KR20230088292A - MR1-restricted Panck T cell and preparing method thereof - Google Patents

Abstract

The present invention relates to MR1-restricted pan cancer killing CD8 ⁺ T lymphocytes, MR1-restricted Panck T cells, and a preparation method thereof. According to the production method of the present invention, new T cells expressing the MR1 receptor having specificity limited to MR1 can be mass-produced, and T cells having these characteristics can be used as a universal anticancer agent for various carcinomas.

Description

MR1-restricted Panck T cell and preparation method thereof {MR1-restricted Panck T cell and preparing method thereof}

The present invention relates to MR1-restricted pan cancer killing CD8 ⁺ T lymphocytes, MR1-restricted Panck T cells, and a preparation method thereof.

MAIT cells (Mucosal associated invariant T cells) are a type of innate immune T lymphocytes that control various immune responses by generating various cytokines. It is known that MAIT cells are abundant in humans but not abundant in mice. MAIT cells have recently attracted attention due to the fact that their T cell receptor (TCR) does not show diversity. TCR is a receptor specifically expressed in T cells and recognizes a peptide fragment bound to a major histocompatibility complex (MHC) molecule as an antigen. Among T cells, TCR is a single cell that does not show diversity, and only two types are known: natural killer T cells (NKT cells) and MAIT cells. In the case of humans, the TCRα chain of MAIT cells is Vα7.2- A combination of Jα33, and in the case of NKT cells, only the Vα24-Jα18 combination is known. Further, in the case of mice, the TCRα chain of MAIT cells is only a combination of Vα19-Jα33, and in the case of NKT cells, the combination is Vα14-Jα18. The TCRs of both cells have different binding antigen-presenting molecules (restriction), and the TCRs of MAIT cells are MR1 (MHC-related molecule-1), which is similar to MHC, an antigen-presenting molecule in normal T cells, and has no diversity. recognizes and binds to, but in the case of NKT, CD1d (cluster of differentiation-1d) similar to MHC is used as an antigen presenting molecule. MAIT cell-specific TCR and MR1 are highly conserved evolutionarily.

MAIT cells have recently been reported and studied as being involved in various diseases, but many studies on their clear mechanisms have not been reported yet. Research using MAIT cells is limited because it is difficult to obtain MAIT from mice, where MAIT itself is rare, and MAIT cells are more abundant in humans than mice, but similarly, there is a limit to obtaining large amounts of cells from biological samples. because there is

Therefore, there is a need for a method for deriving mechanisms and treatment methods for various diseases by developing and mass-producing a cell line with characteristics similar to MAIT cells that can limitedly recognize and bind MRI without diversity. However, studies on the provision of cell lines with such characteristics and their utilization have not yet been widely conducted.

While conducting research on a novel T cell therapeutic agent that restrictively binds to MR1 and can be widely applied to various cancers, the present inventors constructed a method for mass production of new Panck T cells that restrictively bind to MR1 and manufactured The universal anticancer effect of Panck T cells was confirmed and the present invention was completed.

Accordingly, an object of the present invention is to provide a method for producing MR1-restricted T cells, MR1-restricted T cells produced by the production method, and an anti-cancer composition comprising the same.

In order to achieve the above object, the present invention comprises the steps of: 1) preparing cancer cells in which B2M (β ₂ -microglobulin) is knocked out; 2) inducing overexpression of B2M-linked MR1 in the cancer cells of step 1); 3) irradiating radiation to the cancer cells in step 2); 4) co-cultivating the cancer cells prepared in step 3) and the T cells; 5) culturing the T cells after culturing in step 4) in a medium containing one or more cytokines selected from the group consisting of IL-2, IL-7 and IL-15; and 6) co-culturing the cancer cells prepared in step 3) and the T cells cultured in step 5); It provides a method for producing MR1-restricted T cells comprising a.

In addition, the present invention provides MR1-restricted T cells produced by the above method.

In addition, the present invention is i) CD8 + T and 4-1BB +; ii) TCRva7.2-; and iii) CD69 ^high , CD45RO ⁺ , CD161 ^- , PD-1 ^low or CD57 ^low marker expression characteristics, MR1-restricted T cells are provided.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the MR1-restricted T cells.

