MRP3-targeting gene-modified T cells

Abstract

To provide novel therapeutics against advanced liver cancer, since the current treatment of advanced liver cancer with chemical therapy or molecular targeting drugs sometimes comes to insufficient treatment results and peptide vaccines lack instantaneous effect.SOLUTION: The invention provides a T cell receptor protein that specifically binds to a complex of a peptide derived from multidrug resistance protein 3 (MRP3) and HLA-A24 antigen. The invention also provides a method for preparing T cells for gene therapy against cancer characterized by in vitro introduction of the T cell receptor protein or a polynucleotide encoding the protein into T cells derived from a patient. The invention also provides cancer treating agent comprising the T cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to genetically modified T cells, specifically T cells having a T cell receptor that targets multidrug resistance-associated protein 3 (MRP3) and a method for producing the same. The present invention also relates to a therapeutic agent for hepatocellular carcinoma containing the T cells. [Background technology]

[0002] In Japan, the number of deaths from hepatocellular carcinoma (liver cancer, HCC) is approximately 25,000 per year, making it the fourth most common cancer death after lung cancer, colon cancer, and stomach cancer. With advances in treatments for hepatitis C and hepatitis B, the incidence of liver cancer caused by viral hepatitis has been declining, but in recent years there has been an increase in so-called metabolic liver cancer, which is thought to be caused by obesity, diabetes, and fatty liver.

[0003] For the early detection of liver cancer, cancer screening tests for high-risk groups such as hepatitis virus patients are essential, but narrowing down cases with a high risk of developing metabolic liver cancer is difficult, and many cases visit hospitals in an advanced stage.Currently, treatment for advanced liver cancer involves chemotherapy and molecular targeted drugs, but the treatment results are insufficient, and the prognosis for advanced liver cancer is extremely poor, making it one of the most difficult cancers to treat.

[0004] In recent years, immune checkpoint inhibitors and chimeric antigen receptor-based genetically modified T cells (CAR-T) have been clinically applied to the treatment of advanced cancer, and excellent therapeutic results have been reported for some cancer types, but neither of these has been approved for use in liver cancer. The reason for this is that many solid cancers, including liver cancer, do not express cancer-specific antigens on the surface of cancer cells that can be targeted by antibodies.

[0005] The present inventors have conducted basic research into the host immune response to liver cancer and have identified tumor antigens and their T cell epitopes that could be targets for liver cancer immunotherapy (Non-Patent Documents 1-3). Furthermore, as a clinical application of this basic research, we have conducted clinical trials in which peptides derived from antigen epitopes were administered to patients as cancer vaccines, and have reported their effectiveness (Non-Patent Document 4).

[0006] Furthermore, the present inventors have generated T cells into which an alpha-fetoprotein (AFP)-specific T cell receptor gene has been introduced, and have found that these cells have cytotoxic activity against cells that co-express AFP and HLA-A24 (Patent Document 1). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 2017-081836 [Non-patent literature]

[0008] [Non-Patent Document 1] Mizukoshi E et al., J Hepatol. 49: 946-954, 2008 [Non-patent document 2] Mizukoshi E et al., Hepatology. 53:1206-1216, 2011 [Non-patent document 3] Mizukoshi E et al., Hepatology. 57:1448-1457, 2013 [Non-patent document 4] Mizukoshi E et al., Cancer Lett. 369:242-249, 2015 Summary of the Invention [Problem to be solved by the invention]

[0009] As mentioned above, chemotherapy and molecular targeted drug treatments for advanced liver cancer have not always produced satisfactory results. Furthermore, peptide vaccines require multiple steps after administration to the patient, including uptake by dendritic cells and antigen presentation to T cells, resulting in a lack of immediate efficacy. In other words, peptide vaccines are expected to be more effective in preventing recurrence than in treating advanced liver cancer. [Means for solving the problem]

[0010] Based on previous research findings, the present inventors attempted to generate genetically modified T cells using a T cell receptor (TCR) gene that recognizes a target antigen for which peptide vaccination has been shown to be effective. As a result, they obtained a T cell receptor protein that targets a peptide derived from multidrug resistance-associated protein 3 (MRP3), which is presented on the cell surface as a complex with major histocompatibility complexes (MHC), and a polynucleotide encoding the same, and succeeded in generating T cells with cytotoxic activity against cells expressing MRP3. Surprisingly, the T cells of the present invention were found to be effective not only against cancers resistant to chemotherapy or molecular-targeted drug treatment, but also in cases where AFP-specific T cells are not expected to be effective. The present invention was achieved based on these findings.

