JP2024051333A - Cytotoxic T cells and method for producing same - Google Patents

Abstract

[Problem] To provide TCR gene modified T cells that target cancer cells that present peptides derived from hTERT. [Solution] The TCR gene modified T cells have a polypeptide containing an α chain variable region and a polypeptide containing a β chain variable region of a T cell receptor protein that specifically binds to a complex of a peptide derived from human telomerase reverse transcriptase (hTERT) and the HLA-A24 antigen. [Selected Figures] None

Description

The present invention relates to immunotherapy for cancers such as hepatocellular carcinoma, and more specifically to cytotoxic T cells that have cytotoxicity against cancer cells, a method for producing the same, and a cancer therapeutic agent that contains the cytotoxic T cells.

Genetically modified T cells are artificial lymphocytes that have been given high specificity for cancer antigens by introducing and forcibly expressing a T cell receptor (TCR) that recognizes cancer antigens bound to the antigen-presenting apparatus of cancer cells, or a fusion protein (chimeric antigen receptor (CAR) gene) made of the target antigen recognition portion of an antibody that recognizes cancer cells and a lymphocyte activation molecule. The former are called TCR gene modified T cells (TCR-T), and the latter chimeric antibody receptor gene modified T cells (CAR-T).

In particular, the therapeutic method using TCR-T is a treatment method that allows the immune response that was effective in one individual to be utilized in another individual. The above method is used to treat patients with advanced melanoma using tumor-associated antigens (TAA)-specific TCRs (Non-Patent Documents 1 and 2), and is currently being applied to clinical trials of various patients (Non-Patent Document 3), with good clinical results expected.

The present inventors have previously identified an HLA-A24-restricted tumor antigen peptide that can be administered as a vaccine to hepatocellular carcinoma patients (Patent Document 1). On the other hand, the present inventors have discovered a technique for rapidly cloning and expressing individual TCRs from lymphocytes in order to screen for TCR genes suitable for use in therapeutic methods using TCR-T, and have also reported on the application of the above peptide to lymphocytes derived from patients who have been administered the vaccine (Non-Patent Document 4).

Furthermore, the present inventors have obtained AFP-specific TCR genes from T cells in peripheral blood of hepatocellular carcinoma patients who received an α-fetoprotein (AFP)-derived peptide vaccine, as well as from healthy donors, using the TCR cloning system described in Non-Patent Document 4, and have created AFP-specific TCR gene-introduced T cells, which have been evaluated and compared in function (Patent Document 2). The AFP-specific TCR gene-introduced T cells disclosed in Patent Document 2 have cytotoxic activity against cells that express both α-fetoprotein and HLA-A24.

On the other hand, telomerase reverse transcriptase (TERT), which constitutes a telomerase subunit, contributes to maintaining telomere length and genome stability, and is known to be expressed at extremely low levels or not at all in normal somatic cells. However, increased expression of TERT occurs in various types of cancer, and this change is thought to be involved in the immortalization of cancer cells. It is known that mutations in the promoter region are involved in increased TERT expression. These mutations cause new binding sites for the ETS transcription factor, resulting in increased expression of TERT.

Non-Patent Document 5 discloses that HLA-A ^* 2402-restricted cytotoxic T cell (CTL) epitopes of human TERT (hTERT) in hepatocellular carcinoma patients were identified and hTERT-specific CTL responses were analyzed. Furthermore, Non-Patent Document 6 discloses the preparation of an hTERT-derived peptide vaccine and its efficacy.

International Publication No. WO2005/083074 JP 2017-081836 A

Morgan RA et al., Science 2006, Vol. 314, pp. 126-129 Johnson LA et al., Blood 2009, Vol.114, No.3, pp.535-546 Linnemann C et al., J Invest Dermatol 2011, Vol.131, pp.1806-1816 Kobayashi E et al., Nature Medicine, 2013, Vol.19, No.11, pp.1542-1546 Mizukoshi E et al., HEPATOLOGY 2006;43: 1284-1294 Mizukoshi E et al., Cancer Letters 364:98-105, 2015.

However, no TCR gene-modified T cells were known to have cytotoxic activity against cancer cells with increased hTERT expression, and the problem of cancer therapy targeting cancer cells that present hTERT-derived peptides remained unsolved.

In light of the above-mentioned circumstances, the present inventors used a rapid TCR cloning system developed by the present inventors' group (Kobayashi E et al., Nat Med 2013;19:1542-1546) to obtain hTERT-specific TCR genes from T cells in peripheral blood of subjects administered a telomerase reverse transcriptase (hTERT)-derived peptide vaccine, and produced hTERT-specific TCR gene-introduced T cells, which were then evaluated and compared in function. These TCR gene-introduced cells had excellent cytotoxic activity against cells expressing both hTERT and HLA-A24. The present invention was made based on this finding.

That is, the present invention includes the following.
(1) A polypeptide including an α chain variable region and a β chain variable region of a T cell receptor protein that specifically binds to a complex of a peptide derived from human telomerase reverse transcriptase (hTERT) and an HLA-A24 antigen,
The α chain variable region is a polypeptide of the following (i) or (ii):
(i) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19.
(ii) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19. The β-chain variable region is a polypeptide of the following (iii) or (iv):
(iii) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37.
(iv) a polypeptide having an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37.
TCR gene-modified T cells.
(2) The TCR gene modified T cell described in (1), characterized in that the alpha chain variable region and the beta chain variable region are expressed from a polynucleotide introduced into the T cell.
(3) The TCR gene modified T cell described in (1), characterized in that a polynucleotide encoding the alpha chain variable region and a polynucleotide encoding the beta chain variable region are introduced in vitro into T cells of a patient suffering from cancer.
(4) The TCR gene modified T cell according to (3), characterized in that the patient's cancer cells are cancer cells expressing the above complex.
(5) The polynucleotide encoding the α chain variable region is
[1] A nucleic acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary nucleic acid sequence thereof; or
[2] A polynucleotide encoding the β chain variable region comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary base sequence thereto.
[3] A nucleic acid sequence represented by any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34, and 36, or a complementary nucleic acid sequence thereof; or
[4] A TCR gene modified T cell according to (2), characterized in that it comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34 and 36, or a complementary base sequence thereto.
(6) The α chain variable region and the β chain variable region are in the following combination:
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence; or a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
The TCR gene modified T cell according to (1), characterized in that it is any one of the following:
(7) The TCR gene modified T cell according to (1), wherein the polypeptide consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17 and 19 in (ii) above is an amino acid sequence containing the CDR3 sequences shown in SEQ ID NOs: 38 to 46, respectively.
(8) The TCR gene modified T cell according to (1), wherein the polypeptide consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35 and 37 in (iv) above is an amino acid sequence containing the CDR3 sequences shown in SEQ ID NOs: 47 to 55, respectively.
(9) The TCR gene modified T cell according to (1), characterized in that the human telomerase reverse transcriptase (hTERT) derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
(10) The TCR gene modified T cell according to (1), characterized in that it has cytotoxicity against cancer cells expressing the above complex.