In addition, the present invention provides a cancer treatment method comprising administering the MR1-restricted T cells to a subject in need thereof.

In addition, the present invention provides the use of the MR1-restricted T cells for cancer treatment.

In addition, the present invention provides the use of the MR1-restricted T cells in the manufacture of a therapeutic agent for cancer.

According to the production method of the present invention, new T cells expressing the MR1 receptor having specificity limited to MR1 can be mass-produced, and T cells having these characteristics can be used as a universal anticancer agent for various carcinomas.

1 is a schematic diagram showing a process for producing, expanding and proliferating MR1-restricted T cells (hereinafter referred to as 'Panck T cells') according to the present invention.
2 is a diagram showing the results of confirming the expression of MR1 and B2M in SW480 and the knockout of B2M protein in SW480, SK-OV3, A375, A549, THP-1, and K562 cancer cells.
3 is a diagram showing the results of confirming the expression of MR1 and B2M after overexpression of B2M-linked MR1 was induced in SW480, SK-OV3, A375, and A549 cells in which the B2M protein was knocked out.
Figure 4a confirms MHC I expression through binding with anti-HLA-ABC (MHC 1) antibody in SW480 B2M KO / MR1 OV and SK-OV3 / MR1 OV cell lines that are not B2M KO, and MR1-restricted expression It is a diagram showing the results of confirming whether or not the cancer cells are produced. 4B is a diagram showing the result of confirming MHC 1 expression in SK-OV3-B2m-ko/MR1, SK-OV3-B2m-ko, and wtSK-OV3.
5 is a view showing the results of confirming the expression of MR1 and immune T cells in ascites-derived cells of ovarian cancer patients.
Figure 6 is a diagram showing the results of confirming the CD8+, 4-1BB+, TCRva7.2(-) expression characteristics of Panck T cells prepared by the method of the present invention and confirming their purity.
Figure 7 is a diagram showing the results of comparing total T cell production and Panck T cell production capacity according to various cytokine treatments in the production process of the present invention.
8 is a diagram showing the results of confirming the phenotype of Panck T cells through FACS analysis.
9 is a diagram showing the results of confirming the cancer cell killing effect of Panck T cells and its mechanism.
10 is a diagram showing the results of confirming changes in the cytokines IFN-g and TNF after processing Panck T cells in various cancer cell lines.
11 and 12 are diagrams showing the results of confirming the efficacy of LCL tumor removal after administering Panck T cells to the LCL tumor model.
13 is a PBS control (Control), Panck T cells (Panck T 50M-2) isolated and produced from normal people, and Panck T cells (Panck T 50M-1) isolated and produced from ovarian cancer patients Tumor growth inhibition ability in experimental groups It is a diagram showing the result of checking.
14 is a diagram showing the results of confirming the tumor growth inhibition ability and the ratio of CD8 + T cells in a PBS control group (Control) and an experimental group of Panck T cells (Panck-1_#14) isolated and produced from an ovarian cancer patient.
15 is a diagram showing the results of confirming the TCR (T cell receptor) diversity of Panck T cells.
16 is a diagram showing the selection process of TCR clones from Panck T cells, and FIG. 17 is a diagram showing the results of clones showing positive TCR signal activity in the selection of TCR clones.
18 is a diagram showing the results of confirming cancer cell killing activity after treating SW480, A375, and SK-OV3 cancer cells with Panck T cells prepared from various donors.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing MR1-restricted T cells, and MR1-restricted pan cancer killing CD8 ⁺ T lymphocytes, referred to as 'Panck T cells' in the present invention, can be produced in large quantities. Since the Panck T cells of the present invention can universally bind to MR1 (MHC Class related Protein) expressed in various carcinomas, unlike anti-cancer immunity T cells that function only customized according to the carcinoma type and HLA (Huma Leukocyte Antigen), the HLA type It can be used as a treatment for various carcinomas regardless of cancer.