[0011] That is, the present invention provides the following. 1. A T cell receptor protein that specifically binds to a complex of a peptide derived from multidrug resistance-associated protein 3 (MRP3) and the HLA-A24 antigen. 2. The MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide encoding the α chain variable region is a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 2; and The polynucleotide encoding the β-chain variable region is a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 6. The T cell receptor protein described in 1 above. 3. The MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the α chain variable region has the amino acid sequence set forth in SEQ ID NO: 10; and The β chain variable region has the amino acid sequence set forth in SEQ ID NO: 14. The T cell receptor protein described in 1 above. 4. The MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide encoding the α chain variable region is a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:3; and The polynucleotide encoding the β-chain variable region is a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 7. The T cell receptor protein described in 1 above. 5. The MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the α chain variable region has the amino acid sequence set forth in SEQ ID NO: 11; and The β chain variable region has the amino acid sequence set forth in SEQ ID NO: 15. The T cell receptor protein described in 1 above. 6. The MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide encoding the α chain variable region is a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:4; and The polynucleotide encoding the β-chain variable region is a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 8. The T cell receptor protein described in 1 above. 7. The MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the α chain variable region has the amino acid sequence set forth in SEQ ID NO: 12; and The β chain variable region has the amino acid sequence set forth in SEQ ID NO: 16. The T cell receptor protein described in 1 above. 8. A method for producing T cells for gene therapy against cancer, comprising introducing the T cell receptor protein described in any one of 1 to 7 above or a polynucleotide encoding the same into patient-derived T cells in vitro. 9. A vector comprising a polynucleotide encoding the T cell receptor protein described in any one of 1 to 7 above. 10. A T cell comprising the vector of claim 9. 11. T cells containing a T cell receptor protein according to any one of 1 to 7 above, which has been introduced in vitro. 12. A cancer therapeutic agent comprising the T cells according to claim 10 or 11. 13. The cancer therapeutic agent according to 12 above, which is administered in combination with T cells containing a T cell receptor protein that specifically binds to a complex of an alpha-fetoprotein (AFP)-derived peptide and an HLA-A24 antigen. 14. The cancer therapeutic agent according to 12 or 13 above, for treating cancer resistant to anticancer drugs and / or progressive cancer. 15. The cancer therapeutic agent according to any one of 12 to 14 above, which has cytotoxic activity against cancer stem cells. [Effects of the Invention]

[0012] The genetically modified T cells of the present invention can recognize solid tumors and exert cytotoxic activity. Because they can exert antitumor effects immediately after administration, they are considered to be more effective and may provide more effective therapeutic effects when combined with conventional anticancer therapies. [Brief explanation of the drawings]

[0013] [Figure 1] PBMCs derived from hepatocellular carcinoma (HCC) patients and healthy individuals were stimulated with the MRP3765 peptide for 3 weeks to induce CTLs, and the percentage of MRP3-specific T cells was then measured by flow cytometry. [Figure 2] The cytotoxic activity of the genetically modified T cells of the present invention against HepG2 cells (A) and KM cells (B) is shown. CMV: negative control. [Figure 3]Cytotoxic activity of AFP-specific gene-modified T cells against HepG2 cells. CMV: negative control. [Figure 4] A: Cytotoxic activity of AFP-specific gene-modified T cells and MRP3-specific gene-modified T cells against KM cells. B: Expression of MRP3 and AFP in KM cells. The vertical axis shows the relative value when the expression level in HepG2 cells is set to 1. [Figure 5] This shows the expression of MRP3 in HepG2 cells and KM cells. The vertical axis shows the relative value, with the expression level in HepG2 cells set to 1. In KM cells, MRP3 expression was confirmed in both the CD90+ and CD90- fractions. [Figure 6] Figure 1 shows the cytotoxic activity of the genetically modified T cells of the present invention against KM CD90+ cells. CMV: negative control. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides a T cell receptor protein that specifically binds to a complex of a peptide derived from multidrug resistance-associated protein 3 (MRP3) and an HLA-A24 antigen.

[0015] Multi-drug resistance-related protein 3 (MRP3) is a carrier-type transport protein belonging to the ABC transporters involved in ATP-dependent transport of substances. The amino acid sequence of human MRP3 and the nucleotide sequence of the gene encoding it are available from the database of the National Center for Biotechnology Information (NCBI) in the United States, for example, as GenBank Accession No. AAD01430.1 and Gene ID: 8714, respectively.

[0016] The present inventors' group has previously identified several peptides derived from MRP3 that are HLA-A24-restricted and are most commonly found in Japanese people. When the effectiveness of these peptides as peptide vaccines was examined, three peptides were identified: one consisting of nine amino acids at positions 765 to 373 in the amino acid sequence of MRP3 (VYSDADIFL, SEQ ID NO: 1); one consisting of nine amino acids at positions 503 to 511 (LYAWEPSFL, SEQ ID NO: 50); and one consisting of nine amino acids at positions 692 to 700 (AYVPQQAWI, SEQ ID NO: 51) (these peptides are referred to herein as MRP3 peptides, respectively). 765 , MRP3 503 , and MRP3 692 ) has been found to be effective.

[0017] The present inventors have used the above peptide as a peptide vaccine against hepatocellular carcinoma and found that MRP3-specific cytotoxic T lymphocytes (CTLs) were induced in patients receiving the vaccine (Mizukoshi E et al., J Hepatol. 49: 946-954, 2008).

[0018] In this study, the present inventors isolated and expanded the induced CTLs, analyzed their gene sequences, and confirmed that by introducing the resulting gene, highly cytotoxic MRP3-specific T cells can be obtained from cells derived from another individual. These T cells are capable of expressing on their cell surface a T cell receptor protein that specifically binds to a complex of an MRP3-derived peptide and HLA-A24 antigen. The MRP3-derived peptide may be, but is not limited to, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 50, or SEQ ID NO: 51.

[0019] In one embodiment, the T cell receptor protein of the present invention is an MRP3-derived peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide encoding the α chain variable region is a polynucleotide having the nucleotide sequence set forth in SEQ ID NO: 2; and The polynucleotide encoding the β chain variable region is a polynucleotide having the nucleotide sequence shown in SEQ ID NO:6.

[0020] In one embodiment, the T cell receptor protein of the present invention is characterized in that the MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the α chain variable region has the amino acid sequence set forth in SEQ ID NO: 10; and The β chain variable region has the amino acid sequence shown in SEQ ID NO:14.

[0021] In another embodiment of the T cell receptor protein of the present invention, the MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide encoding the α chain variable region is a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:3; and The polynucleotide encoding the β chain variable region is a polynucleotide having the nucleotide sequence shown in SEQ ID NO:7.

