T cell receptor and use thereof

Abstract

Provided is an HLA-independent T cell receptor. This major histocompatibility complex class I-related molecule 1 (MR1)-restricted T cell receptor (TCR) comprises a TCRα chain that includes a CDR3 having a specific amino acid sequence and is encoded by a specific nucleotide sequence and a TCRβ chain that includes a CDR3 having a specific amino acid sequence and is encoded by a specific nucleotide sequence. This TCR specifically recognizes an antigen expressed in breast cancer cells.

Description

T cell receptors and their uses

[0001] This disclosure relates to T cell receptors.

[0002] T cells play a central role in cellular immunity in animals, including humans. T cell receptors (TCRs) expressed on the surface of T cells mediate the recognition and binding of antigens. Conventionally, TCR-T cell therapy has been attempted to treat cancer by expressing TCRs that respond to cancer cells in patient T cells and administering them to cancer patients. As T cell receptors, for example, Non-Patent Documents 1 and 2 disclose T cell receptors that are restricted by Major Histocompatibility Complex (MHC) class I-like related molecule 1 (MR1).

[0003] Rapoport, AP et al. Nat Med. 2015 August; 21(8): 914-921.Chapuis, AG et al. Nat Med. 2019 July; 25(7): 1064-1072.

[0004] Conventional TCR-T cell therapies use TCRs that respond to cancer cells in a way that depends on human leukocyte antigen (HLA) class I, which is a major histocompatibility complex (MHC) in humans. HLA class I is a highly polymorphic molecule, and it is known that each individual possesses different alleles. Therefore, TCR-T cell therapies using specific HLA-dependent TCRs could not be applied to patients who did not possess the allele of that HLA. For this reason, HLA-independent T cell receptors are desired in order to expand the range of applications for immunotherapy.

[0005] This disclosure can be implemented in the following forms:

[0006] (1) According to one embodiment of the present disclosure, a T cell receptor is provided. This T cell receptor is a T cell receptor that is restricted by major histocompatibility class I-like related molecule 1 and comprises any one of the following (a) to (d), and specifically recognizes an antigen expressed on breast cancer cells: (a) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and a TCRα chain encoded by the nucleotide sequence shown in SEQ ID NO: 11 or a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 11 or a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 11, and, (b) A TCRβ chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 2, and the TCRβ chain is encoded by a nucleotide sequence shown in SEQ ID NO: 12 or a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 12 or a nucleotide sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 12, (b) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 3, and the TCRα chain is encoded by a nucleotide sequence shown in SEQ ID NO: 13 or a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 13 or a nucleotide sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 13, and(c) A TCRβ chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 4, and the TCRβ chain is encoded by a nucleotide sequence shown in SEQ ID NO: 14 or an amino acid sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 14 or an amino acid sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 14, (c) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 5, and the TCRα chain is encoded by a nucleotide sequence shown in SEQ ID NO: 15 or an amino acid sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 15 or an amino acid sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 15, and A TCRβ chain comprising CDR3 having the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 6, and a TCRβ chain encoded by the base sequence shown in SEQ ID NO: 16, or a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 16, or a base sequence in which one or more bases are deleted, substituted, or added to the base sequence shown in SEQ ID NO: 16.(d) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 7, and the TCRα chain is encoded by a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 17 or an amino acid sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 17; and a TCRβ chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 8, and the TCRβ chain is encoded by a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 18 or an amino acid sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 18. This form of T cell receptor is HLA-independent.

[0007] (2) The T cell receptor described in (1) above may include any of (a) to (c) above.

[0008] (3) In the T cell receptor described in (1) or (2) above, the breast cancer cells may be MCF7 or MDA-MB-231.

[0009] (4) A polynucleotide is provided which encodes the T cell receptor described in any one of the above items (1) to (3).

[0010] (5) A vector containing the polynucleotide described in (4) above is provided.

[0011] (6) Cells expressing the T cell receptor described in any one of the above items (1) to (3) are provided.

[0012] (7) The cells described in (6) above may lack the expression of endogenous T cell receptors.

[0013] (8) According to another aspect of the present disclosure, a prophylactic or therapeutic agent for breast cancer is provided. The prophylactic or therapeutic agent for breast cancer contains the T cell receptor according to any one of (1) to (3) above, or the polynucleotide according to (4) above, or the vector according to (5) above, or the cell according to (6) or (7) above.

[0014] Note that the present disclosure can be realized in various forms. For example, it can be realized in the form of a pharmaceutical composition for preventing or treating breast cancer containing the above-mentioned T cell receptor, polynucleotide, vector or cell as an active ingredient, or a method for preventing or treating breast cancer including the step of administering any one of the above-mentioned T cell receptor, polynucleotide, vector or cell to a patient.

[0015] Explanatory diagrams showing the results of hMR1 tetramer staining. Histograms showing the surface expression of HLA class I-like molecules. Histograms showing the increase in MR1 surface expression in MCF7 cells. Histograms showing the expression of MR1 in various cell lines. Histograms showing the expression of HLA-E and CD1 molecules in various cell lines. Explanatory diagrams showing the reactivity when expressed in mouse T cells and human PBMCs. Explanatory diagrams showing the reactivity against MCF7 cells in which each factor was knocked out. Explanatory diagrams showing the reactivity against MCF7 cells in which each factor was knocked out. Explanatory diagrams showing the cytotoxicity against MCF7 cells. Explanatory diagrams showing the reactivity against various cancer cell lines. Explanatory diagrams showing the amount of IL2 production depending on the presence or absence of an MR1 ligand. Histograms showing the surface expression of MR1 in MCF7 cells. Explanatory diagrams showing the amount of IL2 production. Explanatory diagrams showing the reactivity of TCR against breast cancer cells and normal breast cells. Explanatory diagrams showing the protocol of Experiment 2. Explanatory diagrams showing the imaging of luciferase-expressing cells in mice. Explanatory diagrams showing the measurement results of the number of tumor cells in mice.

[0016] As shown in the examples described later, the inventors of this invention identified HLA class I-independent TCRs from CD8-positive tumor-infiltrating lymphocytes (TILs) of two breast cancer patients. When these were expressed in BW T cells, it was confirmed that they responded to breast cancer cells independently of HLA and exhibited cytotoxic activity, thus leading to the completion of the present invention.

