hybrid-TCR introduction of PRAME#98 hybrid-TCR into human PBMC and confirmation of expression
  • Human PBMC were stimulated with retronectin (TAKARA bio) and OKT3 (Invitrogen) and cultured in GT-T551 medium (TAKARA bio) containing 0.6% human serum, after which the PRAME#98 hybrid-TCR gene was introduced using a retrovirus and cultured.
  • the cells were stained with HLA-A2402 PRAME p301-309 tetramerized with PE-labeled streptavidin, APC-labeled anti-human CD4 antibody (Biolegend,), and PE-Cy7-labeled anti-human CD8 antibody (Biolegend,) and measured using FACS Foltessa (BD).
  • Effector cells and target cells were mixed at 5x10 ⁇ 4 each and cultured in a CO2 incubator for 20 hours, and the supernatant was collected. 20 microliters of this supernatant was used to measure the amount of IFNg in the supernatant using an IFNg ELISA kit (MabTec). The measurement method followed the manufacturer's protocol. After antigen stimulation, the majority of MAGE CAR T cells differentiate into Tem, but in hybrid-TCR T cells, Tn and Tscm remain in parallel with Tem differentiation, indicating that differentiation into Tcm is also observed (Test Example 8). (a) shows the experimental schedule. (b) shows the memory phenotype of the cells before antigen stimulation. (c) shows the memory phenotype of each cell after antigen stimulation.
  • (b) shows the average value for each group, and (c) shows the measurement results for individual mice.
  • the results of in vivo antitumor activity measurements are shown (Test Example 12).
  • (a) shows the experimental schedule.
  • (b) shows the results of tumor volume measurements.
  • (c) to (e) show the results of cell staining of tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the results of measuring the cytotoxicity of alpha/beta cells into which hybrid-TCR was introduced are shown (Test Example 18).
  • the horizontal axis shows effector cells, and the lower part of each graph shows target cells.
  • the legend shows the ratio of effector cells to target cells.
  • the effector cells are shown in the legend.
  • This shows the results of measuring the cytotoxicity of alpha/beta cells into which hybrid-TCR was introduced (Test Example 20).
  • the effector cells are shown in the legend.
  • the results of in vivo antitumor activity measurements are shown (Test Example 21).
  • the legend indicates the effector cells administered.
  • Identity of amino acid sequences refers to the degree of agreement between the amino acid sequences of two or more comparable amino acid sequences. Thus, the greater the identity between two amino acid sequences, the greater the identity or similarity of those sequences.
  • the level of identity of amino acid sequences is determined, for example, using the sequence analysis tool FASTA, using 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.
  • conservative substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine is a conservative substitution.
  • amino acid residues having acidic side chains such as aspartic acid and glutamic acid
  • amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine
  • amino acid residues having non-polar 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
  • amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.
  • TCR refers to T cell receptor
  • CDR is an abbreviation for Complementarity Determining Region , and is also called complementarity determining region.
  • CDR is a region present in the variable region of immunoglobulin, and is deeply involved in specific binding of an antibody to an antigen.
  • Light chain CDR means a CDR present in the light chain variable region of immunoglobulin
  • heavy chain CDR means a CDR present in the heavy chain variable region of immunoglobulin.
  • variable region refers to a region including CDR1 to CDR3 (hereinafter simply referred to as "CDRs1-3").
  • CDRs1-3 CDR1 to CDR3
  • the order in which these CDRs1-3 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 continuously or via other amino acid sequences referred to as framework regions (FRs) described below.
  • FRs framework regions
  • heavy chain variable region refers to a region in which the above-mentioned heavy chain CDRs1-3 are arranged
  • light chain variable region refers to a region in which the above-mentioned light chain CDRs1-3 are arranged.
  • each variable region other than the above CDR1-3 are called framework regions (FR) as described above.
  • FR framework regions
  • the region between the N-terminus of the variable region and the above CDR1 is defined as FR1
  • the region between CDR1 and CDR2 as FR2
  • the region between CDR2 and CDR3 as FR3
  • the region between CDR3 and the C-terminus of the variable region as FR4.
  • hybrid TCR (sometimes referred to as "hybrid TCR of the present invention” in the present specification) 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.
  • 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 when 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 recognition epitope. It may also be a combination of multiple T cell recognition epitopes.
  • 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.
  • Particularly preferred antigens include MAGE-A4 and PRAME.