(11) A step of introducing into human T cells in vitro a polypeptide comprising an α-chain variable region and a polypeptide comprising a β-chain variable region of a T cell receptor protein that specifically binds to a complex of a peptide derived from human telomerase reverse transcriptase (hTERT) and an HLA-A24 antigen, or a polynucleotide encoding these polypeptides:
The α chain variable region is a polypeptide of the following (i) or (ii):
(i) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19.
(ii) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19. The β-chain variable region is a polypeptide of the following (iii) or (iv):
(iii) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37.
(iv) A method for producing a TCR gene-modified T cell comprising a polypeptide having an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37.
(12) The method for producing TCR gene-modified T cells according to (11), wherein the human T cells are T cells derived from a patient suffering from cancer.
(13) The method for producing TCR gene-modified T cells according to (12), characterized in that the cancer cells of the patient are cancer cells that express the complex.
(14) The polynucleotide encoding the α chain variable region is
[1] A nucleic acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary nucleic acid sequence thereof; or
[2] A polynucleotide encoding the β chain variable region comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary base sequence thereto.
[3] A nucleic acid sequence represented by any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34, and 36, or a complementary nucleic acid sequence thereof; or
[4] A method for producing a TCR gene-modified T cell according to (11), characterized in that the TCR gene-modified T cell comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34 and 36, or a complementary base sequence thereof.
(15) The α chain variable region and the β chain variable region are selected from the following combinations:
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence; or a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
The method for producing a TCR gene-modified T cell according to (11), characterized in that the TCR gene-modified T cell is any one of the following:
(16) The method for producing TCR gene-modified T cells according to (11), wherein the polypeptides in (ii) above having an amino acid sequence that has 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17 and 19 are amino acid sequences that contain the CDR3 sequences shown in SEQ ID NOs: 38 to 46, respectively.
(17) The method for producing TCR gene-modified T cells according to (11), wherein the polypeptides in (iv) above consisting of amino acid sequences having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35 and 37 are amino acid sequences containing the CDR3 sequences shown in SEQ ID NOs: 47 to 55, respectively.
(18) The method for producing TCR gene-modified T cells according to (11), characterized in that the human telomerase reverse transcriptase (hTERT)-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1.
(19) The method for producing TCR gene-modified T cells according to (11), characterized in that the TCR gene-modified T cells have cytotoxicity against cancer cells expressing the complex.

(20) A method for treating a T cell receptor protein comprising the steps of: (a) detecting a human telomerase reverse transcriptase (hTERT)-derived peptide in a human T cell receptor protein; and (b) detecting a human T cell receptor protein in a human T cell receptor protein. The method comprises the steps of: (a) detecting a human telomerase reverse transcriptase (hTERT)-derived peptide in a human T cell receptor protein;
The α chain variable region is a polypeptide of the following (i) or (ii):
(i) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19.
(ii) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19. The β-chain variable region is a polypeptide of the following (iii) or (iv):
(iii) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37.
(iv) An expression vector which is a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35 and 37.
(21) The polynucleotide encoding the α chain variable region is
[1] A nucleic acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary nucleic acid sequence thereof; or
[2] A polynucleotide encoding the β chain variable region comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary base sequence thereto.
[3] A nucleic acid sequence represented by any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34, and 36, or a complementary nucleic acid sequence thereof; or
[4] The expression vector according to (20), characterized in that it comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34 and 36, or a complementary base sequence thereof.
(22) The α chain variable region and the β chain variable region are selected from the following combinations:
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence; or a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
The expression vector according to (20), which is any one of the following:
(23) The expression vector according to (20), wherein the polypeptide having an amino acid sequence having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17 and 19 in (ii) above is an amino acid sequence containing the CDR3 sequences shown in SEQ ID NOs: 38 to 46, respectively.
(24) The expression vector according to (20), wherein the polypeptide having an amino acid sequence having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35 and 37 in (iv) above is an amino acid sequence containing the CDR3 sequences shown in SEQ ID NOs: 47 to 55, respectively.
(25) A cancer therapeutic agent comprising the TCR gene-modified T cell described in any one of (1) to (10) above.

It has been revealed that the TCR gene-modified T cells of the present invention, unlike conventional TCR gene-modified T cells that target AFP, target hTERT and exhibit strong cytotoxic activity. Therefore, the TCR gene-modified T cells of the present invention are highly effective in cancer therapy targeting hTERT.

Furthermore, according to the method for producing TCR gene-modified T cells of the present invention, unlike conventional TCR gene-modified T cells that target AFP, it is possible to produce TCR gene-modified T cells that target hTERT and exhibit strong cytotoxic activity. Therefore, according to the method for producing TCR gene-modified T cells of the present invention, it is possible to produce TCR gene-modified T cells that are extremely effective in cancer treatment targeting hTERT.

FIG. 1 is a characteristic diagram showing the results of measuring the cytotoxic activity of CTL: 12-78 against HepG2 cells. Photographs showing co-culture of CMV gene-modified T cells or TCR gene-modified T cells (12-78) with HepG2 cells. FIG. 1 is a characteristic diagram showing the results of measuring the cytotoxic activity of CTL:12-91 against MT cells. Photographs showing co-culture of CMV gene-modified T cells or TCR gene-modified T cells (12-91) with HepG2 cells.