[0022] In another embodiment of the T cell receptor protein of the present invention, the MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the α chain variable region has the amino acid sequence set forth in SEQ ID NO: 11; and The β chain variable region has the amino acid sequence shown in SEQ ID NO:15.

[0023] In another embodiment of the T cell receptor protein of the present invention, the MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide encoding the α chain variable region is a polynucleotide having the nucleotide sequence set forth in SEQ ID NO:4; and The polynucleotide encoding the β chain variable region is a polynucleotide having the nucleotide sequence shown in SEQ ID NO:8.

[0024] In another embodiment of the T cell receptor protein of the present invention, the MRP3-derived peptide is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1, the α chain variable region has the amino acid sequence set forth in SEQ ID NO: 12; and The β chain variable region has the amino acid sequence shown in SEQ ID NO:16.

[0025] In one embodiment of the present invention, the junction sequence comprising the CDR3 of the α chain and the junction sequence comprising the CDR3 of the β chain in the T cell receptor protein of the present invention are the following combination: SEQ ID NO: 18 and SEQ ID NO: 22, SEQ ID NO: 19 and SEQ ID NO: 23, or SEQ ID NO: 20 and SEQ ID NO: 24 It could be.

[0026] In one embodiment of the present invention, the CDRs in the T cell receptor protein of the present invention are the following combination: CDR1, CDR2, and CDR3 of the α chain are SEQ ID NOs: 26, 27, and 28, respectively, and CDR1, CDR2, and CDR3 of the β chain are SEQ ID NOs: 29, 30, and 31, respectively; or CDR1, CDR2, and CDR3 of the α chain are SEQ ID NOs: 32, 33, and 34, respectively, and CDR1, CDR2, and CDR3 of the β chain are SEQ ID NOs: 35, 36, and 37, respectively; or The CDR1, CDR2, and CDR3 of the α chain can be SEQ ID NOs: 38, 39, and 40, respectively, and the CDR1, CDR2, and CDR3 of the β chain can be SEQ ID NOs: 41, 42, and 43, respectively.

[0027] The present invention also provides a method for producing T cells for gene therapy against cancer, which method comprises introducing the above-mentioned T cell receptor protein of the present invention or a polynucleotide encoding the same into patient-derived T cells in vitro.

[0028] Specifically, the above-mentioned method of the present invention can be achieved by introducing polynucleotides encoding the variable regions of the α chain and β chain of the above-mentioned T cell receptor protein of the present invention, which specifically binds to a complex of an MRP3-derived peptide and an HLA-A24 antigen, or polynucleotides complementary thereto, into patient-derived T cells in vitro.

[0029] Gene transfer may be performed using any method known in the art, and depending on the method, the polynucleotide actually transferred may be DNA or RNA.

[0030] As will be understood by those skilled in the art, 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. T cell receptors (TCRs) expressed on the cell membrane can recognize antigens bound to MHC molecules. This is known to be because the α and β chains (or sometimes 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 contains complementarity-determining regions (CDR1 to CDR3) between framework regions (FR1 to FR4), and CDR3 is thought to bind to the antigen.

[0031] A feature of the present invention is that a T cell receptor protein that specifically binds to a complex of an MRP3-derived peptide and an HLA-A24 antigen is expressed on the surface of patient-derived T cells, and the method of the present invention is an ex vivo method in which the above-mentioned polynucleotide is introduced in vitro into T cells obtained from a patient and the resulting cells are then returned to the patient. Polynucleotides that can be suitably used in the method of the present invention are specifically described below, and each polynucleotide is characterized in that both the α chain and the β chain are introduced into the same T cell, and the TCR generated by this combination specifically binds to the above-mentioned complex.

[0032] In the methods of the present invention, T cells isolated from blood cells obtained from a patient can be used as T cells to be transfected with a polynucleotide, but more conveniently, blood cells such as PBMCs can be used as they are. Alternatively, PBMCs can be used as they are after a T cell activation procedure, for example, by stimulating them with an anti-CD3 antibody, an anti-CD28 antibody, interleukin-2 (IL-2), or the like during culture.

[0033] In one embodiment, the polynucleotide encoding the α chain variable region in the method of the present invention is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 2 to 4. On the other hand, in one embodiment, the polynucleotide encoding the β chain variable region in the method of the present invention is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NOs: 6 to 8.

[0034] For target binding, a combination in which the α chain variable region is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 2 and the β chain variable region is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6; A combination in which the α chain variable region is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 3 and the β chain variable region is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7, and A combination in which the α chain variable region is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 4 and the β chain variable region is a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 8. It is preferable that:

[0035] As described above, the polynucleotide to be introduced may be in the form of RNA, for example, when a retroviral vector is used, etc. Therefore, polynucleotides having RNA sequences complementary to the nucleotide sequences shown as DNA sequences in SEQ ID NOS: 2 to 8 may also be used in the methods of the present invention.

[0036] The T cells of the present invention into which the polynucleotide defined above has been introduced have a log γ in the range of -0.5 to -3.0, preferably -1 to -3.0. 10 (EC 50 The T cells of the present invention have the ability to specifically lyse target cells (peptide-pulsed C1RA24 cells) that express MRP3-derived peptides and HLA-A24 at an E / T ratio of 50:1 or greater. Alternatively, the T cells of the present invention have the effect of killing 20% ​​or more of target cells that endogenously express MRP3 at an E / T ratio of 50:1 or greater.

