T-cell receptor and use thereof

Abstract

To provide an HLA-independent T cell receptor.SOLUTION: A T cell receptor (TCR) with Major Histocompatibility Complex Class I-related molecule 1 (MR1) restriction comprises a TCRα chain including CDR3 having a specific amino acid sequence and encoded by a specific base sequence and a TCRβ chain including CDR3 having a specific amino acid sequence and encoded by a specific base sequence and specifically recognizes antigens expressed in breast cancer cells.SELECTED DRAWING: Figure 6

Description

[Technical field]

[0001] The present disclosure relates to T cell receptors. [Background technology]

[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 antigen recognition and binding. Conventionally, TCR-T cell therapy has been attempted to treat cancer by expressing TCRs that react to cancer cells in patient T cells and administering them to cancer patients. As a T cell receptor, for example, Non-Patent Documents 1 and 2 disclose a T cell receptor restricted to major histocompatibility complex (MHC) class I-like related molecule 1 (MR1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Rapoport, AP et al. Nat Med. 2015 August; 21(8): 914-921. [Non-Patent Document 2] Chapuis, AG et al. Nat Med. 2019 July; 25(7): 1064-1072. Summary of the Invention [Problem to be solved by the invention]

[0004] TCRs used in conventional TCR-T cell therapy react to cancer cells depending on the class I of human leukocyte antigens (HLA), which is a major histocompatibility complex (MHC) in humans. HLA class I is a highly polymorphic molecule, and it is known that each has different alleles. Therefore, TCR-T cell therapy using a specific HLA-dependent TCR cannot be applied to target patients who do not have that HLA allele. For this reason, from the perspective of expanding the scope of application of immunotherapy, HLA-independent T cell receptors are desired. [Means for solving the problem]

[0005] The present disclosure can be realized in the following forms.

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

[0007] (2) The T cell receptor according to (1) above may contain any one of (a) to (c).

[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) There is provided a polynucleotide encoding the T cell receptor described in any one of (1) to (3) above.

[0010] (5) There is provided a vector comprising the polynucleotide described in (4) above.

[0011] (6) There is provided a cell expressing a T cell receptor described in any one of (1) to (3) above.

[0012] (7) The cell according to (6) above may lack expression of an endogenous T cell receptor.

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

[0014] The present disclosure can be realized in various forms, for example, as a pharmaceutical composition for preventing or treating breast cancer, which contains the above-mentioned T cell receptor, polynucleotide, vector, or cell as an active ingredient, or as a method for preventing or treating breast cancer, which includes a step of administering any of the above-mentioned T cell receptor, polynucleotide, vector, or cell to a patient. [Brief description of the drawings]

[0015] [Figure 1] An explanatory diagram showing the results of hMR1 tetramer staining. [Diagram 2] Histogram showing surface expression of HLA class I-like molecules. [Diagram 3] Histogram showing increased MR1 surface expression in MCF7 cells. [Figure 4] Histogram showing the expression of MR1 in various cell lines. [Diagram 5] Histogram showing the expression of HLA-E and CD1 molecules in various cell lines. [Figure 6] An explanatory diagram showing reactivity when expressed in mouse T cells and human PBMCs. [Figure 7] FIG. 1 is an explanatory diagram showing the reactivity to MCF7 cells in which each factor was knocked out. [Figure 8] FIG. 1 is an explanatory diagram showing the reactivity to MCF7 cells in which each factor was knocked out. [Figure 9] FIG. 1 is an explanatory diagram showing cytotoxicity against MCF7 cells. [Figure 10] FIG. 1 is an explanatory diagram showing reactivity against various cancer cell lines. [Figure 11]FIG. 1 is an explanatory diagram showing the amount of IL2 produced in the presence or absence of MR1 ligand. [Figure 12] Histogram showing surface expression of MR1 in MCF7 cells. [Figure 13] FIG. 1 is an explanatory diagram showing the amount of IL2 produced. [Figure 14] Illustrative diagram showing TCR reactivity to breast cancer cells and normal breast cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] As shown in the Examples below, the present inventors 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 reacted to breast cancer cells independent of HLA and exhibited cytotoxic activity, which led to the completion of the present invention.