[0017] According to one embodiment of the present disclosure, a T cell receptor (TCR) that is restricted by major histocompatibility gene (MHC) class I-like related molecule 1 (MR1) is provided, comprising any one of the following (a) to (d), and specifically recognizing an antigen expressed on breast cancer cells: (a) A TCRα chain comprising CDR3 having the amino acid sequence shown in SEQ ID NO: 1 (CAAIDSNYQLIW) or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, wherein the TCRα chain is encoded by the nucleotide sequence shown in SEQ ID NO: 11 or a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 11, or a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 11, and Furthermore, a TCRβ chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 2 (CASKERSGSGDGEQYF), or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 2, and a TCRβ chain encoded by the base sequence shown in SEQ ID NO: 12, or a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 12, or a base sequence in which one or several bases are deleted, substituted, or added to the base sequence shown in SEQ ID NO: 12,(b) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 3 (CALPSRLMF), or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 3, and a TCRα chain encoded by the base sequence shown in SEQ ID NO: 13, or a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 13, or a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 13, and Furthermore, a TCRβ chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 4 (CASSLTSIYEQFF), or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence in which one or several amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 4, and a TCRβ chain encoded by the base sequence shown in SEQ ID NO: 14, or a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 14, or a base sequence in which one or several bases are deleted, substituted, or added to the base sequence shown in SEQ ID NO: 14,(c) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 5 (CIVRVGPGYGGSQGNLIF), or an amino acid sequence that is 95% or more identical to the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 5, wherein the TCRα is encoded by the base sequence shown in SEQ ID NO: 15, or a base sequence that is 90% or more identical to the base sequence shown in SEQ ID NO: 15, or a base sequence in which one or more bases are deleted, substituted, or added to the base sequence shown in SEQ ID NO: 15. A TCRβ chain comprising a chain and a CDR3 having the amino acid sequence shown in SEQ ID NO: 6 (CASSFYGSETQYF) or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 6, and a TCRβ chain encoded by the base sequence shown in SEQ ID NO: 16 or a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 16, or a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 16,(d) A TCR α-chain comprising a CDR3 having the amino acid sequence shown by SEQ ID NO: 7 (CAASMGNTPLVF), or an amino acid sequence having 95% or more identity with the amino acid sequence shown by SEQ ID NO: 7, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 7, and being encoded by a base sequence shown by SEQ ID NO: 17, or a base sequence having 90% or more identity with the base sequence shown by SEQ ID NO: 17, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown by SEQ ID NO: 17; and a TCR β-chain comprising a CDR3 having the amino acid sequence shown by SEQ ID NO: 8 (CSARVEKLFF), or an amino acid sequence having 95% or more identity with the amino acid sequence shown by SEQ ID NO: 8, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown by SEQ ID NO: 8, and being encoded by a base sequence shown by SEQ ID NO: 18, or a base sequence having 90% or more identity with the base sequence shown by SEQ ID NO: 18, or a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown by SEQ ID NO: 18.

[0018] In the present disclosure, "CDR" means Complementarity Determining Region. CDRs (CDR1, CDR2, CDR3) are present in the variable region of TCR and are involved in specific binding to antigens restricted by MHC. Note that the α-β heterodimer TCR has a TCR α-chain and a TCR β-chain. Therefore, the TCR α-chain and the TCR β-chain each contain a CDR. The CDRs in the present disclosure can be identified by analysis using the software provided by IMGT (IMGT is a registered trademark).

[0019] In this disclosure, "identity" of an amino acid sequence or base sequence means the maximum degree of identity (%) of the sequences obtained by aligning two sequences to be compared, introducing gaps as necessary. The identity of an amino acid sequence or base sequence can be calculated, for example, using blastn from NCBI BLAST (http: / / blast.ncbi.nlm.nih.gov / ), which implements the BLAST algorithm. The identity of the amino acid sequences described in this disclosure is preferably 96% or higher, more preferably 97% or higher, even more preferably 98% or higher, and particularly preferably 99% or higher. The identity of the base sequences described in this disclosure is preferably 92% or higher, more preferably 94% or higher, even more preferably 96% or higher, and particularly preferably 98% or higher. In this disclosure, "one or several amino acids" is preferably 1 to 3 amino acids, more preferably 1 to 2 amino acids, and even more preferably 1 amino acid. Furthermore, in this disclosure, "one or several bases" is preferably 1 to 10 bases, more preferably 1 to 8 bases, even more preferably 1 to 6 bases, even more preferably 1 to 4 bases, and even more preferably 1 to 2 bases.

[0020] Amino acid substitutions are preferably conservative substitutions between amino acids having similar side chains. Examples of conservative substitutions include substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine; substitutions between amino acid residues having acidic side chains, such as aspartic acid and glutamic acid; substitutions between amino acid residues having non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; substitutions between amino acid residues having non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; substitutions between amino acid residues having β-branched side chains, such as threonine, valine, and isoleucine; and substitutions between amino acid residues having aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine. Furthermore, base substitutions are preferably substitutions into degenerate sequences in which the encoded amino acid remains unchanged. It is also preferable that base deletions, substitutions, or additions do not result in a frameshift.

[0021] The TCR gene consists of a gene fragment that forms a V domain, which generates molecular diversity called the signal sequence, variable region (V), diversity region (D), and joining region (J), and a gene fragment that forms a C domain, which consists of a constant region (C) including an extracellular constant region, a transmembrane region, and an intracellular region. In IMGT nomenclature, the α-chain variable region (Vα) is identified by the TRAV number, the α-chain joining region (Jα) by the TRAJ number, the β-chain variable region (Vβ) by the TRBV number, and the β-chain joining region (Jβ) by the TRBJ number (IMGT is a registered trademark). These numbers can be identified using the IMGT database.

[0022] The TCRs of this disclosure preferably include one of (a) to (d) above, from (a) to (c). Each of the TCRs of this disclosure preferably includes the following sequences. A TCR including (a) above is TRAV1-2 ＊ 01 F, TRAJ33 ＊ 01 F, TRBV6-1＊ 01 F, TRBJ2-7 ＊ It is preferable to include 01 F. The TCR containing the above (b) is TRAV19 ＊ 01 F, TRAJ29 ＊ 01 F, TRBV2 ＊ 01 F, TRBJ2-1 ＊ It is preferable to include 01 F. The TCR containing the above (c) is TRAV26-1 ＊ 01 F, TRAJ42 ＊ 01 F, TRBV5-4 ＊ 01 F, TRBJ2-5 ＊ It is preferable to include 01 F. The TCR containing the above (d) is TRAV13-1 ＊ 02 (F), TRAJ29 ＊ 01 F, TRBV20-1 ＊ 01 F, TRBJ1-4 ＊ It is preferable to include 01 F.

[0023] The TCR of the present disclosure has MR1 restriction. As used herein, "MR1 restriction" means recognizing an antigen presented by the MR1 molecule. In general, mucosal associated invariant T (MAIT) cells are known as MR1-restricted T cells. MAIT cells are one of the typical MR1-restricted T cells that react to vitamin derivatives (5-OP-RU). <00001l0> As shown in the examples described later, the TCR of the present disclosure specifically recognizes an antigen expressed in breast cancer cells. Examples of breast cancer cell lines include MCF-7 and MDA-MB-231. In addition, as shown in the examples described later, the TCR of the present disclosure is suppressed from decreasing in function by ligands other than specific antigens. Since the TCR of the present disclosure is independent of HLA, the applicable target in TCR-T cell therapy for breast cancer can be expanded.