  • Particularly preferred antigen peptides include peptides derived from MAGE-A4 or PRAME. Of 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)).
  • An antigen peptide-MHC complex is a complex of an antigen peptide and MHC (major histocompatibility complex), and is not particularly limited as long as it is in a form that presents the antigen peptide on immune cells.
  • 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,
  • antigen peptide-MHC complexes include HLA-A0201 MAGE-A4p230-239 peptide-MHC complexes and HLA-A24 PRAMEp301-309 peptide-MHC complexes.
  • the antibody used in the hybrid TCR of the present invention has binding ability to an antigen peptide-MHC complex. That is, the antibody recognizes both the antigen peptide and MHC.
  • the presence or absence of said binding ability can be measured according to or in accordance with the method of Test Example 2 of WO2022/124282.
  • the ratio of the fluorescence intensity in the case of peptide pulse to the fluorescence intensity in the case of non-peptide pulse (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 particularly preferably 8 or more, it can be determined that the peptide has binding ability to the MHC complex.
  • the dissociation constant of the antibody used in the hybrid TCR of the present invention with respect to any of the antigen peptide-MHC complexes is preferably 1 ⁇ M or less, more preferably 500 nM or less, even more preferably 200 nM or less, even 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 of WO2022/124282.
  • the light chain variable region is a light chain variable region possessed by the above antibody and includes light chain CDR1, light chain CDR2, and light chain CDR3, and the heavy chain variable region is a heavy chain variable region possessed by the above antibody and includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3.
  • a portion of the light chain CDR1, light chain CDR2, light chain CDR3, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 recognizes an antigen peptide, and another portion recognizes MHC.
  • the antigen 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)
  • 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).
  • the light chain variable region is preferably a light chain variable region comprising the amino acid sequence shown 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 shown in SEQ ID NO: 13
  • the heavy chain variable region is preferably a heavy chain variable region comprising the amino acid sequence shown 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 shown in SEQ ID NO: 14.
  • the mutation is preferably an amino acid substitution, more preferably a conservative amino acid substitution.
  • the antigen 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)
  • 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).
  • the light chain variable region is preferably a light chain variable region comprising the amino acid sequence shown 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 shown in SEQ ID NO: 15, and the heavy chain variable region is preferably a heavy chain variable region comprising the amino acid sequence shown 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 shown in SEQ ID NO: 16.
  • the mutation is preferably an amino acid substitution, more preferably a conservative amino acid substitution.
  • Polypeptide A preferably contains a leader sequence of the light chain variable region.
  • Polypeptide B preferably contains a leader sequence of the heavy chain variable region. The leader sequence makes it easier to position the hybrid TCR of the present invention on the cell membrane.
  • 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.
  • the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.
  • 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.
  • the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.
  • the position of the leader sequence is not particularly limited, but is preferably located on the N-terminal side of the variable region. That is, the leader sequence of the light chain variable region is preferably located on the N-terminal side of the light chain variable region, and the leader sequence of the heavy chain variable region is preferably located on the N-terminal side of the heavy chain variable region.
  • the leader sequence and the variable region may be directly linked, or indirectly linked (e.g., via another sequence such as a linker).
  • the linker is not particularly limited, but examples thereof include a linker containing glycine alone or glycine and serine.
  • the number of amino acid residues of the linker is, for example, 1 to 30, preferably 1 to 10, and more preferably 1 to 5.
  • the leader sequence and the variable region are directly linked.
  • TCR is a heterodimer consisting of a first subunit and a second subunit.
  • subunits that constitute TCR include ⁇ chain, ⁇ chain, ⁇ chain, and ⁇ chain.
  • the first subunit and the second subunit in the hybrid TCR of the present invention are not particularly limited as long as they are a combination that constitutes TCR as a heterodimer, and examples include a combination in which the first subunit is an ⁇ chain and the second subunit is a ⁇ chain, a combination in which the first subunit is a ⁇ chain and the second subunit is an ⁇ chain, a combination in which the first subunit is a ⁇ chain and the second subunit is a ⁇ chain, and a combination in which the first subunit is a ⁇ chain and the second subunit is a ⁇ chain.