[0017] According to one embodiment of the present disclosure, there is provided a T cell receptor (TCR) that is major histocompatibility complex (MHC) class I-like related molecule 1 (MR1) restricted, the T cell receptor comprising any one of the following (a) to (d), and specifically recognizing an antigen expressed in breast cancer cells: (a) a TCR α chain comprising a CDR3 having an 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 several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, and which is encoded by a 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 several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 11; and and a TCR β chain comprising a 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 in the amino acid sequence shown in SEQ ID NO: 2, the TCR β chain being encoded by the 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 several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 12; (b) a TCR α chain comprising a CDR3 having an amino acid sequence (CALPSRLMF) 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 several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 3, and which 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 several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 13; and and a TCR β chain comprising a CDR3 having an amino acid sequence (CASSLTSIYEQFF) 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 several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4, the TCR β chain being encoded by a nucleotide sequence shown in SEQ ID NO: 14, or a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 14, or a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 14; (c) a TCR α chain comprising a CDR3 having an amino acid sequence shown in SEQ ID NO: 5 (CIVRVGPGYGGSQGNLIF) or an amino acid sequence having 95% or more identity to the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5, and which is encoded by a nucleotide sequence shown in SEQ ID NO: 15, a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 15, or a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 15 a TCR β chain comprising 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 several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 6, and which is encoded by the nucleotide sequence shown in SEQ ID NO: 16, or a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 16, or a nucleotide sequence in which one or several bases have been deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 16; (d) A TCR α chain comprising a CDR3 having an amino acid sequence (CAASMGNTPLVF) shown in SEQ ID NO: 7, or an amino acid sequence having 95% or more identity to the amino acid sequence shown in 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 in SEQ ID NO: 7, and which is encoded by a nucleotide sequence shown in SEQ ID NO: 17, or a nucleotide sequence having 90% or more identity to the nucleotide sequence shown in SEQ ID NO: 17, or a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 17. and a TCR β chain having a CDR3 having the amino acid sequence shown in SEQ ID NO: 8 (CSARVEKLFF) or an amino acid sequence having 95% or more identity to the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 8, wherein the TCR β chain is encoded by the base sequence shown in SEQ ID NO: 18, or a base sequence having 90% or more identity to the base sequence shown in SEQ ID NO: 18, or a base sequence in which one or more bases have been deleted, substituted or added in the base sequence shown in 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 MHC-restricted antigens. An α-β heterodimer TCR has a TCRα chain and a TCRβ chain. Therefore, the TCRα chain and the TCRβ chain each contain a CDR. The CDRs in the present disclosure can be identified by analysis using software provided by IMGT (IMGT is a registered trademark).

[0019] In the present disclosure, the "identity" of an amino acid sequence or a base sequence means the maximum identity (%) of the sequences obtained by aligning two sequences to be compared, with a gap introduced as necessary. The identity of an amino acid sequence or a base sequence can be calculated, for example, using blastn of NCBI BLAST (http: / / blast.ncbi.nlm.nih.gov / ) which implements the BLAST algorithm. The identity of the amino acid sequence described in the present disclosure is preferably 96% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. The identity of the base sequence described in the present disclosure is preferably 92% or more, more preferably 94% or more, even more preferably 96% or more, and particularly preferably 98% or more. In the present 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. In addition, in the present disclosure, "one or several bases" preferably means 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] The amino acid substitution is preferably a conservative substitution with an amino acid having a similar side chain. Examples of conservative substitution include substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine, substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid, substitution between amino acid residues having non-charged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine, substitution between amino acid residues having non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan, substitution between amino acid residues having β-branched side chains such as threonine, valine, and isoleucine, and substitution between amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine. In addition, the base substitution is preferably a substitution into a degenerate sequence that does not change the encoded amino acid. In addition, it is preferable that frameshift does not occur due to deletion, substitution, or addition of bases.

[0021] TCR genes are composed of gene fragments forming a V domain that generates molecular diversity, called a signal sequence, a variable region (V), a diversity region (D), and a joining region (J), which are cleaved when the TCR gene is expressed on the cell membrane surface, and a gene fragment forming a C domain consisting of a constant region (C) including an extracellular constant region, a transmembrane region, and an intracellular region. In the IMGT nomenclature, the α chain variable region (Vα) is identified by a TRAV number, the α chain joining region (Jα) is identified by a TRAJ number, the β chain variable region (Vβ) is identified by a TRBV number, and the β chain joining region (Jβ) is identified by a TRBJ number (IMGT is a registered trademark). These numbers can be identified using the IMGT database.

[0022] The TCR of the present disclosure preferably includes any one of the above (a) to (c) among the above (a) to (d). The TCR of the present disclosure preferably includes the following sequences. The TCR including the above (a) is TRAV1-2 * 01F, TRAJ33* 01F, TRBV6-1 * 01F, TRBJ2-7 * 01 F. The TCR containing (b) is preferably TRAV19 * 01 F, TRAJ29 * 01F, TRBV2 * 01F, TRBJ2-1 * 01 F. The TCR containing (c) is preferably TRAV26-1 * 01 F, TRAJ42 * 01F, TRBV5-4 * 01F, TRBJ2-5 * 01 F. The TCR containing (d) is preferably TRAV13-1 * 02(F), TRAJ29 * 01F, TRBV20-1 * 01F, TRBJ1-4 * It is preferred that it contains 01F.

[0023] The TCR of the present disclosure has MR1 ​​restriction. In this specification, "MR1 restriction" means recognizing an antigen presented by an 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 a vitamin derivative (5-OP-RU).