[0037] Gene introduction can be carried out by appropriately utilizing methods commonly used in the art, and the introduction method is not particularly limited. For example, the gene of the present invention can be introduced by incorporating it into a vector such as a viral vector (e.g., retrovirus, lentivirus, or adenovirus), or a non-viral vector (e.g., a plasmid or a bacterial vector), and then infecting or incorporating it into the intended cells. Alternatively, the gene can be introduced using electroporation, transposon, or the like.

[0038] When multiple genes are introduced, each gene can be placed under the control of an independent promoter and inserted into the same or separate vectors, or the genes can be linked via intervening sequences to form a single expression cassette using a single promoter.

[0039] Examples of intervening sequences that can be used in the latter method include, but are not limited to, an IRES (internal ribosome entry site) sequence and a 2A peptide sequence (Szymczak et al., Expert Opin. Biol. Ther., 2005, 5: 627-638). The 2A peptide is a virus-derived peptide sequence of approximately 20 amino acid residues that is recognized and cleaved by intracellular proteases. Therefore, multiple genes linked by the 2A peptide are cleaved after being transcribed and translated in the cell.

[0040] Alternatively, the T cell receptor protein can be expressed on the cell surface by synthesizing the T cell receptor protein from the polynucleotide using a protein synthesis system and then introducing it into patient-derived T cells. Methods for introducing proteins into target cells include, for example, the method described in Shimono K et al., Protein Sci. 2009 Oct;18(10):2160-71. doi: 10.1002 / pro.230.

[0041] T cells produced by the methods of the present invention can act specifically against target cells expressing MRP3-derived peptides and HLA-A24 antigens. Because MRP3-derived peptides are presented together with MHC antigens on MRP3-expressing hepatocellular carcinoma cells, they can exhibit specific cytotoxicity against tumor cells expressing HLA-A24, a common MHC antigen. As will be understood by those skilled in the art, T cells specific for tumor cells expressing MHC antigens other than HLA-A24 can also be produced as appropriate using the description herein and common technical knowledge in the art.

[0042] The present invention also provides a polynucleotide encoding the T cell receptor protein of the present invention. Furthermore, it also provides a vector comprising such a polynucleotide. The polynucleotide encoding the T cell receptor protein is composed of polynucleotides encoding the variable regions and polynucleotides encoding the constant regions of both the α chain and the β chain.

[0043] In one embodiment, the present invention provides a vector comprising a polynucleotide encoding the α chain variable region and / or a polynucleotide encoding the β chain variable region of a T cell receptor protein that specifically binds to a complex of a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 and an HLA-A24 antigen.

[0044] It has already been reported that by introducing both polynucleotides encoding the α and β chains of TCR, the α and β chain polypeptides expressed in the introduced cells can form an αβ-TCR heterodimer, thereby conferring the desired specificity (Dembic Z. et al. (1986) Transfer of specificity by murine alpha and beta T-cell receptor genes. Nature 320:232-8).

[0045] In one embodiment, in the vector of the present invention, the polynucleotide encoding the alpha chain variable region may be a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2, and the polynucleotide encoding the beta chain variable region may be a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 6.

[0046] In one embodiment, in the vector of the present invention, the polynucleotide encoding the alpha chain variable region may be a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 3, and the polynucleotide encoding the beta chain variable region may be a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 7.

[0047] In one embodiment, in the vector of the present invention, the polynucleotide encoding the alpha chain variable region may be a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 4, and the polynucleotide encoding the beta chain variable region may be a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 8.

[0048] The present invention also provides T cells comprising the above-described vector. The present invention also provides T cells comprising the above-identified T cell receptor proteins introduced in vitro. The T cells of the present invention are genetically modified T cells, more specifically cytotoxic T cells (CTLs), and can be separated from other cells or mixed with other cells, for example, in the form of PBMCs containing CTLs.

[0049] More specifically, the T cells of the present invention may be T cells containing a T cell receptor protein introduced in vitro, wherein the alpha chain variable region of the T cell receptor protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10, and the beta chain variable region of the T cell receptor protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 14.

[0050] Furthermore, the T cells of the present invention may be T cells containing a T cell receptor protein introduced in vitro, in which the alpha chain variable region of the T cell receptor protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11, and the beta chain variable region of the T cell receptor protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 15.

[0051] Furthermore, the T cells of the present invention may be T cells containing a T cell receptor protein introduced in vitro, in which the alpha chain variable region of the T cell receptor protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12, and the beta chain variable region of the T cell receptor protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 16.

[0052] The present invention also provides a cancer therapeutic agent comprising the above-described T cells. The T cells of the present invention can target any cancer cells, including hepatocellular carcinoma, as long as they express an MRP3 antigen epitope. MRP3 has been reported to be expressed on the cell surface of cancer cells, such as lung cancer, prostate cancer, pancreatic cancer, and breast cancer, and the therapeutic agent of the present invention may be effective against these cancer types. Therefore, cancers to be treated with the therapeutic agent of the present invention may include, but are not limited to, liver cancers such as hepatocellular carcinoma, and solid cancers such as lung cancer, prostate cancer, pancreatic cancer, and breast cancer. Furthermore, the cancers to be treated range from mild to severe, and the therapeutic agent can be suitably used for patients diagnosed with stage III or IV cancer.

[0053] As described in the Examples, the present inventors have confirmed that the T cells of the present invention have cytotoxic activity against cancer cells that are resistant to molecular targeted drugs and chemotherapy, and furthermore, have cytotoxic activity against CD90-positive cancer stem cells, which are in a poorly differentiated state and are prone to metastasis, and are a form of mesenchymal cells.

[0054] Therefore, the therapeutic agent of the present invention can be suitably used for the treatment of cancers resistant to anticancer drugs such as molecular targeted drugs and chemotherapy and / or advanced cancers. Furthermore, the therapeutic agent of the present invention may also have cytotoxic activity against cancer stem cells.