The following describes in detail an embodiment of the present invention.

The TCR gene modified T cells of the present invention have the function of specifically binding to a complex of a peptide derived from human telomerase reverse transcriptase (hTERT, NCBI Reference Sequence: NG_009265.1) and the HLA-A24 antigen. Here, human telomerase reverse transcriptase is a catalytic subunit of telomerase, and constitutes a telomerase complex together with the telomerase RNA component (TERC). The complex of a peptide derived from hTERT and the HLA-A24 antigen is known to be expressed in liver cancer, spleen cancer, gastric cancer, rectal cancer, etc.

The inventors' group has previously identified several peptides derived from hTERT that are HLA-A24-restricted and are most commonly found in Japanese people. When the effectiveness of these peptides as peptide vaccines was examined, it was found that a peptide consisting of nine amino acids from positions 461 to 469 in the amino acid sequence of hTERT (VYGFVRACL: SEQ ID NO: 1) was highly effective.

In using this hTERT-derived peptide as a peptide vaccine against hepatocellular carcinoma and verifying its clinical efficacy, the inventors found that hTERT-specific cytotoxic T lymphocytes (CTLs, referred to herein as "cytotoxic T cells" or simply "T cells") were induced and in a higher proportion in patients administered the peptide vaccine compared to patients not administered the vaccine and healthy individuals. The inventors isolated and expanded the induced CTLs and analyzed the gene sequences of the α and β chains that constitute the T cell receptor (TCR). They also confirmed that by introducing the obtained genes, it was possible to obtain hTERT-specific highly cytotoxic T cells (TCR gene-modified T cells) from cells derived from another individual.

In other words, the TCR gene modified T cells of the present invention are T cells into which the variable regions of the α and β chains of a T cell receptor protein that specifically binds to a complex of an hTERT-derived peptide and an HLA-A24 antigen have been introduced. Introducing the variable regions of the α and β chains into T cells includes both a form in which a polynucleotide encoding the variable region of the α chain or a complementary polynucleotide thereof and a polynucleotide encoding the variable region of the β chain or a complementary polynucleotide thereof are introduced, and a form in which a T cell receptor protein consisting of a polypeptide containing the variable region of the α chain and a polypeptide containing the variable region of the β chain is taken up into T cells.

There are several types of T cells with different functions, which can be classified according to the surface antigens expressed on the cell surface. Among them, cytotoxic T cells can be simply described as CD8 ⁺ cells. It is also known that the T cell receptor (TCR) expressed on the cell membrane can recognize antigens bound to MHC molecules, and this is because the α and β chains (or γ and δ chains) that make up the TCR each have a variable region similar to that of an antibody that can specifically bind to the antigen-MHC complex. The variable region has complementarity determining regions (CDR1 to CDR3) between framework regions (FR1 to FR4), and it is believed that CDR3 binds to the antigen.

As shown in the Examples below, multiple CTLs have been identified that have excellent cytotoxic activity against cells expressing a complex of an hTERT-derived peptide and the HLA-A24 antigen. The variable regions of the α and β chains of the T cell receptor protein of the present invention are based on the variable regions of the α and β chains contained in these CTLs.

The base sequences and amino acid sequences encoding the variable regions of the α chains contained in these CTLs are summarized in Table 1 below.

The nucleotide sequences and amino acid sequences encoding the variable regions of the β chains contained in these CTLs are summarized in Table 2 below.

As can be seen from the above table, for example, CTL name: 1-57 has an α chain variable region defined in SEQ ID NOs: 2 and 3, and a β chain variable region defined in SEQ ID NOs: 20 and 21. For CTLs other than CTL name: 1-57, the amino acid sequences of the α chain and β chain variable regions and the nucleotide sequences encoding these variable regions can also be understood based on Tables 1 and 2. The TCR gene-modified T cells of the present invention are preferably those into which a combination of the α chain variable region and the β chain variable region identified in these specified CTLs has been introduced. That is, combinations of the α chain variable region and the β chain variable region include a combination of a polypeptide (α chain variable region) consisting of the amino acid sequence shown in SEQ ID NO: 3 with a polypeptide (β chain variable region) consisting of the amino acid sequence shown in SEQ ID NO: 21; a combination of a polypeptide (α chain variable region) consisting of the amino acid sequence shown in SEQ ID NO: 5 with a polypeptide (β chain variable region) consisting of the amino acid sequence shown in SEQ ID NO: 23; a combination of a polypeptide (α chain variable region) consisting of the amino acid sequence shown in SEQ ID NO: 7 with a polypeptide (β chain variable region) consisting of the amino acid sequence shown in SEQ ID NO: 25; a combination of a polypeptide (α chain variable region) consisting of the amino acid sequence shown in SEQ ID NO: 9 with a polypeptide (β chain variable region) consisting of the amino acid sequence shown in SEQ ID NO: 27; ) and a polypeptide (variable region of the β chain) consisting of the amino acid sequence shown in SEQ ID NO: 29; a combination of a polypeptide (variable region of the α chain) consisting of the amino acid sequence shown in SEQ ID NO: 13 and a polypeptide (variable region of the β chain) consisting of the amino acid sequence shown in SEQ ID NO: 31; a combination of a polypeptide (variable region of the α chain) consisting of the amino acid sequence shown in SEQ ID NO: 15 and a polypeptide (variable region of the β chain) consisting of the amino acid sequence shown in SEQ ID NO: 33; a combination of a polypeptide (variable region of the α chain) consisting of the amino acid sequence shown in SEQ ID NO: 17 and a polypeptide (variable region of the β chain) consisting of the amino acid sequence shown in SEQ ID NO: 35; and a combination of a polypeptide (variable region of the α chain) consisting of the amino acid sequence shown in SEQ ID NO: 19 and a polypeptide (variable region of the β chain) consisting of the amino acid sequence shown in SEQ ID NO: 37.