More specifically, 1) preparing cancer cells in which B2M (β ₂ -microglobulin) was knocked out; 2) inducing overexpression of B2M-linked MR1 in the cancer cells of step 1); 3) irradiating radiation to the cancer cells in step 2); 4) co-cultivating the cancer cells prepared in step 3) and the T cells; 5) culturing the T cells after culturing in step 4) in a medium containing one or more cytokines selected from the group consisting of IL-2, IL-7 and IL-15; and 6) co-culturing the cancer cells prepared in step 3) and the T cells cultured in step 5); It provides a method for producing MR1-restricted T cells comprising a.

Step 1) is a step of preparing cancer cells in which B2M (β ₂ -microglobulin) is knocked out. The cancer cells used in the above method are cancer cells used as stimulants for producing Panck T cells, and may be used without limitation in any type, and may include both solid cancer and hematological cancer. For example, cancer cells used in the present invention include acute lymphocytic cancer, acute myeloid leukemia, acinar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, rectal cancer, eye cancer, head and neck cancer, oral cancer, chronic lymphocytic leukemia, chronic bone marrow cancer, esophageal cancer, colon cancer, Cervical cancer, glioma, Hodgkin's lymphoma, pharynx cancer, kidney cancer, larynx cancer, gun cancer, lung cancer, melanoma, pancreatic cancer, peritoneal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, cervical cancer, ureter It may be one type of cancer cell selected from the group consisting of cancer, bladder cancer, colon cancer, and ovarian cancer. In the present invention, melanoma, colorectal cancer, ovarian cancer or lung cancer cell lines in which MR1 is expressed at a low level can be preferably used.

By knocking out B2M through step 1) above, it is possible to inhibit the induction of HLA-T cells by MHC I by limiting the MHC I ligand, and cancer cells that inhibit the formation of MHC class I and endogenous MR1 are produced. do. For B2M knockout, genetic engineering methods known in the art can be used without limitation, and in the present invention, the B2M gene was knocked out using CRISPR/CAS9.

Step 2) is a step of inducing overexpression of B2M-linked MR1 in the cancer cells of step 1), overexpressing MR1 again in cancer cells in which endogenous B2M has been completely removed, excluding the influence of MHC I and restricting MR1 Cancer cells presenting specific expression are prepared. The cancer cells prepared as described above are characterized in that the expression of MHC I is significantly inhibited and strongly expresses only MR1, unlike cancer cells in which only MR1 is overexpressed without knocking out B2M.

Methods known in the art may be used without limitation to induce overexpression of the B2M-linked MR1, and in a preferred embodiment of the present invention, a plasmid containing the B2M-linked MR1 gene is transfected into cells to induce overexpression.

In the present invention, step 3) is a step of irradiating the cancer cells of step 2) with 70 to 130 Gy gamma rays, more preferably 80 to 110 Gy gamma rays, 1 to 2 days before stimulating T cells with cancer cells. In one embodiment of the present invention, 100 Gy gamma rays were irradiated to prepare cancer cells for T cell stimulation. The cancer cells for stimulating T cells are cancer cells in which the expression of MHC I is significantly inhibited and only MR1 is strongly expressed, and cancer cells stimulated by radiation.

In the present invention, as the first step of the induction step for producing Panck T cells, 4) co-culturing the cancer cells prepared by the above method with T cells is included. Co-cultivation may be characterized by culturing the cancer cells and T cells at a cell ratio of 1:1 to 1:5, more preferably at a cell ratio of 1:3 to 1:4 to stimulate T cells by cancer cells. can induce

Thereafter, 5) culturing the T cells after culturing in step 4) in a medium containing one or more cytokines selected from the group consisting of IL-2, IL-7 and IL-15; can include The addition of the cytokine may be repeatedly performed with a time difference, for example, it may be added on the 2nd and 5th day of co-culture of cancer cells and T cells. The added cytokine may include one of IL-2, IL-7, or IL-15, two of these, or a combination of both of these, and in the present invention, as a preferred example, IL-2 alone as a cytokine; IL-7 and IL-15; Alternatively, T cell stimulation was induced by adding IL2, IL-7 and IL-15 to the medium. Among the cytokines, 20 to 80 IU of IL-2, and 1 to 10 ng/ml of IL-7 and IL-15 may be added to the medium, and in one embodiment of the present invention, 50 IU of rIL-2 and 5 ng/ml of rIL-7 ml and 5 ng/ml of rIL-15 were added to the medium to induce stimulation.