[0055] The above-mentioned therapeutic agents may be used alone or in combination with other drugs and treatment methods that can be used in the treatment of cancer. Usable other drugs and treatment methods include, but are not limited to, molecular targeted drugs such as sorafenib, chemotherapy, radiofrequency ablation therapy, surgical therapy, hepatic arterial (chemo)embolization therapy, radiation therapy, heavy ion therapy, radioisotope therapy, hepatic arterial infusion chemotherapy, peptide vaccine therapy, and other immune cell therapies. The therapeutic agent of the present invention and the above-mentioned other drugs may be administered simultaneously or separately, and may be administered by the same or different administration routes.

[0056] As an example of a combination with other drugs, the therapeutic agent of the present invention can be administered in combination with T cells containing a T cell receptor protein that specifically binds to a complex of an AFP-derived peptide and the HLA-A24 antigen. AFP is known to be highly expressed on the surface of various cancer cells and has therefore been investigated as a target for anticancer therapy. However, drugs targeting the AFP gene are ineffective against cancer cells that do not express AFP. As shown in the Examples, the present inventors have confirmed that the T cells of the present invention have cytotoxic activity against cancer cells that do not express AFP but do express MRP3.

[0057] T cells containing a T cell receptor protein that specifically binds to a complex of an AFP-derived peptide and an HLA-A24 antigen include, but are not limited to, the T cells disclosed in JP 2017-081836 A. In this case, the T cells of the present invention and the T cells that target AFP may be administered simultaneously or separately. When administered simultaneously, the T cells may be mixed in advance and then administered.

[0058] The therapeutic agents described above can also be formulated into pharmaceutical compositions, either alone or in combination with other active ingredients. In addition to the therapeutic agents of the present invention and other active ingredients, pharmaceutical compositions can contain pharmaceutically acceptable carriers, buffers, stabilizers, and the like commonly used in the art, depending on the administration route. Carriers include, but are not limited to, physiological saline, phosphate-buffered saline, glucose solution, and buffered saline. Salts, sugars, sugar alcohols, and the like can also be used as additives. The therapeutic agents and pharmaceutical compositions of the present invention are intended to be administered in liquid form, and therefore must be formulated in a form that maintains the stability of the active ingredient, CTL.

[0059] 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, or by injection or infusion into the affected area or in the vicinity of the affected area.

[0060] The dosage and frequency of administration of the therapeutic agent and pharmaceutical composition of the present invention vary depending on the patient's weight, sex, age, severity of the disease, etc., and are not particularly limited. For example, when the T cells of the present invention are used as an active ingredient, the dosage and frequency of administration are about 1 × 10 6 ~Approx. 1×10 12 pieces, preferably about 1 x 10 8 ~Approx. 1×10 11 It contains a range of cells and can be administered once to several times a day, every two days, every three days, every week, every two weeks, every month, every two months, every three months, or every six months. [Example]

[0061] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0062] Example 1: Induction of MRP3-specific CTLs and TCR cloning MRP3 of SEQ ID NO: 1, which contains an HLA-A24-restricted CTL epitope and has been confirmed to induce CTLs 765 Peripheral blood mononuclear cells (PBMCs) were collected from 12 HLA-A24 positive hepatocellular carcinoma patients who received peptide vaccines and from 10 HLA-A24 positive healthy donors.

[0063] The obtained PBMCs were analyzed by MRP3 765 After inducing CTLs by stimulation with the peptide for 3 weeks, the cells were incubated with anti-CD8 antibody (Beckman Coulter, Inc.) and MRP3 765 The cells were stained with -MHC (HLA-A24) tetramer (Medical and Biological Laboratories, Inc.) for 30 minutes at room temperature in the dark, washed, and then analyzed by flow cytometry.

[0064] As a result, as shown in Figure 1, 3.74% of cells derived from hepatocellular carcinoma patients (A) and 1.21% of cells derived from healthy donors (B) expressed MRP3. 765 The results showed that the cells were CD8-positive and MRP3-specific T cells with specificity for MRP3, confirming the presence of the desired MRP3-specific T cells.

[0065] MRP3 obtained above 765-MHC tetramer-binding T cells were detected and collected as single cells using a FACSAria II sorting system (BD Bioscience, San Diego, CA, USA) according to the method described in Kobayashi E et al., Nature Medicine 19:1542-1546, 2013 and Nakagawa H et al., Gastroenterology. 152:1395-1406, 2017. TCR α and β chain cDNAs were then amplified by 5' RACE and RT-PCR and sequenced. The cDNAs were then analyzed in a repertoire, and antigen specificity was confirmed using the hTEC10 system.

[0066] Table 1 shows the cDNA sequences of the α and β chains of representative TCRs (TCR#: 12-47, 12-38, and 12-92) obtained from cells derived from a hepatocellular carcinoma patient, and a TCR (TCR#: HD6-89) obtained from cells derived from a healthy donor, with their respective SEQ ID NOs. Table 2 shows the sequences of the α chain variable region (VJ region) of the obtained TCRs and the SEQ ID NOs of the junction sequence including its CDR3. Table 3 shows the sequences of the β chain variable region (VDJ region) of the obtained TCRs and the SEQ ID NOs of the junction sequence including its CDR3. Table 4 shows the CDR sequences and SEQ ID NOs of the obtained TCRs. Repertoire analysis was performed using the Immunogenetics database website (http: / / www.imgt.org / ).