However, the variable regions of the α and β chains of the T cell receptor protein according to the present invention are not limited to those consisting of the amino acid sequences shown in Tables 1 and 2, and also include peptides consisting of amino acid sequences having 90% or more sequence identity to the odd-numbered amino acid sequences among SEQ ID NOs: 2 to 37, preferably amino acid sequences having 95% or more sequence identity, and more preferably amino acid sequences having 98% or more sequence identity, and exhibiting binding activity to a complex of an hTERT-derived peptide and an HLA-A24 antigen.

The polynucleotide encoding the variable regions of the α and β chains of the T cell receptor protein according to the present invention is not limited to polynucleotides consisting of the base sequences shown by even numbers among SEQ ID NOs: 2 to 37 shown in Tables 1 and 2, but may be a polynucleotide consisting of a base sequence including degeneracy of the genetic code with respect to these base sequences. The polynucleotide encoding the variable regions of the α and β chains of the T cell receptor protein according to the present invention also includes polynucleotides consisting of base sequences having 70% or more sequence identity, preferably 80% or more sequence identity, more preferably 90% or more sequence identity, even more preferably 95% or more sequence identity, and even more preferably 98% or more sequence identity with respect to the base sequences shown by even numbers among SEQ ID NOs: 2 to 37, and encoding peptides that exhibit binding activity to a complex of an hTERT-derived peptide and an HLA-A24 antigen.

The identity value between amino acid sequences can be calculated using the BLASTN or BLASTX program that implements the BLAST algorithm (default settings). The identity value is calculated by calculating the number of amino acid residues that are completely identical when a pair of amino acid sequences is subjected to pairwise alignment analysis, and calculating the percentage of the total amino acid residues compared. The identity value between base sequences in a pair of polynucleotides is calculated in the same way.

Even if the variable regions of the α and β chains of the T cell receptor protein according to the present invention are composed of amino acid sequences different from those shown in Tables 1 and 2, the variable regions of the α and β chains are preferably amino acid sequences that maintain the CDR3 contained in the amino acid sequences shown in Tables 1 and 2. That is, it is preferable that the amino acid sequences having 90% or more sequence identity, preferably 95% or more sequence identity, and more preferably 98% or more sequence identity to the amino acid sequences shown by odd numbers among SEQ ID NOs: 2 to 37 are amino acid sequences in which amino acids other than the partial sequence corresponding to CDR3 are substituted, deleted, or inserted. Since CDR3 is involved in binding to an antigen, it is considered that there is a high probability that the binding activity to the complex of the hTERT-derived peptide and HLA-A24 antigen can be maintained by making the amino acid sequences of the variable regions of the α and β chains amino acid sequences that maintain the CDR3 contained in the amino acid sequences shown in Tables 1 and 2.
The CDR3 sequences in the variable regions of the α-chain and β-chain contained in the CTL are summarized in Table 3 below.

The TCR gene modified T cells according to the present invention can be prepared, for example, by introducing the above-mentioned polynucleotide encoding the variable region of the α chain and the polynucleotide encoding the variable region of the β chain into T cells using standard methods. The introduced polynucleotide can be in the form of RNA, for example, when a retroviral vector or the like is used. Thus, although the base sequences indicated by odd numbers in SEQ ID NOs: 2 to 37 are shown as DNA strands, polynucleotides rewritten as RNA strands can also be used in the method of the present invention.

The T cells that are the source of the TCR gene modified T cells according to the present invention are not particularly limited, but can be, for example, T cells isolated from cells in the blood obtained from a patient, or more simply, blood cells such as peripheral blood mononuclear cells (PBMCs) can be used as they are. Alternatively, PBMCs can be used as they are after a T cell activation procedure in which stimulation is added during culture with anti-CD3 antibodies, anti-CD28 antibodies, interleukin 2 (IL-2), etc.

The above-mentioned polynucleotides encoding the variable region of the α chain and the polynucleotides encoding the variable region of the β chain can be introduced by appropriately utilizing methods commonly used in the art, and the introduction method is not particularly limited. For example, the above-mentioned polynucleotides encoding the variable region of the α chain and the polynucleotides encoding the variable region of the β chain can be incorporated into a vector (i.e., an expression vector according to the present invention) such as a viral vector such as a retrovirus, lentivirus, or adenovirus, or a non-viral vector such as a plasmid or a bacterial vector, and then introduced by infecting or incorporating the cells into which they are intended to be introduced. Alternatively, the above-mentioned polynucleotides encoding the variable region of the α chain and the polynucleotides encoding the variable region of the β chain can be introduced using electroporation, transposon, or the like.

When introducing the polynucleotides encoding the variable region of the α chain and the variable region of the β chain, each polynucleotide can be placed under the control of an independent promoter and inserted into the same vector or separate vectors. Alternatively, the polynucleotides encoding the variable region of the α chain and the polynucleotides encoding the variable region of the β chain can be linked via an intervening sequence to form a single expression cassette using a single promoter.

Intervening sequences that can be used in the latter method include, but are not limited to, IRES (internal ribosome entry site) sequences, 2A peptide sequences, etc. (Szymczak et al., Expert Opin. Biol. Ther., 2005, 5: 627-638). The 2A peptide is a peptide sequence of about 20 amino acid residues derived from a virus, and is recognized and cleaved by intracellular proteases. Therefore, the variable region of the α chain and the variable region of the β chain linked by the 2A peptide are cleaved after being transcribed and translated in the cell.

It has already been reported that by introducing a polynucleotide encoding the variable region of the α chain and a polynucleotide encoding the variable region of the β chain, the variable region of the α chain and the variable region of the β chain expressed in the introduced cells form an αβ-TCR heterodimer, thereby providing the desired specificity (Dembic Z. et al. (1986) Transfer of specificity by murine alpha and beta T-cell receptor genes. Nature 320:232-8).