[0024] The TCR of the present disclosure specifically recognizes an antigen expressed in breast cancer cells, as shown in the examples below. Examples of breast cancer cell lines include MCF7 and MDA-MB-231. In addition, as shown in the examples below, the TCR of the present disclosure is inhibited from functioning impaired by ligands other than the specific antigen. As shown in the examples below, the TCR of the present disclosure is HLA-independent, and therefore can be applied to a wider range of TCR-T cell therapy targets for breast cancer.

[0025] The TCR of the present disclosure may be chemically modified as long as it can recognize an antigen expressed in breast cancer cells. The C-terminus of each chain of the TCR of the present disclosure is not particularly limited, and may be, for example, a carboxyl group (-COOH), a carboxylate (-COO-), an amide (-CONH2), or an ester (-COOR). Here, R in the ester may be, for example, a C group such as methyl, ethyl, n-propyl, isopropyl, or n-butyl. 1-6 Alkyl groups; for example, cyclopentyl, cyclohexyl, etc. 3-8 Cycloalkyl groups; for example, C phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl group; α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 It may be an aralkyl group; a pivaloyloxymethyl group, etc. Furthermore, in each chain of the TCR of the present disclosure, a carboxyl group or a carboxylate other than that at the C-terminus may be amidated or esterified. The esterification is not particularly limited, but may be, for example, the above-mentioned C-terminal ester, etc. Furthermore, in each chain of the TCR of the present disclosure, the amino group of the amino acid residue at the N-terminus may be protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1-6 Alkanoyl C 1-6 those protected by protective groups (e.g., C-acyl groups, etc.) in the N-terminus of the glutamine residue that may be cleaved in vivo and be formed by pyroglutamylation; those protected by protective groups (e.g., C-formyl groups, acetyl groups, etc.) in the side chains of amino acids in the molecule (e.g., -OH, -SH, amino groups, imidazole groups, indole groups, guanidino groups, etc.) in the formyl groups, acetyl groups, etc. 1-6 Alkanoyl group etc. 1-6 The TCR of the present disclosure may be protected with a cytosine residue (e.g., an acyl group). In addition, the TCR of the present disclosure may be bound to an intracellular domain such as CD3-Zeta chain, CD28, or CD137.

[0026] The TCR of the present disclosure may be one to which a known protein tag, protein or peptide such as a signal sequence, or a labeling substance is added, so long as it can recognize an antigen expressed in breast cancer cells. Examples of protein tags include, but are not limited to, biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, etc. Examples of signal sequences include, but are not limited to, nuclear localization signals, etc.

[0027] The method for producing the TCR of the present disclosure is not particularly limited, and may be produced as follows. For example, the TCR of the present disclosure may be produced by a method including a step of culturing a host transformed with a polynucleotide encoding the TCR of the present disclosure. The host is not particularly limited, and may be, for example, a peripheral blood mononuclear cell, preferably a lymphocyte, more preferably a T cell, etc. The method for transformation and culture is not particularly limited, and may be, for example, a known method for producing a TCR.

[0028] According to another aspect of the present disclosure, there is provided a polynucleotide encoding the TCR of the present disclosure. The polynucleotide of the present disclosure is not particularly limited as long as it contains the TCR of the present disclosure in an expressible state.

[0029] In the polynucleotide of the present disclosure, it is preferable that the region encoding the α chain or the like and the region encoding the β chain or the like are expressed as a single polypeptide so that the coding regions are linked via a linker that is cleaved after expression, so that the TCR of the present disclosure can be expressed more efficiently. For the same reason, it is also preferable that the polynucleotide of the present disclosure comprises a region encoding a short-chain RNA that suppresses the expression of endogenous TCR. Examples of the short-chain RNA include, but are not limited to, siRNA and miRNA.

[0030] The polynucleotide of the present disclosure may also be introduced into a vector. That is, according to another embodiment of the present disclosure, a vector containing the polynucleotide of the present disclosure is provided. The vector is not particularly limited, but is preferably a viral vector, and more preferably a retroviral vector. The vector of the present disclosure may contain other sequences in addition to the polynucleotide of the present disclosure. The other sequences are not particularly limited, but include, for example, a secretory signal peptide coding sequence, 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, a drug resistance gene coding sequence, and the like. Among these, it is preferable to include a reporter protein coding sequence from the viewpoint that the cells into which the polynucleotide of the present disclosure has been introduced can be relatively easily detected, selected, concentrated, and the like, for example, by FACS analysis, etc.