[0067] [Table 1]

[0068] [Table 2]

[0069] [Table 3]

[0070] [Table 4]

[0071] [Example 2] Preparation of MRP3-specific genetically modified T cells MRP3 of SEQ ID NO: 1 765 CD3 positive selection was performed using an automated magnetic cell separator on PBMCs from HLA-A24-positive hepatocellular carcinoma patients who had been administered peptides as peptide vaccines, and from frozen, preserved PBMCs from healthy individuals, and the CD3 positive fraction was sorted.

[0072] After sorting, 1 x 10 6 CD3-positive cells were stimulated in a culture medium containing 35 U / ml of IL-2 (Peprotech, Inc.) and CD3 / CD28 Dynabeads.

[0073] Separately, for cell transduction, a TCR expression vector was constructed by linking the cDNAs (SEQ ID NOS: 2-8) encoding the variable regions of the TCR α and TCR β chains, the sequences of which were determined in Example 1, with cDNAs encoding the constant regions (obtained by cloning from human PBMCs) via the viral P2A sequence (Leisegang M et al., J. Mol. Med. 2008, July, 86(7), p. 855). This vector was then cloned into the pMXs-IRES-GFP vector (Cosmo Bio Co., Ltd.) and transfected into the Phoenix-A retrovirus packaging cell line (National Gene Vector Biorepository, Indianapolis, USA). The codons in the constant regions of each TCR were optimized.

[0074] The resulting viral supernatant was filtered and added to a 24-well plate that had been coated overnight with Retronectin (Takara Bio Inc., Shiga, Japan) diluted to 50 μg / ml and washed with 2% BSA / PBS (500 μl / well). The plate was then centrifuged at 1900 × g at 32°C for 2 hours to spin-load the retrovirus.

[0075] After centrifugation, the stimulated CD3-positive cells were washed and cultured at 5 × 10 5 The resulting solution was adjusted to cells / ml, and the cells were seeded onto a spin-loaded plate with the retrovirus, and infected with the retrovirus to introduce the obtained polynucleotide encoding the TCR as RNA.

[0076] The seeded plate was centrifuged at 1000 xg at 32°C for 10 minutes and cultured in an incubator at 37°C. The next day, the CD3-positive cells were transferred to a new plate prepared in the same manner and cultured in an incubator at 37°C. The next day, the culture was scaled up by adding 10 ml of culture medium supplemented with 35 U / ml of IL-2 to a cell culture flask.

[0077] Seven days after the start of stimulation of CD3-positive cells, CD3 / CD28 Dynabeads were added and restimulated. On the 10th day, the gene-modified T cells were thoroughly suspended and a small amount was taken out. After centrifugation to remove the CD3 / CD28 Dynabeads, the cells were incubated with anti-CD8 antibody (Beckman Coulter, Inc.) and MRP3. 765 The cells were stained using -MHC (HLA-A24) tetramer (Medical and Biological Laboratories, Inc.) for 30 minutes at room temperature in the dark, and the generation of genetically modified T cells expressing the desired TCR was confirmed by flow cytometry.

[0078] Example 3: Evaluation of cytotoxic activity using C1RA24-Luc cells The cytotoxic activity of the MRP3-specific gene-modified T cells (effector cells) of the present invention was evaluated using C1RA24-Luc cells, which were derived from C1R-A24 cells (a subline of C1R lymphoma expressing HLA-A24, kindly provided by Dr. Masafumi Takiguchi, Center for AIDS Research, Kumamoto University) transfected with Kusabira orange luciferase. These cells, along with K562 cells (human chronic myeloid leukemia cells; purchased from the Cell Bank, RIKEN BioResource Center), used to suppress nonspecific cytotoxicity, were maintained in RPMI-1640 medium (Wako Pure Chemical Industries Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (BioWest SAS, Nuaille, France) and 1% penicillin / streptomycin.

[0079] First, 4 to 16 hours before the assay, C1RA24-Luc cells (500,000 cells) were transfected with the tumor antigen peptide MRP3. 765 The cells were pulsed with 10 μg / ml of peptide and cultured in an incubator at 37°C and 5% CO2 for 4 to 16 hours. On the day of the assay, the pulsed C1RA24-Luc cells and K562 cells were collected and the cell numbers were adjusted, and the two cells were mixed so that the number of K562 cells was 40 times the number of C1RA24-Luc cells.

[0080] Meanwhile, the MRP3-specific gene-modified T cells prepared in Example 2 were collected, the cell numbers were adjusted, and a 2-fold dilution series was prepared in a 96-well plate. A mixture of C1RA24-Luc cells and K562 cells was added as target cells to the prepared plate and co-cultured at 37°C for 4 hours (effector cell:target cell ratios of 50:1, 25:1, 12.5:1, and 6.25:1). As a control, the epitope derived from cytomegalovirus, CMV pp65 328 We used CMV-specific gene-modified T cells, which were generated in the same manner as the MRP3-specific gene-modified T cells, using a T cell receptor gene that recognizes CMV.

[0081] After incubation, the plate was centrifuged to remove the supernatant, and PBS and Steady-Glo Luciferase Assay System were added. The mixture was incubated at room temperature for 10 minutes and then photometrically measured.

[0082] As a result, it was confirmed that both MRP3-specific genetically modified T cells expressing TCRs derived from hepatocellular carcinoma patients and TCRs derived from healthy individuals had significant cytotoxic activity against C1RA24-Luc cells presenting the MRP3 peptide compared to the control (CMV-specific genetically modified T cells) (data not shown).