The TCR gene modified T cells according to the present invention are not limited to those obtained by the method of introducing a polynucleotide encoding the variable region of the α chain and a polynucleotide encoding the variable region of the β chain into T cells as described above. The TCR gene modified T cells according to the present invention can also be obtained by a method of synthesizing the variable region of the α chain and the variable region of the β chain and incorporating the synthesized αβ-TCR heterodimer consisting of the variable region of the α chain and the variable region of the β chain into T cells. The method of synthesizing the variable region of the α chain and the variable region of the β chain is not particularly limited, but for example, a cell-free protein synthesis system can be applied. The method of incorporating the synthesized αβ-TCR heterodimer into T cells is also not particularly limited, but examples of the method include a known method for introducing a protein into a target cell (for example, the method described in Shimono K et al., Protein Sci. 2009 Oct;18(10):2160-71. doi: 10.1002/pro.230).

The TCR gene modified T cells according to the present invention described above can act specifically on target cells expressing hTERT peptide and HLA-A24 antigen, and exhibit cytotoxic activity against the target cells. Therefore, the TCR gene modified T cells according to the present invention can be used as a therapeutic agent for cancers expressing hTERT peptide and HLA-A24 antigen. Cancers to be treated are not particularly limited, and examples thereof include cancers in which hTERT expression is enhanced. Examples of such cancers include hepatocellular carcinoma, pancreatic cancer, prostate cancer, gastric cancer, rectal cancer, and renal cell carcinoma.

The therapeutic agent containing the TCR gene-modified T cells according to the present invention may be used alone or in combination with other drugs and treatment methods that can be used to treat cancer. Other drugs and treatment methods that can be used include, but are not limited to, molecular targeted drugs such as sorafenib, chemotherapy, radiofrequency ablation therapy, surgical therapy, hepatic artery (chemo)embolization therapy, radiation therapy, heavy particle therapy, radioisotope therapy, hepatic arterial infusion chemotherapy, peptide vaccine therapy, other immune cell therapy, and the like, for hepatocellular carcinoma. The therapeutic agent of the present invention and the other drugs described above may be administered simultaneously or separately, and may be administered by the same administration route or by different administration routes.

The above-mentioned therapeutic agent can also be made into a pharmaceutical composition, either alone or in combination with other active ingredients. Ingredients that can be contained in the pharmaceutical composition include, in addition to the therapeutic agent of the present invention and other active ingredients, pharma- ceutically acceptable carriers, buffers, stabilizers, etc. that are usually mixed in the art depending on the administration form. Carriers include, but are not limited to, saline, phosphate-buffered saline, glucose solution, and buffered saline. Salts, sugars, sugar alcohols, etc. can also be used as additives. The therapeutic agent and pharmaceutical composition of the present invention are intended to be administered in liquid form due to the nature of the present invention, and therefore need to be in a form that can maintain the stability of the active ingredient, CTL.

The therapeutic agent and pharmaceutical composition of the present invention can be administered locally or systemically, and the administration route is not particularly limited, but can be, for example, intravenous administration. Alternatively, it can be administered by injection or infusion into the affected area or in the vicinity of the affected area.

The dosage and frequency of administration of the therapeutic agent and pharmaceutical composition of the present invention vary depending on the patient's body weight, sex, age, severity of the disease, etc., and are not particularly limited. For example, the T cells of the present invention are used as the active ingredient and each dosage contains about 1 x ¹⁰ to about 1 x ¹⁰ cells, preferably about 1 x ¹⁰ to about 1 x ¹⁰ cells, and can be administered once to several times a day, every 2 days, every 3 days, every week, every 2 weeks, every month, every 2 months, every 3 months, or every 6 months.

The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

Cell lines
The following cells were used: C1R-A24 (a C1R lymphoma subline expressing HLA-A24; provided by Dr. Masafumi Takiguchi, AIDS Research Center, Kumamoto University), K562 (human chronic myeloid leukemia cells; purchased from the Cell Bank, Cell Engineering Division, RIKEN BioResource Center), HepG2 (human hepatoma cell line; purchased from the Cell Bank, Cell Engineering Division, RIKEN BioResource Center), and MT and KM cells, which are human hepatoma cell lines established by the present inventor. C1R-A24 and K562 cells were maintained in RPMI-1640 (Wako Pure Chemical Industries Ltd., Osaka, Japan) containing 10% fetal bovine serum (BioWest SAS, Nuaille, France) and supplemented with 100 μg/mL streptomycin and 100 units/mL penicillin. C1R-A24 was selected in a medium containing 500 μg/mL hygromycin B. HepG2 cells were maintained in DMEM (Wako Pure Chemical Industries Ltd.) containing 10% fetal bovine serum and supplemented with 100 μg/mL streptomycin and 100 units/mL penicillin. MT and KM cells were maintained in DMEM (Wako Pure Chemical Industries Ltd.) containing 10% fetal bovine serum and supplemented with 100 μg/mL streptomycin and 100 units/mL penicillin.

[Induction of hTERT-specific CTL and TCR cloning]
As the hTERT-derived peptide vaccine, hTERT peptide (SEQ ID NO: 1) was used. In this example, peripheral blood mononuclear cells (PBMCs) were collected from 14 human leukocyte antigen (HLA)-A24 patients who were enrolled in a phase I clinical trial (UMIN000003511) of hTERT-derived peptide vaccine therapy and who had been previously diagnosed by measuring blood pressure and pulse and checking the overall condition.

First, put 1 ml of saline into a peptide vial using a 5 ml locking syringe and mix thoroughly. Then, collect the dissolved peptide solution using a 1 ml syringe. Then, connect this syringe to a three-way stopcock, remove the cap on the other side, and fill the inside with peptide solution. Take 1 ml of Montanide ISA-51 into a 5 ml locking syringe and connect it to the three-way stopcock to which the peptide solution is connected. Then, mix thoroughly until completely emulsified, collect the peptide vaccine in one syringe, and remove it from the three-way stopcock. Attach a 21G injection needle and drop a drop on the surface of the saline to confirm that it does not spread. Connect it to the three-way stopcock again and fill the peptide vaccine into three tuberculin syringes, dividing them into 500 μl each. After preparation in this way, the drug is administered subcutaneously to the left or right armpit of each subject (0.03-3.0 mg per administration, for a total of three administrations. Three subjects received a dose of 0.03 mg, three subjects received a dose of 0.3 mg, and eight subjects received a dose of 3.0 mg).