[0031] According to another embodiment of the present disclosure, a cell expressing the TCR of the present disclosure is provided. Examples of the cell expressing the TCR of the present disclosure include a cell containing the polynucleotide of the present disclosure and a cell containing a vector into which the polynucleotide of the present disclosure has been introduced. Such a vector may contain two vectors, a first vector containing a polynucleotide encoding the TCRα chain and a second vector containing a polynucleotide encoding the TCRβ chain. The cell expressing the TCR of the present disclosure may also be a cell induced to differentiate from a pluripotent stem cell into which the polynucleotide or vector of the present disclosure has been introduced. From the viewpoint of increasing the expression amount of the TCR of the present disclosure and suppressing mispairing with the endogenous TCR chain, it is preferable that the cell expressing the TCR of the present disclosure lacks the expression of endogenous TCR. For example, it is preferable that the expression of the endogenous TCRα chain and TCRβ chain is suppressed by short RNA such as siRNA or miRNA.

[0032] In the cell expressing the TCR of the present disclosure, the TCR of the present disclosure is preferably expressed on the cell membrane, and the TCR of the present disclosure is preferably expressed in a state where its variable region is exposed outside the cell membrane. The cell expressing the TCR of the present disclosure is not particularly limited, but for example, peripheral blood mononuclear cells, preferably lymphocytes, more preferably T cells, and even more preferably CD8 positive T cells can be mentioned. The cell expressing the TCR of the present disclosure is preferably a cell derived from a patient who is to be treated or prevented from breast cancer. In addition, when using insect cells, eukaryotic cells, mammalian cells, etc. as the cell, it is preferable to simultaneously express CD3 or to combine the extracellular domain and the intracellular signaling domain, etc.

[0033] According to another embodiment of the present disclosure, a preventive or therapeutic agent for breast cancer is provided. The preventive or therapeutic agent for breast cancer in the present disclosure (hereinafter also referred to as "agent of the present disclosure") includes the TCR of the present disclosure, or a polynucleotide encoding the TCR of the present disclosure, or a vector containing the polynucleotide of the present disclosure, or a cell expressing the TCR of the present disclosure. Examples of breast cancer include MCF7 and MDA-MB-231. The subject of the prevention or treatment of breast cancer is preferably a human, but may also be other mammals (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.).

[0034] The content of the active ingredient in the agent of the present disclosure can be appropriately set taking into consideration the type of breast cancer to be treated, the desired therapeutic effect, the administration method, the treatment period, the patient's age, and the patient's body weight, etc.

[0035] The administration form of the agent of the present disclosure is not particularly limited as long as the desired effect can be obtained, but parenteral administration (e.g., intravenous injection, intramuscular injection, subcutaneous administration) is preferable. The manufacturing method of the agent of the present disclosure is not particularly limited, and it can be manufactured according to a conventional method by mixing an active ingredient and a pharma- ceutically acceptable carrier, etc.

[0036] Dosage forms for parenteral administration include injection preparations (e.g., drip injections, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections), etc. For example, an injection preparation may be prepared by suspending the cells expressing the TCR of the present disclosure in an injection saline solution, and adding a solubilizing agent, a buffer, a pH adjuster, an isotonic agent, a pain-reducing agent, a preservative, a stabilizer, etc. as necessary. Carriers used in formulating the agent of the present disclosure are not particularly limited, and examples thereof include excipients, binders, disintegrants, lubricants, colorants, flavoring agents, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, bulking agents, wetting agents, surface activators, dispersants, buffers, preservatives, pain-reducing agents, etc.

[0037] The dosage of the agent of the present disclosure may be determined by a physician based on various factors, such as the route of administration, the type of breast cancer, the degree of symptoms, the age, sex, and weight of the patient, the severity of the disease, pharmacological findings such as pharmacokinetics and toxicological characteristics, the presence or absence of a drug delivery system, and whether the agent is administered as part of a combination of other drugs. The administration schedule of the agent of the present disclosure may be determined taking into account factors similar to the dosage, and may be, for example, administered once a day to once a month at the above-mentioned daily dosage. The agent of the present disclosure may be used in combination with other pharmaceutical compositions intended for the prevention or treatment of breast cancer. EXAMPLES

[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] 1. Materials and Methods (1) Reagents Acetyl-6-FP (Cayman Chemicals) was dissolved at 2.0 mg / mL in 10 mM NaOH. 5-A-RU (Toronto Research Chemicals) was combined with 50 μM methylglyoxal (Sigma) prior to each assay to obtain 5-OP-RU. 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 from eBioscience. APC-labeled human HLA-A, B, C mAb (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 from BioLegend. Human MR1 tetramer was from MBL. pMXs-IRES-GFP retroviral expression vector (RTV-013) was from Cell Biolabs.