  • particularly preferred are a combination in which the first subunit is an ⁇ chain and the second subunit is a ⁇ chain, and a combination in which the first subunit is a ⁇ chain and
  • the constant region of the first/second subunit is a portion other than the variable region of the first/second subunit (a region that includes CDRs and whose amino acid sequence differs depending on the type of antigen), and is not particularly limited as long as it has the function of the constant region of the first/second subunit (e.g., the function of penetrating a cell membrane and positioning the variable region of the first/second subunit in an appropriate orientation on the cell surface).
  • the constant region of the ⁇ chain 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.
  • the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.
  • the constant region of the ⁇ chain 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.
  • the mutation is preferably an amino acid substitution, and more preferably a conservative amino acid substitution.
  • the light chain variable region is usually located on the N-terminal side of the first subunit constant region.
  • the heavy chain variable region is usually located on the N-terminal side of the second subunit constant region.
  • 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 directly linked.
  • 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 directly linked.
  • the linker is the same as the linker that can link the leader sequence and the variable region described above.
  • Polypeptides A/B may 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, 4-1BBL domain, ICOSL domain, etc.)), or in one embodiment, may not contain such sequences/regions.
  • 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, 4-1BBL domain, ICOSL domain, etc.)
  • a costimulatory factor
  • 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 polypeptides A/B 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.
  • 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, even more preferably 0 to 40, particularly preferably 0 to 30, and particularly preferably 0 to 25.
  • the hybrid TCR of the present invention may be chemically modified.
  • the C-terminus of the polypeptide constituting the hybrid TCR of the present invention may be any of a carboxyl group (-COOH), a carboxylate ( -COO- ), an amide ( -CONH2 ) or an ester (-COOR).
  • examples of R in the ester include C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl; C3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; C6-12 aryl groups such as phenyl and ⁇ -naphthyl; C1-2 phenyl- C1-2 alkyl groups such as benzyl and phenethyl; C7-14 aralkyl groups such as ⁇ -naphthyl- C1-2 alkyl groups such as ⁇ -naphthylmethyl; and pivaloyloxymethyl groups.
  • C1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl, and n-butyl
  • C3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl
  • C6-12 aryl groups such
  • the carboxyl group (or carboxylate) of the polypeptide constituting the hybrid TCR of the present invention other than the C-terminus may be amidated or esterified.
  • examples of the esters that can be used include the C-terminus esters described above.
  • the polypeptides constituting the hybrid TCRs of the present invention also include those in which the amino group of the N-terminal amino acid residue is protected with a protecting group (e.g., a C1-6 acyl group, such as a formyl group, a C1-6 alkanoyl group such as an acetyl group), those in which the N-terminal glutamine residue that may be generated by cleavage in the body is pyroglutamylated, and those in which substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino groups, imidazole groups, indole groups, guanidino groups, etc.) are protected with an appropriate protecting group (e.g., a C1-6 acyl group, such as a formyl group, a C1-6 alkanoyl group such as an acetyl group).
  • a protecting group e.g., a C1-6 acyl group, such as a formyl group
  • the hybrid TCR of the present invention may have a known protein tag, signal sequence, or other protein or peptide added thereto.
  • protein tags include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, and fluorescent protein tag.
  • the hybrid TCR of the present invention may be in the form of a pharma- ceutically acceptable salt with an acid or base.
  • the salt is not particularly limited as long as it is a pharma- ceutically acceptable salt, and either an acid salt or a basic salt can be used.
  • acid 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; amino acid salts such as aspartate and glutamate; and the like.
  • 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.
  • the hybrid TCR of the present invention may be in the form of a solvate.
  • the solvent is not particularly limited as long as it is pharma- ceutically acceptable, and examples of the solvent include water, ethanol, glycerol, acetic acid, etc.
  • the present invention relates to a polynucleotide (sometimes referred to as the "polynucleotide of the present invention” in this specification) encoding the hybrid TCR of the present invention. This will be described below.
  • the polynucleotide of the present invention may contain other sequences in addition to the coding sequence of the hybrid TCR of the present invention.
  • the polynucleotide of the present invention preferably contains the hybrid TCR of the present invention in an expressible state. Examples of other sequences include a promoter sequence, an enhancer sequence, a repressor sequence, an insulator sequence, a replication origin, a reporter protein (e.g., a fluorescent protein, etc.) coding sequence, and a drug resistance gene coding sequence.
  • 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 virus vector (e.g., an adenovirus or a retrovirus).
  • the vector may be, for example, a cloning vector or an expression vector.