[0025] The TCRs of this disclosure may be chemically modified insofar as they can recognize antigens expressed on breast cancer cells. The C-terminus of each chain of the TCRs of this disclosure is not particularly limited, but may include, for example, a carboxyl group (-COOH), a carboxylate (-COO-), or an amide (-CONH ２ ) or ester (-COOR). Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. １－６ Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. ３－８ Cycloalkyl groups; for example, phenyl, α-naphthyl, etc. ６－１２ Aryl group; for example, phenyl-C such as benzyl and phenethyl. １－２ Alkyl groups; such as α-naphthylmethyl and α-naphthyl-C １－２ C such as alkyl groups ７－１４ An aralkyl group; a pivaloyloxymethyl group, etc. may also be used. Furthermore, each chain of the TCR in this disclosure may have amidated or esterified carboxyl group or carboxylate other than the C-terminus. This esterification is not particularly limited, but may include, for example, the C-terminus ester mentioned above. Furthermore, each chain of the TCR in this disclosure may have a protecting group (e.g., a formyl group, an acetyl group, etc.) on the amino group of the N-terminal amino acid residue. １－６ C such as Alkanoyl １－６ Protected by acyl groups, etc., the N-terminal glutamine residue which can be cleaved and produced in vivo is pyroglutamine-oxidized, and substituents on the side chains of amino acids within the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by protecting groups (e.g., formyl group, acetyl group, etc.) １－６ C such as alkanoyl groups １－６ The TCR may be protected by an acyl group, etc. Furthermore, the TCR of this disclosure may have an intracellular domain such as a CD3-Zeta chain, CD28, or CD137 attached to it.

[0026] The TCRs of this disclosure may be proteins or peptides such as known protein tags or signal sequences, or labeled substances, insofar as they can recognize antigens expressed on breast cancer cells. Examples of protein tags are not particularly limited, but include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, etc. Examples of signal sequences are not particularly limited, but include nuclear localization signals, etc.

[0027] The method for producing the TCRs of this disclosure is not particularly limited and may be as follows. For example, the TCRs of this disclosure may be produced by a method that includes the step of culturing a host transformed with a polynucleotide encoding the TCRs of this disclosure. The host is not particularly limited and may include, for example, peripheral blood mononuclear cells, preferably lymphocytes, more preferably T cells. The transformation and culturing methods are not particularly limited and may include, for example, known methods for the production of TCRs.

[0028] Other forms of this disclosure provide polynucleotides encoding the TCR of this disclosure. The polynucleotides of this disclosure are not particularly limited in that they contain the TCR of this disclosure in an expressible state.

[0029] The polynucleotides of this disclosure preferably have a region encoding an α chain, etc., and a region encoding a β chain, etc., expressed as a single polypeptide, with the coding regions linked via a linker that is cleaved after expression, so that the TCRs of this disclosure can be expressed more efficiently. Also, for the same reason, the polynucleotides of this disclosure preferably include a region encoding a short RNA that suppresses the expression of endogenous TCRs. The short RNA is not particularly limited, but examples include siRNA and miRNA.

[0030] Furthermore, the polynucleotides of this disclosure may be introduced into a vector. That is, according to other forms of this disclosure, a vector containing the polynucleotides of this disclosure is provided. The vector is not particularly limited, but is preferably a viral vector, and more preferably a retroviral vector. The vector of this disclosure may contain other sequences in addition to the polynucleotides of this disclosure. Other sequences are not particularly limited, but include, for example, secretion signal peptide coding sequences, promoter sequences, enhancer sequences, repressor sequences, insulator sequences, replication bases, reporter protein (e.g., fluorescent protein) coding sequences, drug resistance gene coding sequences, etc. Among these, it is preferable to include a reporter protein coding sequence from the viewpoint that cells into which the polynucleotides of this disclosure have been introduced can be detected, selected, and concentrated relatively easily by, for example, FACS analysis.

[0031] Other forms of this disclosure provide cells expressing the TCR of this disclosure. Examples of cells expressing the TCR of this disclosure include cells containing the polynucleotide of this disclosure and cells containing a vector into which the polynucleotide of this disclosure has been introduced. Such a vector may include two vectors: a first vector containing a polynucleotide encoding the TCRα chain and a second vector containing a polynucleotide encoding the TCRβ chain. Alternatively, cells expressing the TCR of this disclosure may be cells differentiated from pluripotent stem cells into which the polynucleotide or vector of this disclosure has been introduced. Cells expressing the TCR of this disclosure preferably lack the expression of endogenous TCR from the viewpoint of increasing the expression level of the TCR of this disclosure and suppressing mispairs with endogenous TCR chains. For example, it is preferable that the expression of endogenous TCRα and TCRβ chains is suppressed by short-chain RNA such as siRNA or miRNA.

[0032] Cells expressing the TCR of this disclosure preferably express the TCR on the cell membrane, and preferably express the TCR with its variable region exposed outside the cell membrane. Cells expressing the TCR of this disclosure are not particularly limited, but examples include peripheral blood mononuclear cells, preferably lymphocytes, more preferably T cells, and even more preferably CD8-positive T cells. Cells expressing the TCR of this disclosure are preferably derived from patients targeted for the prevention or treatment of breast cancer. When using insect cells, eukaryotic cells, mammalian cells, etc., it is preferable to simultaneously express CD3 or to attach extracellular domains and intracellular signaling domains.

[0033] Other forms of this disclosure provide agents for the prevention or treatment of breast cancer. The agents for the prevention or treatment of breast cancer in this disclosure (hereinafter also referred to as the "agents of this disclosure") include the TCR of this disclosure, or a polynucleotide encoding the TCR of this disclosure, or a vector comprising the polynucleotide of this disclosure, or cells expressing the TCR of this disclosure. Examples of breast cancers include MCF7 and MDA-MB-231. The target of prevention or treatment of breast cancer is preferably human, but may be other mammals (e.g., mouse, rat, hamster, rabbit, cat, dog, cattle, sheep, monkey, etc.).

[0034] The amount of active ingredient in the agent disclosed herein can be appropriately determined considering the type of breast cancer being treated, the desired therapeutic effect, the method of administration, the duration of treatment, the patient's age, and the patient's weight.

[0035] The administration method of the agent of this disclosure is not particularly limited as long as the desired effect is obtained, but parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration) is preferred. The method of manufacturing the agent of this disclosure is not particularly limited and can be manufactured by conventional methods by mixing the active ingredient and a pharmaceutically acceptable carrier, etc.

[0036] Dosage forms for parenteral administration include injectable formulations (e.g., intravenous infusion, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection). For example, an injectable formulation may be prepared by suspending cells expressing the TCR of this disclosure in injectable saline, and adding solubilizers, buffers, pH adjusters, isotonic agents, analgesics, preservatives, stabilizers, etc., as needed. Carriers used in the formulation of the agent of this disclosure are not particularly limited, but examples include excipients, binders, disintegrants, lubricants, colorants, flavoring and deodorizing agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, volume expanders, wetting agents, surface activators, dispersants, buffers, preservatives, analgesics, etc.

[0037] The dosage of the agent disclosed herein may be determined by a physician based on various factors, such as the route of administration, the type of breast cancer, the severity of symptoms, the patient's age, sex, weight, the severity of the disease, pharmacological findings such as pharmacokinetic and toxicological characteristics, whether a drug delivery system is used, and whether it is administered as part of a combination of other drugs. The administration schedule of the agent disclosed herein may be determined by considering similar factors as the dosage, for example, it may be administered once a day to once a month at the above-mentioned daily dose. The agent disclosed herein may also be used in combination with other pharmaceutical compositions for the purpose of preventing or treating breast cancer, etc.