[0083] [Example 4] Evaluation of cytotoxic activity against HepG2 cells The cytotoxic activity of the MRP3-specific gene-modified T cells (effector cells) of the present invention was evaluated using HepG2 cells, a human hepatoma-derived cell line expressing HLA-A24, as target cells in the same manner as in Example 3. Expression of the MRP3 gene in HepG2 cells was confirmed in advance by RT-PCR.

[0084] The day before the assay, 3 x 10 HepG2 cells were plated on an 8-well coverglass chamber. 4 The cells were seeded at 100 cells / well and cultured in phenol red-free RPMI1640 containing 10% FBS and 1% penicillin / streptomycin.

[0085] On the day of the assay, the culture medium of the HepG2 cells was removed, and a staining solution of Calcein Vioret 450 AM Viability Dye was prepared to a final concentration of 10 μM. 500 μl of the solution was added to each well and incubated at 37°C for 30 minutes. After 30 minutes of staining, the staining solution was removed and the cells were washed with 500 μl of the above culture medium per well.

[0086] The MRP3-specific gene-modified T cells (TCR#: 12-47 or HD6-89) prepared in Example 2 and the CMV-specific gene-modified T cells (negative control) similar to those in Example 3 were thoroughly suspended and collected, centrifuged to remove the CD3 / CD28 Dynabeads, and then 6 × 105 The solution was adjusted to a concentration of 10 ... The mixture of HepG2 cells and genetically modified T cells was left to stand for 10 minutes, allowing the genetically modified T cells to settle.

[0087] The cells were photographed every 30 seconds for 10 hours using a confocal microscope, and a 2-minute video was then recorded. To analyze cytotoxicity, screenshots were taken at 0 seconds and every 10 seconds thereafter, and the dead cells and live cells in the 0-second image were counted and the ratios were compared with those in the control group.

[0088] As a result, as shown in Figure 2A, the MRP3-specific gene-modified T cells of the present invention rapidly exhibited cytotoxic activity compared with CMV-specific gene-modified T cells, killing approximately 90% of target cells approximately 5 hours after contact. In contrast, the TCR (TCR#: HD6-89) derived from a healthy individual exhibited low cytotoxic activity approximately 5 hours after contact with target cells, with a cell death rate of approximately 20% (data not shown).

[0089] Example 5 Cytotoxic activity of MRP3-specific gene-modified T cells and AFP-specific gene-modified T cells against KM cells The target cells were KM cells, which are liver cancer cells established by the present inventors' group from an HCC patient who was refractory to sorafenib and intra-arterial chemotherapy, and the cytotoxic activity of MRP3-specific gene-modified T cells was evaluated in the same manner as in Example 4.

[0090] As a result, as shown in Figure 2B, the MRP3-specific gene-modified T cells of the present invention rapidly exhibited cytotoxic activity compared to the CMV-specific gene-modified T cells, killing most of the target cells approximately 5 hours after contact.

[0091] Next, the cytotoxic activity of MRP3-specific gene-modified T cells against KM cells was evaluated in comparison with the activity of AFP-specific gene-modified T cells. As AFP-specific gene-modified T cells, AFP-specific gene-modified T cells were used as disclosed in JP 2017-081836 A. 357 T cells (TCR#: 1-14) containing a T cell receptor protein that specifically binds to a complex of AFP-derived peptide and HLA-A24 antigen were used, which were generated using a TCR gene that recognizes the epitope peptide. As shown in Figure 3, these AFP-specific gene-modified T cells were evaluated in the same manner as in Example 4 and were confirmed to exhibit cytotoxic activity against HepG2 cells, which are known to express AFP.

[0092] The day before the assay, 6 x 10 KM cells were plated on an 8-well cover glass chamber. 4 The cells were seeded at 100 cells / well and cultured in phenol red-free RPMI1640 containing 10% FBS and 1% penicillin / streptomycin.

[0093] On the day of the assay, the culture medium of the KM cells was removed, and a staining solution of Calcein Vioret 450 AM Viability Dye was prepared to a final concentration of 10 μM. 500 μl of this solution was added to each well and incubated at 37°C for 30 minutes. After 30 minutes of staining, the staining solution was removed and the cells were washed with 500 μl of the above culture medium per well.

[0094] As effector cells, MRP3-specific gene-modified T cells (TCR#: 12-47), AFP-specific gene-modified T cells (TCR#: 1-14), and CMV-specific gene-modified T cells (negative control) prepared in Example 2 were thoroughly suspended and collected, centrifuged to remove the CD3 / CD28 Dynabeads, and 6 × 10 5 The concentration was adjusted to 1 / ml, 1 μl of PI and 10 μl of anti-fading agent were added, and the mixture was mixed well, and then added at 500 μl / well to the stained KM cells.

[0095] The mixture of KM cells and genetically modified T cells was left to stand for 10 minutes to allow the genetically modified T cells to settle, and the cells were photographed using a confocal microscope every 30 seconds for 10 hours, after which a 2-minute video was recorded. To analyze cytotoxicity, screenshots were taken of the video at 0 seconds and every 10 seconds thereafter, and the dead cells and live cells in the 0-second image were counted and their ratios were compared.

[0096] As a result, as shown in Figure 4A, the MRP3-specific gene-modified T cells of the present invention exhibited significantly higher cytotoxic activity than the AFP-specific gene-modified T cells, demonstrating their effectiveness against cancers for which conventional treatments have been ineffective. RT-PCR confirmed that the KM cells used in this study for cytotoxic activity expressed the MRP3 gene but not the AFP gene (Figure 4B).