Peripheral blood mononuclear cells (PBMCs) from subjects were isolated by density gradient centrifugation using Ficoll. First, the Lymphoprep tube was centrifuged at 2000 rpm for 1 minute to allow the Ficoll to settle. Next, phosphate-buffered saline (PBS) and heparinized blood were placed into the Lymphoprep tube. Then, the tube was centrifuged at 2000 rpm for 20 minutes at 20°C (brake off). The PBMC layer was then collected using a pipette and placed into a new Falcon tube. PBS was added until the tube was full. Then, the tube was centrifuged at 1600 rpm for 6 minutes at 20°C (brake on). The supernatant was then removed, suspended in a Cell Banker, and stored in a cryostat tube.

In this example, the obtained PBMCs were stained with phycoerythrin (PE)-conjugated hTERT peptide MHC tetramer (MBL Co., LTD., Aichi, Japan) after induction of cytotoxic lymphocytes (CTLs), or with fluorescein isothiocyanate (FITC)-conjugated anti-CD8 antibody (MBL Co., LTD) and 7-AAD (Beckman Coulter, Inc., Indianapolis, IN, USA) after direct staining.

The induced antigen-specific CTLs were then detected and collected as single cells using a FACSAria II (BD Bioscience, San Diego, CA, USA). One-step PCR (RT→1st PCR) and 2nd PCR were then performed for the α-chain variable region and β-chain variable region, respectively. The amplified nucleic acid fragments were purified using the PEG precipitation method and electrophoresed on a 1% agarose gel to confirm amplification and single bands. The nucleotide sequences of the amplified fragments were then determined by an external contractor, and the obtained nucleotide sequences were confirmed as the α-chain variable region and β-chain variable region using IMGT/V-QUEST (the international ImMunoGeneTics information system).

The α-chain variable region and β-chain variable region of interest were then linked to the pMXs-IRES-GFP vector (liner vector), β fragment, and Cα fragment using the Gibson assembly system to create a circularly ligated vector. The vector thus created was transformed into competent cells (JM109) according to standard methods. The transformed JM109 strain was plated on an LB plate, and fragments containing the α-chain variable region and β-chain variable region were amplified by colony PCR using the resulting colonies, after which the amplified fragments were confirmed by electrophoresis on a 1% agarose gel. In addition, the fragments amplified by PCR were purified using the PEG precipitation method, and the base sequence of the amplified fragments was confirmed by outsourcing, after which the fragments containing the α-chain variable region and β-chain variable region were purified in large quantities using the Maxiprep method.

In this way, polynucleotides encoding the α-chain variable region and the β-chain variable region were cloned from CTLs derived from subjects administered the hTERT peptide vaccine. As a result, hTERT peptide-specific CTLs were induced in 5 of the 14 subjects (Pt1, Pt2, Pt3, Pt12, and Pt13).

[Creation of TCR gene-modified T cells]
For transduction into PBMCs, cDNAs encoding the TCR α and TCR β chain variable regions were linked together with cDNAs encoding the constant regions (obtained by cloning from human PBMCs) in the same TCR-derived sequence combination via the viral P2A sequence to construct TCR expression vectors (Leisegang M et al., J. Mol. Med. 2008, July, 86(7), p.855), which were then cloned into the pMXs-IRES-GFP vector (Cosmo Bio Co., Ltd.) and transfected into the Phoenix-A retroviral packaging cell line (National Gene Vector Biorepository, Indianapolis, USA). The codons of the constant regions of each TCR were optimized. The resulting viral supernatant was filtered, placed on a plate coated with 50 μg/ml RetroNectin (Takara Bio Inc., Shiga, Japan), and centrifuged.

Cryopreserved peripheral blood mononuclear cells from healthy donors were thawed and seeded at ^1x106 cells/well and stimulated with culture medium containing 35U/ml IL-2 and CD3/CD28 Dynabeads. Meanwhile, a 24-well plate was coated overnight with Retronectin diluted to 50μg/ml. The plate coated overnight with Retronectin was washed with 500μl/well of 2% BSA/PBS, and culture supernatant containing a retrovirus encoding the target TCR was added. The plate was centrifuged at 1900xg at 32℃ for 2 hours to spin-load the retrovirus.

After centrifugation, the stimulated peripheral blood mononuclear cells were washed and adjusted to 5 x ¹⁰⁵ cells/ml in culture medium supplemented with 35 U/ml IL-2, and seeded on the plate spin-loaded with the retrovirus. The seeded plate was centrifuged at 1000 x g for 10 minutes at 32°C, and the cells were cultured in a 37°C incubator. The following day, the peripheral blood mononuclear cells were transferred to a new plate prepared in the same manner and cultured in a 37°C incubator. The following day, the experiment was scaled up to 10 ml of culture medium supplemented with 35 U/ml IL-2 in a cell culture flask.

Seven days after the start of peripheral blood mononuclear cell stimulation, CD3/CD28 Dynabeads were added for restimulation. Ten days after the start of peripheral blood mononuclear cell stimulation, TCR gene-modified T cells were tetramer stained and TCR expression was evaluated by flow cytometry in the same manner as described above.

[Measurement of cytotoxic activity]
Cytotoxicity against C1RA24 cells
In this experiment, C1RA24-Luc cells, which were C1RA24 cells transfected with Kusabira Orange luciferase, were used as the target cells. C1RA24-Luc cells were pulsed with hTERT peptide 4 to 13 hours before the assay to measure cytotoxicity. For the assay, the two cells were mixed so that the number of K562 cells was 40 times that of the pulsed C1RA24-Luc cells.