[0038] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0039] <Experiment 1> 1. Materials and Methods (1) Reagent Acetyl-6-FP (Cayman Chemicals) was dissolved in 10 mM NaOH at a concentration of 2.0 mg / mL. 5-OP-RU was obtained by combining 5-A-RU (Toronto Research Chemicals) with 50 μM methylglyoxal (Sigma) before each assay. Peridinin chlorophyll protein-cyanine 5.5 (PerCP-Cy5.5)-conjugated human CD3 monoclonal antibody (mAb) (45-0037-41), allophycocyanin (APC)-conjugated human CD8 mAb (17-0088-41), allophycocyanin-cyanine 7 (APC-Cy7)-conjugated Fixable Viability Dye (65-0865-14), and APC-conjugated human CD8α mAb (17-0088-42) were all manufactured by eBioscience. APC-labeled human HLA-A, B, C mAbs (311410), APC-labeled human HLA-E mAb (342605), APC-labeled human CD80 mAb (305219), APC-labeled human / mouse / rat MR1 mAb (361107), APC-labeled human CD1a (300110), CD1b (329109), CD1c (331523), and CD1d (350307) mAbs were used from BioLegend. The Human MR1 tetramer was used from MBL. The pMXs-IRES-GFP retrovirus expression vector (RTV-013) was used from Cell Biolabs.

[0040] (2) BW5147.3 cells expressing the mouse CD8αβ cell line were provided by Professor Ellis L. Reinherz of the Reinherz, Dana Farber Cancer Institute, Harvard Institutes of Medicine. BW5147.3 cells were maintained in Dulbecco Modified Eagle Medium (DMEM culture medium) containing 10% fetal calf serum (FCS), 2-mercaptoethanol (50 μmol / L), streptomycin (100 μg / mL), and penicillin (100 U / mL) in the presence of 0.4 mg / mL hydroxylamine B and 0.4 mg / mL G418. Human CD8α and CD8β cDNA were introduced into BW5147.3 cells using retroviral vectors. In the following description, these cells are referred to as "BW-hCD8αβ ＋ Also called "cells". MCF7 cells were purchased from ATCC and maintained in DMEM culture medium. Human CD80 and β2-microglobulin cDNA were introduced into a retroviral vector (human CD80 ＋ β2-microglobulin ＋MCF7 cells were introduced using MCF7 cells. Patient HLA-A, B, and C cDNA were introduced into MCF7 cells along with green fluorescent protein (GFP) cDNA using a retroviral vector, and GFP-positive cells were sorted as patient HLA-introduced cells using FACSAria II (Beckton Dickinson). Luciferase-expressing MCF7 cells (hereinafter also referred to as "MCF7-Luc cells") were prepared by introducing luciferase cDNA (Promega) and Kusabira-Orange cDNA (MBL) into MCF7 cells using a retroviral vector. To select MCF7-Luc cells, Kusabira-Orange-positive cells were sorted using FACSAria II. The K562, Jurkat, Karpas299, COLO205, A549, MDA-MB-231, MDA-MB-453, MDA-MB468, BT474, and ZR-75-1 cell lines were all taken from the laboratory stock. The A549, MDA-MB-231, MDA-MB-453, MDA-MB468, and BT474 cells were maintained in Dulbecco-modified Eagle medium (DMEM) containing 10% FCS, 50 μmol / l 2-mercaptoethanol, streptomycin (100 μg / ml), and penicillin (100 U / ml). K562, Jurkat, Karpas299, COLO205, and ZR-75-1 cells were maintained in RPMI1640 containing 10% fetal bovine serum (FCS), 50 μmol / l 2-mercaptoethanol, streptomycin (100 μg / ml), and penicillin (100 U / ml). Human mammary epithelial cells (HMECs) were purchased from ATCC and maintained in basal growth medium.

[0041] (3) Identification of TCRs HLA class I-independent TCRs were identified from CD8-positive tumor-infiltrating lymphocytes (TILs) of two breast cancer patients. Specifically, tumor specimens were cut, washed, and then digested with liberase DH solution (Roche). Cells filtered with a cell strainer (Becton Dickinson) were frozen at -80°C in Cellbanker-1 medium (Nippon Zenyaku Kogyo Co., Ltd.) and used as TILs. Flow cytometry analysis and cell sorting were performed according to the guidelines (Cossarizza, A. et al., Eur. J. Immunol. 2019. 49: 1457-1973.). The TILs were thawed and stained with PerCP-Cy5.5-anti-human CD3 antibody, APC-anti-human CD8 antibody, PE-anti-human CD137 antibody, and FITC-anti-human PD-1 antibody. Dead cells were removed using APC-Cy7-Fixable Viability Dye (eBioscience). After staining, cells were analyzed using FACSAria II (Beckton Dickinson). PD-1 ＋ CD137 ＋ CD8 ＋ Single T cells were sorted into 96-well PCR plates. HLA class I or class I-like molecule expression in MCF7 cells was analyzed using FACSCanto (Beckton Dickinson) after staining with either APC-anti-HLA-A, B, C antibodies, anti-CD1a, b, c, d antibodies, or anti-MR1 antibodies. For MR1 expression analysis, MCF7 cells were pulsed overnight with 1 μg / mL Ac-6-FP and stained the following day. TCR cDNA was amplified from single T cells by one-step RT-PCR and second-step PCR. The DNA sequences of the PCR products were then analyzed by direct sequencing. The sequences of the identified TCRs used in subsequent experiments are shown in Table 1 below.

[0042]

[0043] (4) Preparation of HLA-E and TAP1 / 2 knockout MCF7 cells Using the CRISPR-Cas9 KO plasmid (manufactured by Santa Cruz Biotechnology), HLA-E and TAP1 / 2 genes were knocked out from MCF7 cells according to the manufacturer's instructions. TAP1 / 2 gene knockout cells (hereinafter also referred to as "MCF7ΔTAP1 / 2") were stained with anti-HLA-A, B, and C monoclonal antibodies (mAb) (W6 / 32), and HLA class I negative cells were sorted using FACSAria II. HLA-E gene knockout cells (hereinafter also referred to as "MCF7ΔHLA-E") were stained with anti-HLA-E mAb, and HLA-E negative cells were selected.

[0044] (5) Construction of TCR expression vector To express TCR, a TCR expression vector was constructed as described above. Specifically, the TCRβ PCR fragment, a codon-optimized gene encoding the human TCRβ constant region-1 bound to the self-cleaved P2A peptide (Cb1-P2A- fragment), the TCRα PCR fragment, and the codon-optimized TCRα constant region gene (Ca- fragment) were incorporated together into a linear pMXs-IRES-GFP retroviral vector using Gibson Assembly Master Mix (New England Biolabs, E2611). The constructed plasmid vector, pMXs-TCRβ-P2A-TCRα-IRES-GFP, was used for retrovirus production.

[0045] (6) Retrovirus Production To produce a retrovirus for introducing the TCR gene into human PBMCs, Phoenix A cells (provided by Dr. Garry Nolan, Stanford University) were used. The day before transfection, Phoenix A cells (3.7 × 10⁶) ６ The cells were placed in a 10 cm dish and cultured in 10 mL of DMEM culture medium. The TCR expression vector was transfected into Phoenix A cells using FuGENE6 transfection reagent (Promega) according to the manufacturer's instructions. The cells were then exposed to 5% CO2. ２The cells were cultured at 37°C under atmospheric conditions. The cell medium was changed the following day. Two days later, the culture supernatant was collected, filtered through a 0.22 μm filter (Millipore, SLHV033RS), and stored at -80°C until use. In addition, a retrovirus for introducing the TCR gene into mouse T cells was created using Plat E cells (provided by Dr. Toshio Kitamura (University of Tokyo)).