[0040] (2) Cell line BW5147.3 cells expressing mouse CD8αβ were kindly provided by Prof. Ellis L. Reinherz, Dana Farber Cancer Institute, Harvard Institutes of Medicine. BW5147.3 cells were maintained in Dulbecco's modified Eagle's 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 hygromycin B and 0.4 mg / mL G418. cDNAs for human CD8α and CD8β were introduced into BW5147.3 cells using retroviral vectors. In the following description, these cells are referred to as "BW-hCD8αβ". + MCF7 cells were purchased from ATCC and maintained in DMEM medium. Human CD80 and β2-microglobulin cDNA were transfected into the cells using retroviral vectors (human CD80 + Beta-2 microglobulin +MCF7 cells) were used to introduce the cDNA of patient HLA-A, B, and C together with cDNA of green fluorescent protein (GFP) was introduced into MCF7 cells using a retroviral vector, and GFP-positive cells were sorted as patient HLA-introduced cells using FACSAriaII (Beckton Dickinson). Luciferase-expressing MCF7 cells (hereinafter also referred to as "MCF7-Luc cells") were generated 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 FACSAriaII. K562, Jurkat, Karpas299, COLO205, A549, MDA-MB-231, MDA-MB-453, MDA-MB468, BT474, and ZR-75-1 cell lines were all used from laboratory stocks. A549, MDA-MB-231, MDA-MB-453, MDA-MB468, and BT474 cells were maintained in Dulbecco's modified Eagle's 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 calf serum (FCS), 50 μmol / l 2-mercaptoethanol, streptomycin (100 μg / ml), and penicillin (100 U / ml). Human mammary epithelial cells (HMEC) were purchased from ATCC and maintained in basal growth medium.

[0041] (3) Identification of TCR HLA class I-independent TCRs were identified from CD8-positive tumor-infiltrating lymphocytes (TILs) from two breast cancer patients. Specifically, tumor specimens were cut, washed, and then digested with Liberase DH solution (Roche). Cells filtered through a cell strainer (Becton Dickinson) were frozen at -80°C in Cellbanker-1 medium (Nihon 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.). 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 excluded using APC-Cy7-Fixable Viability Dye (eBioscience). After staining, cells were analyzed using a FACSAria II (Beckton Dickinson). PD-1 + CD137 + CD8 + Single T cells were sorted into 96-well PCR plates. The expression of HLA class I or class I-like molecules in MCF7 cells was analyzed using a FACSCanto (Beckton Dickinson) by staining with either APC-anti-HLA-A, B, C antibodies, anti-CD1a, b, c, d antibodies, or anti-MR1 antibodies. When analyzing the expression of MR1, MCF7 cells were pulsed with 1 μg / mL Ac-6-FP overnight and stained the following day. TCR cDNA was amplified from single T cells by one-step RT-PCR and second PCR. The DNA sequence of the PCR product was then analyzed by direct sequencing. The sequences of the TCRs identified and used in subsequent experiments are shown in Table 1 below.

[0042] [Table 1]

[0043] (4) Generation of HLA-E and TAP1 / 2 knockout MCF7 cells The HLA-E and TAP1 / 2 genes were knocked out from MCF7 cells using CRISPR-Cas9 KO plasmid (Santa Cruz Biotechnology) 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, C monoclonal antibody (mAb) (W6 / 32) and HLA class I negative cells were sorted using FACSAriaII. 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, TCR expression vector was constructed as previously described: TCRβ PCR fragment, codon-optimized gene encoding human TCRβ constant region-1 linked with self-cleaving P2A peptide (Cb1-P2A-fragment), TCRα PCR fragment, and codon-optimized TCRα constant region gene (Ca-fragment) were assembled together into linearized 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 retroviral production.

[0045] (6) Retrovirus production Phoenix A cells (kindly provided by Dr. Garry Nolan (Stanford University)) were used to prepare retroviruses for introducing TCR genes into human PBMCs. The day before transfection, Phoenix A cells (3.7 × 10 6) 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 cultured at 37°C under a 5% CO2 atmosphere. The cell medium was replaced the next day. After 2 days, 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 produced using Plat E cells (provided by Dr. Toshio Kitamura (University of Tokyo)).

[0046] (7) Isolation of primary T cells and retroviral transduction of TCR into primary T cells Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (Promocell) density gradient centrifugation of buffy coats from healthy donors. PBMCs were maintained in RPMI 1640 containing 10% FCS, 50 μmol / L 2-mercaptoethanol, 100 μg / mL streptomycin, and 100 U / mL penicillin. For TCR transduction, 1 × 10 6 PBMCs were stimulated in vitro for 2 days with CD3 / CD28 Dynabeads (Invitrogen) in the presence of human IL-2 (PeproTech). PBMCs were harvested and cultured at 5 × 10 in RPMI medium in the presence of human IL-2 (30 U / ml). 5The wells were resuspended at 1000×g / ml. For retroviral transduction, wells of a 24-well plate were coated with 0.3 ml of Retronectin (50 μg / ml) (Takara) overnight at 4°C. The following day, the culture supernatant containing the retrovirus was added to the wells and centrifuged at 1900×g for 2 hours at 32°C according to the manufacturer's guidelines to adsorb the TCR-encoding retrovirus to the wells. The suspended PBMC cells were then added to the retrovirus-loaded adsorbed wells, spun down at 1000×g for 10 minutes at 32°C, and incubated overnight at 37°C with 5% CO2. The PBMCs were then transferred to a freshly prepared retroviral-coated plate and incubated at 37°C with 5% CO2. The following day, the TCR-transduced PBMCs were grown for an additional 2 days in the presence of human IL2 (30 U / ml). The TCR-transduced PBMCs were harvested and stored at -80°C until use. For TCR transduction into mouse splenic T cells, splenic cells were stimulated in vitro for 2 days with mouse T-Activator CD3CD28 Dynabeads (Invitrogen) in the presence of recombinant mouse IL-2 (PeproTech). The stimulated splenic cells were infected with a retrovirus encoding the human TCR in the same manner as for retroviral transduction of TCR into human PBMCs.