  • expression vectors include vectors for prokaryotic cells such as Escherichia coli or actinomycetes, or vectors for eukaryotic cells such as yeast cells, insect cells, or mammalian cells.
  • the polynucleotide of the present invention includes not only DNA and RNA, but also those which have been subjected to known chemical modifications, as exemplified below.
  • the phosphoric acid residue (phosphate) of each nucleotide can be replaced with a chemically modified phosphoric acid residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate.
  • PS phosphorothioate
  • methylphosphonate methylphosphonate
  • phosphorodithioate phosphorodithioate
  • the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide can be replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CH2OCH3 ( 2' -O-MOE), CH2CH2NHC (NH) NH2 , CH2CONHCH3 , CH2CH2CN , etc.).
  • R represents, for example, CH3(2'-O-Me), CH2CH2OCH3 ( 2' -O-MOE), CH2CH2NHC (NH) NH2 , CH2CONHCH3 , CH2CH2CN , etc.
  • the base portion pyrimidine, purine
  • polynucleotide includes not only natural nucleic acids but also bridged nucleic acids (BNAs), locked nucleic acids (LNAs), peptide nucleic acids (PNAs), etc.
  • 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 (e.g., a polynucleotide encoding polypeptide A and a polynucleotide encoding polypeptide B). From the viewpoint of the expression efficiency of the hybrid TCR, one type of polynucleotide encoding polypeptide A and polypeptide B is preferred. In this preferred embodiment, it is preferred that the coding sequence of polypeptide A and the coding sequence of polypeptide B are linked via a coding sequence of a self-cleaving peptide.
  • self-cleaving peptide refers to a peptide sequence with cleavage activity occurring between two amino acid residues within the peptide sequence itself.
  • self-cleaving peptides include 2A peptides or 2A-like peptides.
  • cleavage occurs between glycine and proline residues on these peptides. This occurs due to a "ribosomal skip 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 skip mechanism is known in the art and is used for the expression of multiple proteins encoded by one messenger RNA (mRNA).
  • the self-cleaving peptide used in the present invention can be obtained from a viral 2A peptide or a functionally equivalent 2A-like peptide.
  • it can 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 appropriately mutated as long as its activity is not significantly impaired.
  • the present invention relates to a cell (sometimes referred to as the "cell of the present invention” in this specification) containing the polynucleotide of the present invention. This will be described below.
  • the cells of origin of the cells of the present invention are not particularly limited. If the purpose of using the cells of the present invention is to produce the hybrid TCR of the present invention, the cells of origin include cells that can be used for protein expression (e.g., insect cells, eukaryotic cells, mammalian cells, etc.).
  • the cell is preferably a lymphocyte cell (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 ⁇ -T cell, a ⁇ -T cell, etc.), a NK cell, a NKT cell, etc.).
  • the lymphocyte cell is preferably a cell that expresses the hybrid TCR of the present invention, and in a more specific embodiment, the lymphocyte cell expresses the hybrid TCR of the present invention on the cell membrane.
  • Lymphocyte cells expressing hybrid TCR recognize the antigen peptide-MHC complex with the antibody variable region, then transmit the recognition signal to the inside of T cells, etc., and activate a signal that induces cytotoxic activity, which enables the cell to attack or exert cytotoxic activity against other cells or tissues expressing the antigen.
  • lymphocytes specifically recognize cancer tissue (tumor tissue), they are useful for treating or preventing cancer.
  • types of cancer include blood cancer and solid cancer.
  • blood cancer include various B-cell malignant lymphomas (B-cell acute lymphocytic 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.
  • B-cell malignant lymphomas B-cell acute lymphocytic leukemia, follicular lymphoma, diffuse lymphoma, mantle cell lymphoma, MALT lymphoma, intravascular B
  • solid cancers include lung cancer, colon cancer, ovarian cancer, breast cancer, brain tumors, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, melanoma, neuroblastoma, bladder cancer, etc.
  • 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.
  • a specific marker CD antigen such as CD8
  • cells expressing hybrid TCRs have enhanced cytokine production and are highly cytotoxic. They also have increased sensitivity to antigen recognition and are slower to become exhausted. In in vivo antitumor tests using NOG mice, the tumor disappeared quickly and the effect was sustained.
  • the present invention relates to a pharmaceutical composition (sometimes referred to as the "pharmaceutical composition of the present invention” in this specification) containing the cell of the present invention in which the hybrid TCR of the present invention is expressed on the cell membrane. This will be described below.