[0046] (7) Isolation of primary T cells and introduction of retroviral TCR into primary T cells Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of healthy donors by density gradient centrifugation using Ficoll-Paque (Ficoll is a registered trademark) (Promocell). PBMCs were maintained in RPMI1640 containing 10% FCS, 50 μmol / L 2-mercaptoethanol, 100 μg / mL streptomycin, and 100 U / mL penicillin. For TCR transduction, 1 × 10⁶ ６ PBMCs were stimulated in vitro for 2 days with CD3 / CD28 Dynabeads (Invitrogen) in the presence of human IL-2 (PeproTech). PBMCs were collected and incubated in RPMI culture medium in the presence of human IL-2 (30 U / ml), with 5 × 10⁶ cells. ５ The cells were resuspended at 0.3 ml / ml. To introduce retrovirus, the wells of a 24-well plate were coated overnight at 4°C with 0.3 ml of retronectin (50 μg / ml) (Takara). The following day, the culture supernatant containing the retrovirus was added to the wells, and according to the manufacturer's guidelines, the cells were centrifuged at 1900 x g for 2 hours at 32°C to adsorb the retrovirus encoding the TCR onto the wells. Subsequently, the suspended PBMC cells were added to the wells containing the adsorbed retrovirus, and the cells were spun down at 1000 x g at 32°C for 10 minutes, followed by 5% CO2 adsorption. ２ After culturing overnight at 37°C, the PBMC was transferred to a plate coated with freshly prepared retrovirus and then placed in 5% CO2. ２The cells were cultured at 37°C. The following day, the PBMCs into which the TCR had been introduced were grown for a further two days in the presence of human IL-2 (30 U / ml). After collection, the TCR-introduced PBMCs were stored at -80°C until use. To introduce the TCR into mouse spleen T cells, spleen cells were stimulated in vitro for two days with mouse T-Activator CD3CD28 Dynabeads (Invitrogen) in the presence of recombinant mouse IL-2 (PeproTech). The stimulated spleen cells were infected with a retrovirus encoding human TCR using the same method as for retroviral introduction of TCR into human PBMCs.

[0047] (8) Tetramer staining of BW hCD8αβ+ cells expressing HLA class I-independent TCRs Approximately 2 × 10⁻¹⁶ cells expressing various HLA class I-independent TCRs ５ BW hCD8αβ+ cells were stained with APC-labeled human tetramer (hMR1), hMR1 / 5-OP-RU (5-(2-oxopropylideneamino)-6-D-ribitylaminouracil), or MR1 / 6FP (Acetyl-6-formylpterin) at 30 ng / stain for 20 minutes at room temperature in the dark. The cells were then washed three times with buffer (PBS containing 2% FCS) and resuspended in 200 μl buffer. Data were acquired using BD FACS Canto and analyzed with FlowJo software. TCR-ve BW cells were used as a negative control.

[0048] (9) Detection of MR1 surface expression MCF7 cells (1 × 10 ５ The cells were incubated overnight with different concentrations of 5-OP-RU, Ac-6-FP, or medium alone. The cells were washed and stained with APC-anti-MR1 mAb26.5 on ice for 20 minutes, then washed twice with PBS. The cells were suspended in FACS buffer. Data were acquired using BD FACS Canto and analyzed with FlowJo software.

[0049] (10) Detection of HLA-E and CD1s expression MCF7 cells were stimulated with hIFN-γ 100 U / ml for 48 hours. The cells were washed and stained with APC-anti-HLA-E and CD1a, b, c, d mAbs on ice for 20 minutes, after which the cells were washed twice with PBS. The cells were suspended in FACS buffer. Data were acquired using BD FACS Canto and analyzed with FlowJo software.

[0050] (11) Evaluation of human IFN-γ secretion by ELISA PBMC (1 × 10) into which the gene encoding TCR has been introduced ５ ) treated with human IFN-γ 1 × 10 ５ MCF7 cells were co-cultured in 200 μl of RPMI1640 culture medium in a 96-well plate. After 24 hours of incubation, the supernatant was collected, and IFNγ production was measured as a result of the T cell response using ELISA (R&D Systems, DY285B) according to the manufacturer's instructions.

[0051] (12) Evaluation of irritation to MR1 T cells by ELISA BW-hCD8αβ+ cells transformed with the gene encoding TCR (1 × 10 ５ ) were treated with human IFN-γ in 200 μl of DMEM culture medium in the presence of mouse IL-1 alpha, resulting in 1 × 10⁶ samples. ５ MCF7 cells were co-cultured with MAIT cells in 96-well plates. 5-OP-RU was added to the culture medium to stimulate MAIT cells. Ac-6-FP and anti-MR1 antibody were added for inhibition, and the cells were cultured. After 24 hours of culture, the supernatant was collected, and IL-2 production resulting from MAIT cell stimulation and inhibition was measured using ELISA (R&D Systems, DY285B) according to the manufacturer's instructions.

[0052] (13) Cytotoxic assay For the cytotoxic assay, co-culture of TCR-expressing PBMCs and luciferase-expressing MCF7 cells was performed. As TCRs, MR1-restricted TCR(c) and HLA-B ＊A 59:01 restrictive TCR(e) was introduced into healthy peripheral blood lymphocytes via a retroviral vector. Lymphocytes that had not been introduced with an external TCR were used as a negative control (TCR-ve). MCF7 cells were used, specifically those with HLA-B ＊ Wild-type MCF7 cells that do not express 59:01, HLA-B ＊ MCF7 cells expressing 59:01 and MCF7 cells with MR1 knocked out (ΔMR1 cells) were used. The luciferase gene was introduced into MCF7 cells using a retrovirus and expressed (Yamaguchi, Eur J Immunol). MCF7-Luc cells (1 × 10⁻⁶) ４ The TCR-expressing PBMCs were co-cultured in 96-well plates at the respective effector-to-target (E / T) ratios and incubated for 24 hours. Cytotoxicity against MCF7-Luc cells was evaluated by measuring cell-associated luciferase activity using the Steady-Glo luciferase assay system (Promega, E2520).

[0053] (14) Statistical analysis data are presented as mean ± S.D., and statistical significance is determined by each test. Statistical differences were calculated using Microsoft Excel 2016 software.

[0054] 2. Results and Discussion (1) Analysis of MR-1 Restriction of HLA Class I Independent TCRs Figure 1 is an explanatory diagram showing the results of hMR1 tetramer staining of BW hCD8αβ+ cells expressing TCRs. In Figure 1, flow cytometry dot plots are shown for each TCR, for control (No tetramer), hMR1 / 5-OP-RU, and hMR1 / 6-FP. Figure 1 shows representative results from two independent experiments. As shown in Figure 1, BW cells expressing TCR(a) bound to the 5-OP-RU / MR1 tetramer. On the other hand, in BW cells expressing other TCRs, no binding to the hMR1 tetramer was observed with either hMR1 / 5-OP-RU or hMR1 / 6-FP.

[0055] (2) Characterization of non-conventional HLA class I-like molecules in various cell lines In order to clarify the characteristics of antigen-presenting molecules, the expression of HLA class I-like molecules in MCF7 cells, which are breast cancer cells, and other cell lines was compared.