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

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

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

[0050] (11) Evaluation of human IFN-γ secretion by ELISA PBMCs (1 × 10 5 ) treated with human IFN-γ and 5 MCF7 cells were co-cultured in 200 μl of RPMI 1640 medium in a 96-well plate. After 24 hours of culture, the supernatant was collected and IFNγ production was measured as a result of the T cell response by ELISA (R&D Systems, DY285B) according to the manufacturer's instructions.

[0051] (12) Evaluation of MR1 T cell stimulation by ELISA BW-hCD8αβ cells (1 × 10 5 ) were cultured in 200 μl of DMEM culture medium in the presence of mouse IL-1 alpha and human IFN-γ-treated 1 × 10 5MAIT cells were co-cultured with MCF7 cells in a 96-well plate. 5-OP-RU was added to the medium to stimulate MAIT cells. Ac-6-FP and anti-MR1 antibody were added to inhibit MAIT cells, and the cells were cultured. After 24 hours of culture, the supernatant was collected and IL2 production as a result of MAIT cell stimulation and inhibition was measured by ELISA (R&D Systems, DY285B) according to the manufacturer's instructions.

[0052] (13) Cytotoxicity assay For the cytotoxicity assay, TCR-expressing PBMCs were co-cultured with luciferase-expressing MCF7 cells. * The 59:01-restricted TCR(e) was introduced into peripheral blood lymphocytes of healthy donors via retroviral vectors. In addition, lymphocytes without TCR introduced from outside were used as negative controls (TCR-ve). * Wild-type MCF7 cells that do not express 59:01, HLA-B * MCF7 cells expressing 59:01 and MCF7 cells in which MR1 was knocked out, ΔMR1 cells, were used. The luciferase gene was introduced into the MCF7 cells using a retrovirus and expressed (Yamaguchi, Eur J Immunol). MCF7-Luc cells (1 × 10 4 ) and TCR-expressing PBMCs were co-cultured in 96-well plates at various effector-to-target (E / T) ratios and incubated for 24 h. Cytotoxicity against MCF7-Luc cells was assessed by measuring cell-associated luciferase activity using the Steady-Glo luciferase assay system (Promega, E2520).

[0053] (14) Statistical analysis Data are presented as mean ± SD, and statistical significance was 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 FIG. 1 is an explanatory diagram showing the results of hMR1 tetramer staining of BW hCD8αβ+ cells expressing TCR. In FIG. 1, flow cytometry dot plots are shown for each TCR, control (no tetramer), hMR1 / 5-OP-RU, and hMR1 / 6-FP. In FIG. 1, a representative result of two independent experiments is shown. As shown in FIG. 1, BW cells expressing TCR(a) bound to 5-OP-RU / MR1 tetramer. On the other hand, in BW cells expressing other TCRs, no binding to hMR1 tetramer was observed in either hMR1 / 5-OP-RU or hMR1 / 6-FP.

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

[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) shows HLA-ABC (HLA class I), (B) shows HLA-E, (C) shows CD1 molecules, and (D) shows MR1 ​​molecules. In Figure 2(D), Isotype shows the negative control, and Mock shows mock cells as a control. As shown in Figure 2(A)-(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 molecule) is expressed in tumor cells, and its expression is inducible by IFN-γ (Seliger, Barbara et al. Oncotarget. 2016;7(41): 67360-67372.). As shown in Figure 2(B), when MCF7 cells were stimulated with IFN-γ, HLA-E molecules were expressed. Regarding the expression of MR1, MCF7 cells were stimulated with MR1 ligands 5-OP-RU and Ac-6-FP, and the expression of MR1 was examined in a dose-dependent manner. Ac-6-FP (acetylated derivative of folic acid derivative 6-formylpterin, 6-FP) has strong MR1 binding ability, and this MR1 ​​ligand can induce the expression of MR1 (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 MR1 molecules.