  • the content of the above-mentioned cells in the pharmaceutical composition can be appropriately set 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.
  • the content of the antibody in the pharmaceutical composition can be about 0.001 to 10 parts by weight, assuming that the entire 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.
  • 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 administration or parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration, rectal administration, transdermal administration, topical administration). Since the active ingredient is cells, the preferred administration form is parenteral administration, and more preferably intravenous injection. Dosage forms for oral and parenteral administration and methods for their production are well known to those skilled in the art, and can be produced in accordance with conventional methods by mixing the antibody or cells according to the present invention with a pharma- ceutically acceptable carrier, etc.
  • Dosage forms for parenteral administration include injectable preparations (e.g., drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), topical preparations (e.g., ointments, poultices, lotions), suppositories, inhalants, eye preparations, eye ointments, nasal drops, ear drops, liposomes, etc.
  • injectable preparations are prepared by dissolving or suspending antibodies or cells in distilled water for injection, and solubilizing agents, buffers, pH adjusters, isotonicity agents, soothing agents, preservatives, stabilizers, etc. can be added as necessary.
  • the pharmaceutical composition can also be a lyophilized preparation for preparation immediately before use.
  • the pharmaceutical composition may further contain other drugs that are effective in the diagnosis, treatment, or prevention of disease.
  • the pharmaceutical composition may also contain ingredients such as bactericides, anti-inflammatory agents, cell activators, vitamins, and amino acids, as necessary.
  • Carriers used in the formulation of pharmaceutical compositions include excipients, binders, disintegrants, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, solubilizers, soothing agents, etc., that are commonly used in the relevant technical field.
  • 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.
  • Blood cancers include, for example, 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 diseases, myelodysplastic/myeloproliferative neoplasms (CMML, JMML, CML, MDS/MPN-UC), myelodysplastic syndromes, acute myeloid leukemia, multiple myeloma, etc.
  • B-cell malignant lymphomas B-cell acute lymphoblastic leukemia, f
  • solid cancers include lung cancer, colon cancer, ovarian cancer, breast cancer, brain tumors, stomach cancer, liver cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, melanoma, neuroblastoma, and bladder cancer.
  • 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. "May be afflicted” can be determined by a known diagnostic method.
  • the animal is, for example, a mammal, preferably a human.
  • the dosage of the 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 age, sex, and weight of the patient, the severity of the disease, pharmacological knowledge such as pharmacokinetic and toxicological characteristics, whether or not a drug delivery system is used, and whether or not the pharmaceutical composition is administered as part of a combination of other drugs.
  • the dosage of the pharmaceutical composition can be approximately 1 microgram/kg (body weight) to 10 g/kg (body weight) per day.
  • 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 the same factors as the dosage. For example, the above daily dosage can be administered once a day to once a month.
  • Hybrid-TCR structure and expression on PBMC As shown in Figure 1, hybrid-TCR is a gene-constructed receptor introduced into T cells and expressed, which recognizes cancer-specific peptide-MHC complexes (pMHC complexes) on cancer cells and exerts cytotoxicity against cancer cells. This suppresses cancer in the body and enables cancer treatment.
  • pMHC complexes cancer-specific peptide-MHC complexes
  • the hybrid-TCR created this time is of the retroviral expression type, but lentiviral and transposon expression is also possible ( Figure 2).
  • the antibody recognition sites VH and VL are bound to TCR Cb and TCR Ca, respectively, and the 2A peptide produces a heterodimer to form a CD3 complex.
  • Cb57 and Ca48 were replaced with cysteine. This is thought to form S-S bonds between the introduced heterodimer molecules, stabilizing the structure.
  • Ca116, 119, and 120 were replaced with leucine. This is thought to increase the frequency of intermolecular binding of the introduced heterodimer molecules.
  • VH and VL domains which are the antigen recognition portions 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, to construct hybrid TCRs by binding them to TCR Cb and TCR Ca.
  • the nucleotide and amino acid sequences of the hybrid TCR using MAGE#17 and the hybrid TCR using PRAME#98 are shown below.
  • VH leader/VH/TCR/P2A/VL leader/VL/TCR are linked in this order.
  • Cb57 and Ca48 were replaced with Cysteine.
  • Ca116, 119, and 120 were replaced with Leucine.
  • the CDR sequence is underlined.
  • VH leader/VH/TCR/P2A/VL leader/VL/TCR are linked in this order.