[0056] Figure 2 is a histogram showing the surface expression of HLA class I and HLA class I-like molecules in MCF7 cells with and without IFN-γ treatment. In Figure 2, (A) represents HLA-ABC (HLA class I), (B) represents HLA-E, (C) represents CD1 molecules, and (D) represents MR1 ​​molecules. In Figure 2(D), Isotype represents the negative control, and Mock represents mock cells as a control. As shown in Figures 2(A) to (D), MCF7 cells without IFN-γ treatment expressed HLA class I molecules, but the expression of HLA-E, CD1, and MR1 molecules was not detected. HLA-E (non-classical HLA molecules) are expressed in tumor cells, and their expression can be induced by IFN-γ (Seliger, Barbara et al. Oncotarget. 2016;7(41): 67360-67372). As shown in Figure 2(B), HLA-E molecules were expressed when MCF7 cells were stimulated with IFN-γ. Regarding MR1 expression, MCF7 cells were stimulated with MR1 ligands 5-OP-RU and Ac-6-FP, and MR1 expression was examined in a dose-dependent manner. Ac-6-FP (6-FP, an acetylated derivative of the folic acid derivative 6-formylpterin) has strong MR1 binding activity, and MR1 expression can be induced by this MR1 ​​ligand (Sario M et al. Proc Natl Acad Sci US A. 2020). As shown in Figure 2(D), the MR1 ligand Ac-6-Fp increased the expression of the MR1 molecule.

[0057] Figure 3 is a histogram showing the increase in MR1 surface expression in MCF7 cells. In Figure 3, (A) shows the results for 5-OP-RU at different doses, (B) shows the results for Ac-6-Fp at different doses, and (C) shows the results for β2m-deficient MCF7 cells and MR1-deficient MCF7 cells. As shown in Figure 3(B), the MR1 ligand Ac-6-Fp increased MR1 molecule expression in a dose-dependent manner. On the other hand, as shown in Figure 3(A), 5-OP-RU did not increase MR1 molecule expression. Furthermore, as shown in Figure 3(C), no MR1 molecule expression was observed in β2m-deficient MCF7 cells or MR1-deficient MCF7 cells. This is thought to be due to the fact that MR1 expression is related to β2-microglobulin (Godfrey, Nat Immunol, 16:1114, 2015).

[0058] Figure 4 is a histogram showing the expression of MR1 in various cell lines. Figure 5 is a histogram showing the expression of HLA-E and CD1 molecules in various cell lines. Figure 4(A) shows the results for breast cancer cell lines other than MCF7 cells, and Figure 4(B) shows the results for cancer cell lines other than breast cancer (colon, lung, myeloid, and lymphoid cell lines). As shown in Figure 4, MR1 expression was confirmed by Ac-6-Fp treatment in breast cancer cell lines other than MCF7 cells and other cancer cell lines.

[0059] (3) Expression of TCR in mouse spleen T cells and human PBMCs and their responsiveness to MCF7 cells Previously, we had analyzed the responsiveness by expressing TCR in BW T cells, and this time we investigated the responsiveness when expressed in human PBMCs and mouse T cells.

[0060] Figure 6 is an explanatory diagram showing the reactivity when expressed in mouse T cells and human PBMCs. Figure 6(A) shows the results of co-culture of mouse spleen T cells expressing each TCR by transformation with IFN-γ stimulated MCF7 cells, and Figure 6(B) shows the results of co-culture of healthy human PBMCs expressing each TCR by transformation with IFN-γ stimulated MCF7 cells. IFN-γ production was measured by ELISA. PBMCs without TCR introduction (TCR-ve) were used as a negative control. The sample size was 3.

[0061] As shown in Figure 6, TCR(e) is HLA-B ＊ It responded only to MCF7WT expressing 59:01. This suggests that TCR(e) is HLA-B ＊ This indicates a dependence on 59:01. In contrast, as shown in Figure 6(A), TCRs (a) to (d), when expressed in BW T cells, responded to MCF7 cells independently of HLA. Furthermore, when TCRs (a) to (d) were expressed in mouse T cells and mouse IFN-γ was secreted, they all responded to MCF7 cells. As shown in Figure 6(B), TCR (c) also responded to MCF7 cells when expressed on human PBMCs. In contrast, TCRs (a), (b), and (d) did not respond to MCF7 cells when expressed on human PBMCs. The lack of response of TCRs (a), (b), and (d) to MCF7 cells is presumed to be due to incompatibility with endogenous TCRs.

[0062] (4) Responsiveness to MCF7 cells with each factor knocked out Detailed analysis of TCR(c) was performed. To identify the antigen-presenting molecule of HLA-independent TCR(c), first, HLA-I, β2-microglobulin, and MR1 genes were knocked out from MCF7 cells using the CRISPER / Cas9 system (also referred to as "MCF7ΔHLA-I", "MCF7Δβ2m", and "MCF7ΔMR1", respectively). Next, the HLA-E gene was knocked out from MCF7 cells using the CRISPER / Cas9 system (hereinafter also referred to as "MCF7ΔHLA-E"). As shown in Figure 2(B) above, when MCF7 cells were treated with hIFN-γ, HLA-E expression was confirmed.

[0063] Figures 7 and 8 illustrate the responsiveness of MCF7 cells to each factor knocked out. They show the results of co-culture between healthy human PBMCs expressing each TCR by transformation and IFN-γ stimulated MCF7 cells. Figure 7(A) shows the results of ELISA measurement of TNF-α production, and Figures 7(B) and 8 show the results of ELISA measurement of IFN-γ production. PBMCs without TCR introduction (TCR-ve) were used as a negative control, and T cells stimulated with PMA + ionomycin were used as a positive control. The sample size was 3. HLA-E deletion was confirmed by staining the cells with an anti-HLA-E antibody.

[0064] As shown in Figure 7, TCR(c) secreted TNF-α and IFN-γ and responded to MCF7 cells in an HLA-independent manner, while conventional TCR(e) responded to MCF7 cells in an HLA-dependent manner. TCR(c) did not respond to MCF7 cells lacking β2-microglobulin or the MR1 gene. As shown in Figure 8, TCR(c) responded to MCF7ΔHLA-E cells but not to MCF7ΔMR1 cells.

[0065] (5) Analysis of cytotoxicity The cytotoxicity of each TCR-transfected PBMC against MCF7 cells expressing luciferase was analyzed in vitro.

[0066] Figure 9 is an explanatory diagram showing the cytotoxicity against MCF7 cells. Figure 9(A) shows HLA-B ＊ The results for wild-type MCF7 cells that do not express 59:01 are shown in Figure 9(B), and HLA-B ＊ Figure 9(C) shows the results for MCF7 cells expressing 59:01, and Figure 9(C) shows the results for MCF7 ΔMR1 cells with MR1 knocked out. As shown in Figure 9, TCR(c) was found to damage MCF7 cells in an HLA-independent manner, but showed no damaging effect on MCF7ΔMR1. Therefore, it was shown that TCR(c) damages MCF7 cells in an HLA-independent manner that is restricted by MR1.

[0067] (6) Reactivity to cancer cell lines We investigated which tumor cells HLA-independent TCR(c) responds to.