[0057] Figure 3 is a histogram showing the increase in MR1 surface expression in MCF7 cells. In Figure 3, (A) shows the results of 5-OP-RU at different doses, (B) shows the results of Ac-6-Fp at different doses, and (C) shows the results of β2m-deficient MCF7 cells and MR1-deficient MCF7 cells. As shown in Figure 3(B), the MR1 ligand Ac-6-Fp increased the expression of MR1 molecules in a dose-dependent manner. On the other hand, as shown in Figure 3(A), 5-OP-RU did not increase the expression of MR1 molecules. Also, as shown in Figure 3(C), the expression of MR1 molecules was not observed in β2m-deficient MCF7 cells or MR1-deficient MCF7 cells. This is thought to be due to the fact that MR1 expression is associated with β2-microglobulin (Godfrey, Nat Immunol, 16:1114, 2015).

[0058] FIG. 4 is a histogram showing the expression of MR1 in various cell lines. FIG. 5 is a histogram showing the expression of HLA-E and CD1 molecules in various cell lines. FIG. 4(A) shows the results for breast cancer cell lines other than MCF7 cells, and FIG. 4(B) shows the results for cancer cell lines other than breast cancer (colon, lung, myeloid, and lymphoid cell lines). As shown in FIG. 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 splenic T cells and human PBMCs and their reactivity to MCF7 cells Previously, we have analyzed reactivity by expressing TCR in BW T cells, and we now examine the reactivity when the TCR is 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 splenic T cells expressing each TCR by transformation with IFN-γ stimulated MCF7 cells, and Figure 6(B) shows the results of co-culture of human PBMC from a healthy individual expressing each TCR by transformation with IFN-γ stimulated MCF7 cells. IFN-γ production was measured by ELISA. PBMC without TCR (TCR-ve) were used as a negative control. n=3.

[0061] As shown in FIG. 6, TCR(e) binds to HLA-B * The TCR(e) gene was expressed only in MCF7WT expressing HLA-B 59:01. * 59:01. In contrast, as shown in FIG. 6(A), when TCR(a)-(d) were expressed in BW T cells, they reacted to MCF7 cells independent of HLA. In addition, when TCR(a)-(d) were expressed in mouse T cells and secreted mouse IFN-γ, all of them reacted to MCF7 cells. As shown in FIG. 6(B), TCR(c) also reacted to MCF7 cells when expressed on human PBMC. In contrast, TCR(a), (b), and (d) did not react to MCF7 cells when expressed on human PBMC. The failure of TCR(a), (b), and (d) to react to MCF7 cells is presumably due to incompatibility with endogenous TCR.

[0062] (4) Reactivity to MCF7 cells in which each factor was knocked out Detailed analysis was performed on TCR(c). To clarify the antigen-presenting molecules of HLA-independent TCR(c), first, the HLA-I, β2-microglobulin, and MR1 genes were knocked out from MCF7 cells using the CRISPR / 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 CRISPR / Cas9 system (hereinafter also referred to as "MCF7ΔHLA-E"). As shown in FIG. 2(B) above, when MCF7 cells were treated with hIFN-γ, the expression of HLA-E was confirmed.

[0063] Figures 7 and 8 are explanatory diagrams showing the reactivity to MCF7 cells in which each factor was knocked out. The results of co-culture of PBMC from a healthy individual in which each TCR was expressed by transformation with IFN-γ-stimulated MCF7 cells are shown. Figure 7(A) shows the results of TNF-α production, and Figures 7(B) and 8 show the results of IFN-γ production, measured by ELISA. PBMC without TCR (TCR-ve) were used as a negative control, and T cells stimulated with PMA + ionomycin were used as a positive control. n 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 showed reactivity to MCF7 cells in an HLA-independent manner, whereas conventional TCR(e) reacted to MCF7 cells in an HLA-dependent manner. TCR(c) did not react to MCF7 cells lacking β2-microglobulin or the MR1 gene. As shown in Figure 8, TCR(c) showed reactivity to MCF7ΔHLA-E cells but not to MCF7ΔMR1 cells.

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

[0066] Figure 9 is an explanatory diagram showing the cytotoxicity against MCF7 cells. * The results for wild-type MCF7 cells that do not express 59:01 are shown in Fig. 9(B), and HLA-B * FIG. 9(C) shows the results for MCF7 cells expressing 59:01, and FIG. 9(C) shows the results for MCF7 cells ΔMR1 cells in which MR1 was knocked out. As shown in FIG. 9, TCR(c) was found to cytotoxicize MCF7 cells in an HLA-independent manner, and to have no cytotoxic effect on MCF7ΔMR1. This indicates that TCR(c) cytotoxicizes MCF7 cells in an HLA-independent manner restricted by MR1.

[0067] (6) Reactivity to cancer cell lines We investigated what types of tumor cells HLA-independent TCR(c) reacts with.