  • Cb57 and Ca48 were replaced with Cysteine.
  • Ca116, 119, and 120 were replaced with Leucine.
  • the CDR sequence is underlined.
  • Retroviral vectors for MAGE#17 hybrid TCR and PRAME#98 hybrid TCR were constructed and then introduced into human PBMCs for expression (Fig. 3a, b, c, d, e, f, g, h).
  • Test Example 2 Hybrid-TCR-introduced T cells enhance cytokine production compared to CAR-introduced T cells.
  • SK-MEL-37 cells HLA-A0201 + MAGE-A4 + ), a positive target, were co-cultured with effector cells, and the change over time in the amount of IFNg produced in the medium was measured by ELISA.
  • Hybrid-TCR-introduced T cells have enhanced cytotoxicity compared to CAR-introduced T cells.
  • SK-MEL-37 cells HLA-A0201+ MAGE-A4+
  • effector cells were co-cultured with effector cells, and the change over time in current value due to cytotoxic activity was measured using xCelligence.
  • Test Example 4 Hybrid-TCR-introduced T cells show slower progression of exhaustion than CAR-introduced T cells and 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.
  • Hybrid-TCR cultured without stimulation has a higher ratio of T naive (Tn), T central memory (Tcm), and T effector memory (Tem) compared to CAR and TCR.
  • Tn T naive
  • Tcm T central memory
  • Tem T effector memory
  • the ratio of memory types is almost the same for hybrid-TCR and TCR, which is a clear difference in ratio compared to CAR, which has less Tn and more T effector (Te).
  • Te increases significantly in MAGE#17 CAR and MAGE TCR, and the ratio of Tn decreases significantly.
  • the ratio of Tcm and Tem is low.
  • Test Example 6 Tumors disappeared in the treatment group infused with hybrid-TCR-introduced T cells, and this phenomenon was maintained. NW-MEL-38 cells were borne, and on day 4, effector cells were borne on day 11 for each of the five experimental groups: PBMC, NGMC, MAGE#17 CAR, MAGE#17 hybrid-TCR, and MAGE TCR, and the antitumor effect was examined (Figure 8 a). Tumor suppression did not occur in the PBS and NGMC groups ( Figure 8 b, c, d). In contrast, tumor growth was suppressed in the MAGE#17 CAR-infused group, but the effect was limited, and tumor suppression was insufficient after day 28 after tumor bearing ( Figure 8 b, e).
  • Test Example 7 A hybrid-TCR with enhanced effector function could be produced for the PRAME#98 antibody that recognizes PRAME.
  • This PRAME#98 hybrid-TCR recognized K562 A2402, which is HLA-A2402+ PRAME+, but did not recognize K562, which is A2402- PRAME+.
  • IFNg production was enhanced and effector function was enhanced compared to PRAME#98 CAR, which recognizes in the same way ( Figure 10).
  • 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 microgram neutravidin/0.2 ml PBS at 4°C for one day to immobilize the antigen, then washed with PBS, and reacted with 2 microgram MAGE-A4/HLA-A*0201 monomer/0.2 ml PBS overnight at 4°C to immobilize the antigen, then washed with PBS, and used as the antigen plate.
  • FIG. 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 300IU/ml human recombinant IL-2 (Novartis) and 0.6% autologous plasma.
  • the cells were cultured for 48 hours and then harvested, and the cell count was adjusted to 5x10 ⁇ 5 for the next antigen stimulation. After a total of four antigen stimulations, cell staining and seahorse assay were performed.
  • BD Fortessa X-20
  • the antibodies used were as follows: 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), BV711-labeled anti-human CD45RA antibody (clone HI100, Biolegend).
  • Test Example 9 After antigen stimulation, the spare respiratory capacity level (SRC) of MAGE CAR T cells is reduced, but that of MAGE hybrid-TCR is maintained (MAGE#17 hybrid-TCR)
  • SRC spare respiratory capacity level
  • OCR oxygen consumption rate
  • Test Example 10 MAGE hybrid-TCR T cells have stronger antitumor effects 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 4 days later, and the tumor volume was measured.
  • Four NOG mice were used in each group.
  • 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, and 3x10 ⁇ 5 effector cells were administered via the tail vein, and the tumor volume was measured. Three NOG mice were used in each group.