[0068] Figure 10 is an explanatory diagram showing the responsiveness to various cancer cell lines. Figure 10 shows the results of co-culturing human PBMCs expressing TCR(a) and (c) with IFN-γ stimulated human cancer cells, respectively. MDA-MB-231 and ZR-75-1 breast cancer cells, colo205 colon cancer cells, A549 lung cancer cells, K562 myeloid cells, and Karpas299 lymphoid cells were used as human cancer cell lines and cultured for 24 hours. PBMCs without TCR (TCR-ve) were used as a negative control, and T cells stimulated with PMA + ionomycin were used as a positive control. IFN-γ production was measured by ELISA.

[0069] As shown in Figure 10, TCR(c) expressed in human PBMCs responded not only to MCF7 breast cancer cells but also to MDA-MB231 breast cancer cells. In contrast, it did not respond to other cancer cell lines (Colo205 colon cancer cells, A549 lung cancer cells, K562 myeloid cells, and Karpas299 lymphoid cells). Furthermore, as shown in Figure 4(A) above, the breast cancer cell line ZR-75-1 showed higher surface expression of MR1 than MDA-MB231 cells, but as shown in Figure 10, TCR(c) did not respond to ZR-75-1. Thus, it was found that the HLA-independent TCR of this disclosure responds to antigens specifically expressed in at least MCF7 breast cancer cells and MDA-MB231 breast cancer cells.

[0070] (7) Effects of known microbial MR1 antigens, etc. The effects of known microbial antigens of MR1, 5-OP-RU and Ac-6-FP (Corbett AJ et al. 2020 Front. Immunol. 11:1961), were analyzed. TCR(a) and TCR(c) were retrovirally introduced into BW cells, and CD3 ＋ Cells were selected, expanded in culture, and used for further experiments. TCR-expressing BW cells were co-cultured overnight with IFN-γ stimulated MCF7 cells in the presence / absence of 5-OP-RU, Ac-6-FP, and anti-MR1 antibody (Anti-MR1 ab). The following day, ELISA was performed to measure IL2 secretion by BW cells.

[0071] Figure 11 is an explanatory diagram showing the amount of IL2 produced with and without the MR1 ligand. In Figure 11, "+" indicates the presence of the ligand, and "-" indicates the absence of the ligand. As shown in Figure 11, when MCF7 cells were co-cultured without the addition of the MR1 ligand, IL2 was produced in both TCR(a) and TCR(c). Furthermore, 5-OP-RU increased IL2 production in TCR(a), but did not show an effect of increasing IL2 production in TCR(c). Despite being a non-stimulating ligand for MAIT cells, Ac-6-FP significantly suppressed BW MAIT activity against the agonist ligand (5-OP-RU). Similarly, the anti-MR1 antibody inhibited only the response to the agonist ligand (5-OP-RU). This discriminative function is thought to be because 5-OP-RU contains a ribityl tail that is primarily recognized by TCRs in MAIT cells, while Ac-6-Fp does not (Corbett, A., et al. Nature (2014); Eckle et al., 2014). These agonist and antagonist effects of MR1 ligands were observed in the response of TCR(a) to 5-OP-RU, but not in the response of TCR(a) and TCR(c) to MCF7 cells. This data suggests that antigens expressed in MCF7 cells with HLA-independent TCRs have different binding sites for known microbial antigens, 5-OP-RU or Ac-6-FP.

[0072] (8) Reactivity to MCF7 TAP1 / 2 knockout cells It has been reported that MR1 uses the same molecular assembly mechanism as classical class I molecules (Michael J. Miley, et al. J Immunol 15 June 2003). MAIT cells do not require TAP (transporter associated with antigen processing) for stimulation, and MR1 surface expression is TAP-independent (Huang, S. et al., FASEB J, 22: 1068.11-1068.11, 2008). Therefore, to confirm whether the response of TCR(e) and (c) to MCF7 is TAP-dependent or independent, the TAP1 / 2 genes were deleted from MCF7 cells using the CRISPER / Cas9 system (MCF7ΔTAP1 / 2). MCF7 cells were stimulated overnight with the indicated concentration of Ac-6-FP, and the cells were stained with anti-MR1 antibody to analyze MR1 surface expression. Next, we analyzed the effects of TAP1 / 2 gene deletion on stimulation of MR1 T cells (typical MAIT TCRs and atypical MR1-TCRs). BW cells expressing TCR(e) (typical MAIT TCRs) and TCR(c) (atypical MR1-TCRs) were co-cultured with MCF7 cells and MCF7ΔTAP1 / 2 cells, and IL2 secretion levels were measured by ELISA. The sample size was 3.

[0073] Figure 12 is a histogram showing the surface expression of MR1 in MCF7 cells. As shown in Figure 12, the surface expression of MR1 was reduced to approximately 50% in MCF7ΔTAP1 / 2 cells compared to wild-type MCF7 cells.

[0074] Figure 13 is an explanatory diagram showing the amount of IL-2 produced. As shown in Figure 13, IL-2 production was observed in both TCR(e) and TCR(c), and its production was reduced in MCF7ΔTAP1 / 2 cells. These data indicate that MR1-restricted antigen recognition in TCR(e) and (c) is independent of TAP.

[0075] (9) Reactivity of TCR to normal mammary cells Since TCR(c) reacts only to mammary cancer cell lines, further evaluation was performed using healthy mammary cells to confirm the effect of TCR(c) on normal cells. In this evaluation, human mammary epithelial cells (HMECs), TCR(e), and TCR(c) were used. IFN-γ production was measured by ELISA. PBMCs without TCR introduction (TCR-ve) were used as a negative control, and T cells stimulated with PMA + ionomycin (Phorbol 12-myristate 13-acetate and ionomycin: PMA / IM) were used as a positive control. The n size was 2.

[0076] Figure 14 is an explanatory diagram showing the responsiveness of TCR to breast cancer cells and normal mammary cells. As shown in Figure 14, TCR(c) showed responsiveness to MCF7 cells but not to normal mammary cells. In other words, normal mammary cells did not activate TCR(c). As a result, it is thought that HLA-independent TCR(c) did not respond to normal mammary cells. Note that TCR(e) is HLA(B) ＊ It showed responsiveness to MCF7 cells expressing 59:01).

[0077] <Experiment 2> 1. Materials and Methods As mice, 5-week-old NOD.Cg-Prkdcscid IL-2rgtm1Wjl / SzJ (NSG) female mice were purchased from Charles River Japan Co., Ltd., and tumor cells were administered subcutaneously two weeks later. As tumor cells, MCF7 cells into which the Luciferase gene had been introduced (MCF7-Luc cells) were used. Note that the MCF7-Luc cells are the same as the MCF7-Luc cells described in Experiment 1, and are also described in Cells 2024, 13, 1711. (https: / / doi.org / 10.3390 / cells13201711). As the TCR, TCR(c) described in Experiment 1 was used. As described in Experiment 1 and Cells 2024, 13, 1711 (https: / / doi.org / 10.3390 / cells13201711), the TCR(c) gene was introduced into human peripheral blood lymphocytes using a retroviral vector. Note that TCR(c) corresponds to "TCR10-59" as described in Cells 2024, 13, 1711 (https: / / doi.org / 10.3390 / cells13201711). Human peripheral blood lymphocytes into which the TCR(c) gene (TCR10-59 gene) was introduced were used as TCR(c)-expressing human T cells.