[0068] Figure 10 is an explanatory diagram showing the reactivity 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. Human cancer cell lines, including 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 and cultured for 24 hours. TCR-unintroduced PBMCs (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 FIG. 10, TCR(c) expressed in human PBMC reacted not only to MCF7 breast cancer cells but also to MDA-MB231 breast cancer cells. In contrast, it did not react to other cancer cell lines (Colo205 colon cancer cells, A549 lung cancer cells, K562 myeloid cells, and Karpas299 lymphoid cells). In addition, as shown in FIG. 4(A) above, breast cancer cell line ZR-75-1 showed higher surface expression of MR1 than MDA-MB231 cells, but as shown in FIG. 10, TCR(c) did not react to ZR-75-1. Thus, it was found that the HLA-independent TCR of the present disclosure reacts to an antigen specifically expressed at least in MCF7 breast cancer cells and MDA-MB231 breast cancer cells.

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

[0071] FIG. 11 is an explanatory diagram showing the amount of IL2 production in the presence or absence of MR1 ligand. In FIG. 11, "+" indicates the presence of addition, and "-" indicates the absence of addition. As shown in FIG. 11, when co-cultured with MCF7 cells without the addition of MR1 ligand, IL2 was produced in both TCRs, TCR(a) and TCR(c). In addition, 5-OP-RU increased IL2 production in TCR(a), but did not show any effect of increasing IL2 production in TCR(c). Although Ac-6-FP is a non-stimulatory ligand for MAIT cells, it significantly suppressed BW MAIT activity against the agonist ligand (5-OP-RU). Similarly, anti-MR1 antibody also inhibited only the response to the agonist ligand (5-OP-RU). This discriminatory function is likely due to the fact that 5-OP-RU contains a ribityl tail that is primarily recognized by the TCR of MAIT cells, whereas Ac-6-Fp does not (Corbett, A., et al. Nature (2014); Eckle et al., 2014). This agonistic and antagonistic effect of these MR1 ligands was observed in the response of TCR(a) to 5-OP-RU, whereas it was ineffective in the response of TCR(a) and TCR(c) to MCF7 cells. This data suggests that the antigens expressed on MCF7 cells of the HLA-independent TCR have different binding sites for known microbial antigens, 5-OP-RU or Ac-6-FP.

[0072] (8) Reactivity against 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). To confirm whether the response of TCR(e) and (c) to MCF7 is TAP-dependent or independent, we deleted the TAP1 / 2 genes from MCF7 cells using the CRISPER / Cas9 system (MCF7ΔTAP1 / 2). MCF7 cells were stimulated overnight with the indicated concentrations of Ac-6-FP, and the expression of MR1 surface was analyzed by staining the cells with anti-MR1 antibody. We next analyzed the effect of TAP1 / 2 gene deletion on the stimulation of MR1 T cells (canonical MAIT TCR and atypical MR1-TCR). BW cells expressing TCR(e) (canonical MAIT TCR) and TCR(c) (atypical MR1-TCR) were co-cultured with MCF7 and MCF7ΔTAP1 / 2 cells, and IL2 secretion was measured by ELISA. n=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 in MCF7ΔTAP1 / 2 cells was reduced to about 50% compared to wild-type MCF7 cells.

[0074] Figure 13 is an explanatory diagram showing the amount of IL2 production. As shown in Figure 13, IL2 production was observed in both TCR(e) and TCR(c), and the production was reduced in MCF7ΔTAP1 / 2 cells. These data indicate that MR1-restricted antigen recognition by TCR(e) and (c) is independent of TAP.

[0075] (9) TCR reactivity to normal breast cells Since TCR(c) only reacts with breast cancer cell lines, further evaluation was performed with healthy breast cells to confirm the effect of TCR(c) on normal cells. For this evaluation, human mammary epithelial cells (HMECs), TCR(e) and TCR(c) were used. IFN-γ production was measured by ELISA. TCR-untransduced PBMCs (TCR-ve) were used as negative control, and T cells stimulated with PMA + ionomycin (PMA / IM) were used as positive control. n=2.

[0076] FIG. 14 is an explanatory diagram showing the reactivity of TCR to breast cancer cells and normal breast cells. As shown in FIG. 14, TCR(c) showed reactivity to MCF7 cells but did not show reactivity to normal breast cells. In other words, normal breast cells did not activate TCR(c). As a result, it is considered that HLA-independent TCR(c) did not react to normal breast cells. Note that TCR(e) was activated by the patient's HLA(B * The antibody showed reactivity against MCF7 cells expressing 59:01.

[0077] The present invention is not limited to the above-mentioned embodiment, and can be realized in various configurations without departing from the spirit of the present invention. 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 column can be appropriately replaced or combined in order to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted.

Claims

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

18.

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

3. The T cell receptor of claim 1, wherein the breast cancer cell is MCF7 or MDA-MB-231.

4. A polynucleotide encoding the T cell receptor of claim 1.

5. A vector comprising the polynucleotide of claim 4.

6. A cell expressing the T cell receptor of claim 1.

7. The cell of claim 6 , which lacks expression of an endogenous T cell receptor.

8. 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 claim 7. A preventive or therapeutic agent for breast cancer.

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