  • Test Example 12 MAGE hybrid-TCR T cell administration resulted in more active effector cells remaining in the tumor than CAR T cell administration, and even large solid tumors were suppressed (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, and tumor diameter was measured. On the 37th day (8 days after effector administration) and the 43rd day (14 days after effector administration), cancer tissues were collected, and tumor infiltrating lymphocytes (TIL) were isolated and analyzed by cell staining. Three NOG mice were used in each group.
  • Test Example 13 Introduction of MAGE hybrid-TCR gene into alpha/beta and gamma/delta cells and expression analysis (MAGE#17 hybrid-TCR) Gene transfer, culture and antibody staining into alpha/beta cells were performed as follows. 1x10 ⁇ 6 healthy peripheral blood mononuclear cells were cultured in a 12-well plate immobilized with 5 micrograms of retronectin (Takara bio) and 1 microgram of OKT-3 (Invitrogen), and then cultured in GT-T551 medium (Takara bio) containing 0.6% autologous plasma and 300 IU/ml of IL-2 (Novartis). After 3 and 4 days, gene transfer was performed using retrovirus.
  • Gene transfer, culture and antibody staining into 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 0.67 microgram/ml PTA in a 24-well plate, and one day later were cultured in YM-AB medium containing 25 nanogram/ml IL7 and 25 nanogram/ml IL15, and gene transfer was performed using a retrovirus 3 and 4 days later.
  • 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 Chemicals), and the experimental method was according to the manufacturer's manual. That is, A375 cells were prepared as a positive target for MAGE-A4+A2+, and HCT116 cells were prepared as a negative target for MAGE-A4-A2+, and MAGE hybrid-TCR T cells, CAR T cells, and NGMC were prepared as effector cells. The target cells were 2x10 ⁇ 4, and the effector cells were 20x10 ⁇ 5, 6x10 ⁇ 4, and 2x10 ⁇ 4.
  • the target cells were labeled with BM-HT, washed with 10% FCS/RPMI1640, mixed with the designated effector cells, and co-cultured for 2 hours. EU solution was then mixed, and luminescence was measured using a TriStar2S LB942 Multimode Reader (BER THOLD).
  • PRAME hybrid-TCR gene can be successfully introduced into alpha/beta and gamma/delta cells (PRAME#98 hybrid-TCR)
  • Gene transfer, culture, and antibody staining into alpha/beta cells were performed as follows. 1x10 ⁇ 6 peripheral blood mononuclear cells from healthy individuals were cultured in a 12-well plate immobilized with 5 micrograms of retronectin (Takara bio) and 1 microgram of OKT-3 (Invitrogen), and then cultured in GT-T551 medium (Takara bio) containing 0.6% autologous plasma and 300 IU/mL of IL-2 (Novartis). Gene transfer was performed by the retronectin method 3 and 4 days later.
  • Gene transfer, culture and antibody staining into gamma/delta cells were performed as follows: 1x10 ⁇ 6 peripheral blood mononuclear cells from healthy individuals were cultured in 24-well plates in 1.5mL of YM-AB medium containing 0.67 microgram/mL PTA, and one day later they were cultured in YM-AB medium containing 25 nanogram/mL IL-7 (Biolegend) and 25 nanogram/mL IL-15 (Biolegend), and gene transfer was performed using a retrovirus 3 and 4 days later.
  • 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 show 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, the cells were infected with retroviral vectors carrying hybrid-TCR and CAR genes using the retronectin method, and these genes were introduced. The number of cells was 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 showed almost similar proliferation.
  • 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 show sufficient proliferation (PRAME#98 hybrid-TCR) Peripheral blood mononuclear cells were isolated from healthy volunteers, and gamma/delta cells were cultured using the procedure in Test Example 15. Using 3x10 ⁇ 5 gamma/delta cells on day 3, PRAME hybrid-TCR was introduced by retroviral method, and cultured using the same procedure. The number of cells was counted on days 4, 6, 7, and 8, and the proliferation rate compared to the start of culture was examined. The results are shown in Figure 20. PRAME hybrid-TCR T cells showed almost the same proliferation as NGMC cells.
  • 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)
  • the cytotoxicity test was performed using the N-SPC Non-RI Cytotoxicity Assay Kit (Suzuta Pure Chemical Industries, Ltd.), and the experimental method followed the manufacturer's manual. That is, K562-A24 cells, SK-MEL-124 cells, and MKN-45 cells were prepared as positive targets for PRAME+A24+, and K562 cells and NW-MEL-38 cells were prepared as negative targets for PRAME+A24-, and PRAME hybrid-TCR T cells, CAR T cells, and NGMC were prepared as effector cells.