Meanwhile, the TCR gene-modified T cells prepared as described above were collected, the cell number was adjusted, and a 2-fold dilution series was prepared in a 96-well plate. Then, a mixture of C1RA24-Luc cells and K562 cells was placed in the wells so that the ratio of TCR gene-modified T cells to C1RA24-Luc cells was 50:1, 25:1, 12.5:1, and 6.25:1, and co-cultured at 37°C for 4 hours (medium: RPMI1640 medium containing 10% FBS and 1% penicillin/streptomycin). After incubation, the plate was centrifuged to remove the supernatant, PBS and Steady-Glo were added, and the mixture was incubated at room temperature for 10 minutes and photometrically measured (Steady-Glo Luciferase Assay System).

Cytotoxic activity against HepG2 cells In this experiment, the target cells were HepG2 human hepatoma cell line, whose expression of the hTERT gene had been confirmed by RT-PCR in advance. On the day before the cytotoxic activity measurement assay, HepG2 cells were seeded at 3×10 ⁴ cells/well on an 8-well cover glass chamber (medium: 10% FBS, 1% penicillin/streptomycin-containing phenol red-free RPMI1640 medium). On the day of the assay, the culture medium of the HepG2 cells was removed, and 500 μl/well of Calcein Violet staining solution (Calcein Violet 450 AM Viability Dye) prepared to a final concentration of 10 μM was added, and staining was performed at 37°C for 30 minutes. After staining for 30 minutes, the Calcein Violet staining solution was removed, and the cells were washed with 500 μl/well of culture medium.

On the other hand, the TCR gene-modified T cells prepared as described above were thoroughly suspended and collected after culture, centrifuged, the CD3/CD28 Dynabeads were removed, and the concentration was adjusted to 6 x ¹⁰⁵ cells/ml. In this experiment, CMV gene-modified T cells were also prepared as a negative control, and a suspension of CMV gene-modified T cells was prepared to a concentration of 6 x ¹⁰⁵ cells/ml, similar to the TCR gene-modified T cells.

Then, 1μl of dead cell staining agent: PI (Propidium Iodide) and 10μl of anti-fading agent: Live Antifade Reagent, for live cell imaging were added to the hTERT gene modified T cells and CMV gene modified T cells prepared at ^a concentration of 6×10 5 cells/ml, mixed well, and then added to the stained HepG2 cells at 500μl/well. After that, the mixture was left to stand for 10 minutes to allow the genetically modified T cells to settle. The state of the cells was photographed every 30 seconds for 10 hours using a confocal microscope, and a 2-minute video was created after the photography. For the analysis of cytotoxicity, screenshots were taken of the video at 0 seconds and every 10 seconds thereafter, and live cells were counted in the image at 0 seconds and dead cells were counted in the other images, and compared with the control group.

Cytotoxic activity against MT(KM) cells In this experiment, MT(KM) cells, whose expression of the hTERT gene had been confirmed by RT-PCR in advance, were used as the target cells. Cytotoxic activity against MT(KM) cells was measured in the same manner as for HepG2 cells.

[result]
As the results of this example, the cytotoxic activity against C1RA24 cells, HepG2 cells, and MT (KM) cells are summarized in Table 4.

As shown in Table 4, nine types of TCRs (CTL names 12-78, 12-91, 12-3, 13-4, 13-47, 1-57, 1-80, 1-112, and 3-55) with excellent cytotoxic activity against the tested cells were identified. The amino acid sequences of the α-chain variable region and β-chain variable region in the identified TCRs, and the base sequences encoding these α-chain variable region and β-chain variable region are summarized in Table 5. In Table 5, odd numbers represent base sequences and even numbers represent amino acid sequences.

The obtained base sequence was analyzed using IMGT/V-QUEST (manufactured by the international ImMunoGeneTics information system), and the results are shown in Table 6.

As an example, the results of measuring the cytotoxic activity of CTL: 12-78 against HepG2 cells are shown in Figure 1. In Figure 1, the horizontal axis indicates the treatment time, and the vertical axis indicates the cytotoxic activity (%). In Figure 1, the cytotoxic activity of TCR gene-modified T cells containing 12-78 is shown by a solid line, and for comparison, the cytotoxic activity of CMV gene-modified T cells is shown by a dashed line. In addition, Figure 2 shows a photograph taken 100 minutes after co-culturing CMV gene-modified T cells or TCR gene-modified T cells with HepG2 cells. In Figure 2, the right side is a photograph when TCR gene-modified T cells are co-cultured with HepG2 cells, and the left side is a photograph when CMV gene-modified T cells are co-cultured with HepG2 cells. As shown in Figures 1 and 2, it was revealed that TCR gene-modified T cells containing 12-78 exhibit extremely excellent cytotoxic activity against HepG2 cells.

Figure 3 shows the results of measuring the cytotoxic activity of CTL:12-91 against MT cells. In Figure 3, the horizontal axis indicates the treatment time, and the vertical axis indicates the cytotoxic activity (%). In Figure 3, the cytotoxic activity of TCR gene-modified T cells containing 12-91 is shown by a solid line, and for comparison, the cytotoxic activity of CMV gene-modified T cells is shown by a dashed line. Figure 4 shows photographs taken 350 minutes after co-culture of CMV gene-modified T cells or TCR gene-modified T cells with MT cells. In Figure 4, the right side shows a photograph when TCR gene-modified T cells are co-cultured with MT cells, and the left side shows a photograph when CMV gene-modified T cells are co-cultured with MT cells. As shown in Figures 3 and 4, it was revealed that TCR gene-modified T cells containing 12-91 exhibit extremely excellent cytotoxic activity against MT cells.