[0078] Figure 15 is an explanatory diagram showing the protocol for Experiment 2. The in vivo verification experiment of the antitumor effect is described in Cells 2023, 12, 2059 (https: / / doi.org / 10.3390 / cells12162059). On Day 0, the cell count was 1 × 10⁶. ６ , 5 x 10 ６ , 1 x 10 ７ MCF7-Luc cells were subcutaneously administered to two NSG mice each. Cell count: 1 × 10⁶ ６ The group administered MCF7-Luc cells was designated as Group 1 (individual mice: #1, #2), with a cell count of 5 × 10⁶. ６ The group administered MCF7-Luc cells was designated as Group 2 (individual mice: #3, #4), with a cell count of 1 × 10⁶. ７The group administered MCF7-Luc cells was designated as Group 3 (individual mice: #5, #6). After subcutaneous administration of MCF7-Luc cells, 200 μL of luciferase substrate (luciferin) was administered intraperitoneally to the mice, and the number of tumors was measured by bioluminescence imaging using the IVIS imaging system (IVIS Lumina II version 4.2, PerkinElmer). On Day 7, one mouse each from Groups 1, 2, and 3 (individual mice: #1, #3, #5) was administered 1 × 10⁶ human T cells expressing TCR(c). ７ The TCR(c)-expressing human T cells were administered via the tail vein. After administration of TCR(c)-expressing human T cells, the mice (#2, #6) that had not been administered TCR(c)-expressing human T cells were administered intraperitoneally with the luciferase substrate as described above, and the number of tumor cells was measured by bioluminescence. Thereafter, approximately every week, the luciferase substrate was administered and bioluminescence imaging was performed in the same manner, and the number of tumor cells was measured.

[0079] 2. Results and Discussion Figure 16 is an explanatory diagram showing imaging of luciferase-expressing cells in mice. Figure 17 is an explanatory diagram showing the results of measuring the number of tumor cells in mice. Figure 17(A) shows the results for Group 1, Figure 17(B) shows the results for Group 2, and Figure 17(C) shows the results for Group 3. In Figure 17, the vertical axis shows the value of tumor cell count measured by bioluminescence, the horizontal axis shows the time period after administration of MCF7-Luc cells, the dashed line shows the results for mice that were not administered T cells, and the solid line shows the results for mice that were administered TCR(c)-expressing human T cells on Day 7. In Group 2, one of the two mice (mouse individual: #4) died between subcutaneous administration of MCF7-Luc cells and Day 7. Therefore, on Day 7, the remaining mouse (mouse individual: #3) was administered TCR(c)-expressing human T cells.

[0080] The results shown in Figures 16 and 17 reveal the following: In Group 1, the number of tumor cells in mice (#2) that were not administered TCR(c)-expressing human T cells increased day by day. In contrast, in mice (#1) that were administered TCR(c)-expressing human T cells, the number of tumor cells increased until Day 14, but the increase stopped on Day 21, and the number of tumor cells decreased thereafter. In Group 2, in mice (#3) that were administered TCR(c)-expressing human T cells, the number of tumor cells increased on Day 14 and Day 28, but the number of tumor cells decreased thereafter. In Group 3, the number of tumor cells in mice (#6) that were not administered TCR(c)-expressing human T cells increased day by day. In contrast, in mice (#5) that were administered TCR(c)-expressing human T cells, the number of tumor cells increased until Day 28, but the number of tumor cells decreased thereafter.

[0081] In Groups 1 and 3, the number of tumor cells increased day by day in mice that were not administered TCR(c)-expressing human T cells (#2, #6). However, in Groups 1, 2, and 3, the number of tumor cells began to decrease midway through the study in mice that were administered TCR(c)-expressing human T cells (#1, #3, #5). This suggests that the TCR(c)-expressing human T cells exhibited antitumor activity, leading to the decrease in tumor cell count. In particular, in Groups 2 and 3, the number of tumor cells increased until Day 28 in mice that were administered TCR(c)-expressing human T cells (#3, #5), but then decreased thereafter. Conventionally, in vivo studies using T cells into which antitumor TCR genes have been introduced have reported that when the number of tumor cells increases after administration of antitumor TCR-gene-introduced T cells, the tumor usually continues to grow. In contrast, in this embodiment, the result that the number of tumor cells began to decrease from Day 28 onward after administration of TCR(c)-expressing human T cells indicates that the antitumor effect of TCR(c)-expressing human T cells is excellent, giving great promise for future clinical applications.

[0082] The present invention is not limited to the embodiments described above, and can be realized in various configurations without departing from its spirit. For example, the technical features in the embodiments and examples corresponding to the technical features in each form described in the summary of the invention can be replaced or combined as appropriate in order to solve some or all of the above-described problems, or to achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

Claims

1. A T cell receptor having major histocompatibility class I-like molecule 1 restriction, comprising any one of the following (a) to (d), and specifically recognizing an antigen expressed on breast cancer cells: (a) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, and a TCRα chain encoded by the nucleotide sequence shown in SEQ ID NO: 11 or a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 11 or a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 11, and, (b) A TCRβ chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 2, and the TCRβ chain is encoded by a nucleotide sequence shown in SEQ ID NO: 12 or a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 12 or a nucleotide sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 12, (b) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 3, and the TCRα chain is encoded by a nucleotide sequence shown in SEQ ID NO: 13 or a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 13 or a nucleotide sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 13, and(c) A TCRβ chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 4, and the TCRβ chain is encoded by a nucleotide sequence shown in SEQ ID NO: 14 or an amino acid sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 14 or an amino acid sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 14, (c) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 5 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 5, and the TCRα chain is encoded by a nucleotide sequence shown in SEQ ID NO: 15 or an amino acid sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 15 or an amino acid sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 15, and A TCRβ chain comprising CDR3 having the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence in which one or more amino acids are deleted, substituted, or added to the amino acid sequence shown in SEQ ID NO: 6, and a TCRβ chain encoded by the base sequence shown in SEQ ID NO: 16, or a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 16, or a base sequence in which one or more bases are deleted, substituted, or added to the base sequence shown in SEQ ID NO: 16.(d) A TCRα chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 7, and the TCRα chain is encoded by a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 17 or an amino acid sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO: 17; and a TCRβ chain containing CDR3 having the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 8 or an amino acid sequence in which one or more amino acids are deleted, substituted or added to the amino acid sequence shown in SEQ ID NO: 8, and the TCRβ chain is encoded by a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 18 or an amino acid sequence in which one or more bases are deleted, substituted or added to the nucleotide sequence shown in SEQ ID NO:

18.

2. The T cell receptor according to claim 1, comprising any of (a) to (c) above.

3. The T cell receptor according to claim 1, wherein the breast cancer cells are MCF7 or MDA-MB-231.

4. A polynucleotide encoding the T cell receptor according to claim 1.

5. A vector comprising the polynucleotide described in claim 4.

6. Cells expressing the T cell receptor described in claim 1.

7. The cell according to claim 6, which lacks the expression of endogenous T cell receptors.

8. A preventive or therapeutic agent for breast cancer comprising a T cell receptor according to any one of claims 1 to 3, or a polynucleotide according to claim 4, or a vector according to claim 5, or a cell according to claim 6 or 7.

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