  • the target cells were 1 ⁇ 10 ⁇ 4, and the effector cells were 3 ⁇ 10 ⁇ 4 and 1 ⁇ 10 ⁇ 4.
  • the target cells were labeled with BM-HT, washed with 10% FCS/RPMI1640, mixed with the designated effector cells, and co-cultured for 4 hours, after which EU solution was mixed and luminescence was measured using a TriStar2S LB942 Multimode Reader (BERTHOLD).
  • BERTHOLD TriStar2S LB942 Multimode Reader
  • Test Example 19 PRAME hybrid-TCR T cells and PRAME CAR T cells transfected with alpha/beta cells showed sufficient time-dependent cytotoxicity (PRAME#98 hybrid-TCR)
  • Test Example 20 gamma/delta effector cells showed sufficient time-dependent cytotoxicity (PRAME#98 hybrid-TCR)
  • the time-dependent cytotoxicity test was performed using xCelligence (Agilent) according to the manufacturer's instructions. That is, 20,000 888mel cells (A24+PRAME+) were cultured on an E-plate for 12 hours, and then 20,000 effector cells made from healthy gamma/delta cells were added and cultured, and the cell index was tracked over time.
  • the cell index reflects the number of 888mel cells on the E-plate.
  • the normalized cell index was standardized to the number of 888mel cells immediately before co-culture with the effector cells, which was set to 1.
  • the results are shown in Figure 23.
  • PRAME hybrid-TCR T cells have stronger antitumor effects than PRAME CAR T cells (PRAME#98 hybrid-TCR) 1x10 ⁇ 7 K562-A24 cells were inoculated subcutaneously into NOG mice, and 9 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.
  • PRAME CAR T cell administration showed a tumor growth inhibitory effect compared to NGMC administration, but tumors regrown from day 26. In contrast, in the experimental group administered PRAME hybrid-TCR T cells, tumors almost disappeared by day 22, and tumor regrowth was not observed until day 33.

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Abstract

L'invention concerne un récepteur immunitaire ayant une action inhibitrice de tumeur supérieure. Ce TCR hybride comprend un polypeptide A qui comprend une région variable de chaîne légère d'un anticorps ayant une capacité de liaison par rapport à un complexe peptide antigène-CMH et une première région constante de sous-unité d'un TCR, et un polypeptide B qui comprend une région variable de chaîne lourde de l'anticorps susmentionné et une seconde région constante de sous-unité d'un TCR.
PCT/JP2024/031141 2023-08-31 2024-08-30 Récepteur hybride de lymphocytes t Pending WO2025047925A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018225732A1 (fr) * 2017-06-05 2018-12-13 国立大学法人三重大学 Protéine de liaison à l'antigène reconnaissant un peptide dérivé d'un mage-a4
JP2019500848A (ja) * 2015-10-23 2019-01-17 ユーリカ セラピューティックス， インコーポレイテッド 抗体／ｔ細胞受容体キメラ構築物およびその使用
WO2021020559A1 (fr) * 2019-08-01 2021-02-04 国立大学法人三重大学 Récepteur d'antigène
WO2022124282A1 (fr) * 2020-12-10 2022-06-16 国立大学法人三重大学 Molécule de liaison à prame
JP2022532557A (ja) * 2019-05-08 2022-07-15 メディジーン イミュノテラピーズ ゲーエムベーハー 操作されたｔ細胞

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019500848A (ja) * 2015-10-23 2019-01-17 ユーリカ セラピューティックス， インコーポレイテッド 抗体／ｔ細胞受容体キメラ構築物およびその使用
WO2018225732A1 (fr) * 2017-06-05 2018-12-13 国立大学法人三重大学 Protéine de liaison à l'antigène reconnaissant un peptide dérivé d'un mage-a4
JP2022532557A (ja) * 2019-05-08 2022-07-15 メディジーン イミュノテラピーズ ゲーエムベーハー 操作されたｔ細胞
WO2021020559A1 (fr) * 2019-08-01 2021-02-04 国立大学法人三重大学 Récepteur d'antigène
WO2022124282A1 (fr) * 2020-12-10 2022-06-16 国立大学法人三重大学 Molécule de liaison à prame

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