Claims (25)

The antibody has a polypeptide including an α chain variable region and a polypeptide including a β chain variable region of a T cell receptor protein that specifically binds to a complex of a peptide derived from human telomerase reverse transcriptase (hTERT) and an HLA-A24 antigen,
The α chain variable region is a polypeptide of the following (i) or (ii):
(i) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19.
(ii) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19. The β-chain variable region is a polypeptide of the following (iii) or (iv):
(iii) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37.
(iv) a polypeptide having an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37.
TCR gene-modified T cells. The TCR gene modified T cell according to claim 1, characterized in that the α chain variable region and the β chain variable region are expressed from a polynucleotide introduced into the T cell. The TCR gene modified T cell according to claim 1, characterized in that the polynucleotide encoding the α chain variable region and the polynucleotide encoding the β chain variable region are introduced in vitro into T cells of a patient suffering from cancer. The TCR gene modified T cell according to claim 3, characterized in that the patient's cancer cells are cancer cells that express the complex. The polynucleotide encoding the α chain variable region is
[1] A nucleic acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary nucleic acid sequence thereof; or
[2] A base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary base sequence thereof;
The polynucleotide encoding the β chain variable region is
[3] A nucleic acid sequence represented by any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34, and 36, or a complementary nucleic acid sequence thereof; or
[4] The TCR gene modified T cell according to claim 2, characterized in that it comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34 and 36, or a complementary base sequence thereof. The α chain variable region and the β chain variable region are the following combination:
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence; or a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
The TCR gene modified T cell according to claim 1, characterized in that it is any one of the above. The TCR gene modified T cell according to claim 1, characterized in that the polypeptides consisting of amino acid sequences having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17 and 19 in (ii) above are amino acid sequences containing the CDR3 sequences shown in SEQ ID NOs: 38 to 46, respectively. The TCR gene modified T cell according to claim 1, characterized in that the polypeptides consisting of amino acid sequences having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35 and 37 in (iv) above are amino acid sequences containing the CDR3 sequences shown in SEQ ID NOs: 47 to 55, respectively. The TCR gene modified T cell according to claim 1, characterized in that the human telomerase reverse transcriptase (hTERT) derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. The TCR gene modified T cell according to claim 1, characterized in that it has cytotoxicity against cancer cells expressing the above complex. A step of in vitro introducing into human T cells a polypeptide comprising an α-chain variable region and a polypeptide comprising a β-chain variable region of a T cell receptor protein that specifically binds to a complex of a peptide derived from human telomerase reverse transcriptase (hTERT) and an HLA-A24 antigen, or a polynucleotide encoding these polypeptides:
The α chain variable region is a polypeptide of the following (i) or (ii):
(i) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19
(ii) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19. The β-chain variable region is a polypeptide of the following (iii) or (iv):
(iii) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37.
(iv) A method for producing a TCR gene-modified T cell comprising a polypeptide having an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37. The method for producing TCR gene modified T cells according to claim 11, characterized in that the human T cells are T cells derived from a patient suffering from cancer. The method for producing TCR gene modified T cells according to claim 12, characterized in that the patient's cancer cells are cancer cells that express the complex. The polynucleotide encoding the α chain variable region is
[1] A nucleic acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary nucleic acid sequence thereof; or
[2] A polynucleotide encoding the β chain variable region comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary base sequence thereto.
[3] A nucleic acid sequence represented by any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34, and 36, or a complementary nucleic acid sequence thereof; or
[4] The method for producing TCR gene-modified T cells described in claim 11, characterized in that the TCR gene-modified T cells consist of a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34 and 36, or a complementary base sequence thereof. The α chain variable region and the β chain variable region are selected from the following combinations:
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence; or a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
The method for producing a TCR gene-modified T cell according to claim 11, characterized in that the method is any one of the above. The method for producing TCR gene modified T cells according to claim 11, characterized in that the polypeptides in (ii) above, each having an amino acid sequence that has 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17 and 19, are amino acid sequences that contain the CDR3 sequences shown in SEQ ID NOs: 38 to 46, respectively. The method for producing TCR gene modified T cells according to claim 11, characterized in that the polypeptides consisting of amino acid sequences having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35 and 37 in (iv) above are amino acid sequences containing the CDR3 sequences shown in SEQ ID NOs: 47 to 55, respectively. The method for producing TCR gene modified T cells according to claim 11, characterized in that the peptide derived from human telomerase reverse transcriptase (hTERT) is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1. The method for producing TCR gene modified T cells according to claim 11, characterized in that the TCR gene modified T cells have cytotoxicity against cancer cells expressing the complex. The present invention relates to a method for identifying a T cell receptor protein that specifically binds to a complex of a human telomerase reverse transcriptase (hTERT)-derived peptide and an HLA-A24 antigen, and the method further relates to a method for identifying a T cell receptor protein that specifically binds to a complex of a human telomerase reverse transcriptase (hTERT)-derived peptide and an HLA-A24 antigen.
The α chain variable region is a polypeptide of the following (i) or (ii):
(i) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19.
(ii) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19. The β-chain variable region is a polypeptide of the following (iii) or (iv):
(iii) a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35, and 37.
(iv) An expression vector which is a polypeptide consisting of an amino acid sequence having 90% or more sequence identity to any of the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35 and 37. The polynucleotide encoding the α chain variable region is
[1] A nucleic acid sequence represented by any one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary nucleic acid sequence thereof; or
[2] A polynucleotide encoding the β chain variable region comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, or a complementary base sequence thereto.
[3] A nucleic acid sequence represented by any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34, and 36, or a complementary nucleic acid sequence thereof; or
[4] The expression vector according to claim 20, characterized in that it comprises a base sequence having 90% or more sequence identity to any of the base sequences shown in SEQ ID NOs: 20, 22, 24, 26, 28, 30, 33, 34 and 36, or a complementary base sequence thereof. The α chain variable region and the β chain variable region are selected from the following combinations:
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:21 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:23 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:11 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:13 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:15 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence; or a combination of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:19 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having 90% or more sequence identity to said amino acid sequence;
The expression vector according to claim 20, characterized in that it is any one of the above. The expression vector according to claim 20, characterized in that the polypeptides in (ii) above, each of which has an amino acid sequence having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, and 19, are amino acid sequences containing the CDR3 sequences shown in SEQ ID NOs: 38 to 46, respectively. The expression vector according to claim 20, characterized in that the polypeptides in (iv) above consisting of amino acid sequences having 90% or more sequence identity to the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, 29, 31, 33, 35 and 37 are amino acid sequences containing the CDR3 sequences shown in SEQ ID NOs: 47 to 55, respectively. A cancer therapeutic agent comprising the TCR gene modified T cell according to any one of claims 1 to 10.

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