[0097] Example 6: Evaluation of cytotoxic activity using CD90+ and CD90- fractions of KM cells In Example 5, KM cells were confirmed to express the MRP3 gene and be susceptible to cytotoxicity by the MRP3-specific gene-modified T cells of the present invention. These cells were then separated into stem cell (CD90+) and non-stem cell (CD90-) fractions, and the expression levels of MRP3 mRNA were measured by RT-PCR. As a result, expression of MRP3, a potential target for immunotherapy, was observed even in the CD90+ fraction (Figure 5).

[0098] We then evaluated the cytotoxic activity of MRP3-specific gene-modified T cells against the stem cell fraction. The day before the assay, KM cells were stained with anti-CD90 antibody and sorted into CD90+ and CD90- fractions using a flow cytometer. The CD90+ fraction was then sorted into 7.5 x 10 4 cells / 500 μl / well, CD90- fraction: 1 × 10 5 The cells were seeded in 500 μl / well onto an 8-well cover glass chamber and cultured in phenol red-free RPMI1640 containing 10% FBS and 1% penicillin / streptomycin.

[0099] On the day of the assay, the culture medium of the KM / CD90+ and KM / CD90- cells was removed, and a staining solution of Calcein Vioret 450 AM Viability Dye was prepared to a final concentration of 10 μM. 500 μl of this solution was added to each well and incubated at 37°C for 30 minutes. After 30 minutes of staining, the staining solution was removed and the cells were washed with 500 μl of the above culture medium per well.

[0100] The MRP3-specific gene-modified T cells (TCR#: 12-47) and CMV-specific gene-modified T cells (negative control) prepared in Example 2 were thoroughly suspended and collected, centrifuged to remove the CD3 / CD28 Dynabeads, and then 6 × 10 5 The concentration was adjusted to 1 / ml, 1 μl of PI and 10 μl of anti-fading agent were added, and after mixing well, 500 μl / well of the solution was added to the stained KM / CD90+ and KM / CD90- cells.

[0101] The mixture of KM cells and genetically modified T cells was left to stand for 10 minutes to allow the genetically modified T cells to settle, and the cells were photographed using a confocal microscope every 30 seconds for 10 hours, after which a 2-minute video was recorded. To analyze cytotoxicity, screenshots were taken of the video at 0 seconds and every 10 seconds thereafter, and the dead cells and live cells in the 0-second image were counted and the ratios compared with those of the control group. As a result, as shown in Figure 6, it was demonstrated that the compound exhibited significant cytotoxic activity against CD90+ cells compared to the control, and that it can also act effectively against cancer stem cells. [Industrial Applicability]

[0102] The genetically modified T cells of the present invention can be used as a cell preparation for cancer immunotherapy. The therapeutic agent of the present invention can be effectively used against cancers that are resistant to conventional molecular targeted drugs and chemotherapy, as well as against cancer stem cells, and can serve as a new therapeutic agent for intractable cancers.

Claims

1. A vector comprising a polynucleotide encoding a T cell receptor protein that specifically binds to a complex of a multidrug resistance-associated protein 3 (MRP3)-derived peptide and an HLA-A24 antigen, wherein the T cell receptor protein has any of the following characteristics (i) to (iii): (i) the polynucleotide encoding the α chain variable region is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2, and the polynucleotide encoding the β chain variable region is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 6; (ii) the α chain variable region has the amino acid sequence set forth in SEQ ID NO: 10 and the β chain variable region has the amino acid sequence set forth in SEQ ID NO: 14; and (iii) CDR1, CDR2, and CDR3 of the α chain are SEQ ID NOs: 26, 27, and 28, respectively, and CDR1, CDR2, and CDR3 of the β chain are SEQ ID NOs: 29, 30, and 31, respectively. A vector having

2. A T cell comprising the vector of claim 1.

3. A T cell receptor protein having any of the following characteristics (i) to (iii): (i) the polynucleotide encoding the α chain variable region is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2, and the polynucleotide encoding the β chain variable region is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 6; (ii) the α chain variable region has the amino acid sequence set forth in SEQ ID NO: 10 and the β chain variable region has the amino acid sequence set forth in SEQ ID NO: 14; and (iii) CDR1, CDR2, and CDR3 of the α chain are SEQ ID NOs: 26, 27, and 28, respectively, and CDR1, CDR2, and CDR3 of the β chain are SEQ ID NOs: 29, 30, and 31, respectively. T cells transduced in vitro.

4. A cancer therapeutic agent comprising the T cell according to claim 2 or 3.

5. The cancer therapeutic agent according to claim 4, which is administered in combination with T cells containing a T cell receptor protein that specifically binds to a complex of an alpha-fetoprotein (AFP)-derived peptide and an HLA-A24 antigen.

6. The cancer therapeutic agent according to claim 4 or 5, for treating cancer resistant to anticancer drugs and / or progressive cancer.

7. The cancer therapeutic agent according to any one of claims 4 to 6, which has cytotoxic activity against cancer stem cells.

8. A method for producing T cells for gene therapy against cancer, comprising introducing a T cell receptor protein or a polynucleotide encoding the same into patient-derived T cells in vitro, wherein the T cell receptor protein has any of the following characteristics (i) to (iii): (i) the polynucleotide encoding the α chain variable region is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 2, and the polynucleotide encoding the β chain variable region is a polynucleotide having the nucleotide sequence shown in SEQ ID NO: 6; (ii) the α chain variable region has the amino acid sequence set forth in SEQ ID NO: 10 and the β chain variable region has the amino acid sequence set forth in SEQ ID NO: 14; and (iii) CDR1, CDR2, and CDR3 of the α chain are SEQ ID NOs: 26, 27, and 28, respectively, and CDR1, CDR2, and CDR3 of the β chain are SEQ ID NOs: 29, 30, and 31, respectively. A method comprising:

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