As described above, cytokine stimulation can be appropriately selected and treated according to the purpose, and in order to increase the total number of activated T cells obtained, preferably selected from the group consisting of IL-2, IL-7 and IL-15 A combination of two or more types, a medium containing both of them can be used, and in the case of obtaining a high ratio of CD8+/CD62L+ memory T cells among the total T cells obtained, a medium containing IL-2 alone can be used. .

In the present invention, the step of inducing stimulation of T cells through co-culture with the addition of cancer cells and cytokines prepared through steps 1) to 3) may be repeatedly performed as necessary. Therefore, the present invention, after stimulation by cancer cells and cytokines through step 5) is completed, 5-1) co-cultivating the cancer cells prepared through step 3) and T cells cultured in step 5); and 5-2) culturing the T cells after culturing in step 5-1) in a medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, and IL-15; It may additionally include repeating 1 to 5 times. The repeating step may be repeated 1, 2, 3, 4 or 5 times, but is not limited thereto, and is preferably repeated 2 or 3 times.

The cytokine added at this time may include one of IL-2, IL-7 or IL-15, two of them, or a combination of both. Among the cytokines, 20 to 80 IU of IL-2, and 1 to 10 ng/ml of IL-7 and IL-15 may be added to the medium, and in one embodiment of the present invention, 50 IU of rIL-2 and 5 ng/ml of rIL-7 Stimulation was induced by adding one or more of ml and 5 ng/ml of rIL-15 to the medium.

After the induction of T cells by the cancer cell stimulation and cytokine stimulation as described above, culturing the T cells in a medium containing IL-2 may be further included after step 5).

In this step, T cell stimulation is additionally induced by including a medium containing only IL-2 as a cytokine, and 1/2 of the culture medium is exchanged with a new medium containing IL-2 every 2 to 4 days, and the culture is carried out. carry out At this time, IL-2 may be added to the medium in an amount of 20 to 80 IU, preferably 40 to 60 IU.

The present invention includes 6) co-cultivating the cancer cells prepared in step 3) and the cultured T cells in step 5). In this step, stimulation-induced T cells after culturing in step 5) are stimulated once again with cancer cells that strongly express only MR1 and the expression of MHC I prepared in step 3) is significantly inhibited.

Steps 4) to 6), which are stimulation steps in the present invention, may be performed for 8 to 20 days, preferably 10 to 15 days, and more preferably 11 to 13 days. Therefore, step 6), the last step of the stimulation induction step, may be performed on the 12th day after the initiation of the initial stimulation of the co-culture of cancer cells and T cells, and the stimulation step ends after overnight stimulation.

Such a stimulation step is shown in FIG. 1 of the present invention. T cells after the stimulation phase are compared with T cells before stimulation induction, and 4-1BB expression is confirmed.

Through the above steps, “T cells”, which are MR1-restricted pan cancer killing CD8 ⁺ T lymphocytes exhibiting CD8+ and 4-1BB+ characteristics, can be prepared.

In addition, the present invention includes an additional expansion step to increase the yield of Panck T cells, which are CD8+ T cells that specifically express the MR1 receptor.

In the present invention, in order to increase the purity, a TCRva7.2+ sort out step to exclude the presence of MAIT cells can be additionally performed, through which CD8 (+) and TCRva7.2 (-) T cells are gated, Only CD8+ 4-1BB+ T cells can be isolated and used for the expansion step of the present invention.

Therefore, the present invention 7) isolating CD8+, 4-1BB+ and TCRva7.2(-) cells from among the cells prepared in step 5); and 8) culturing the cells separated in step 7); It may include an expansion step comprising, and step 8) may be characterized by culturing the cells isolated in step 7) in a medium containing irradiated allo-PBMC, IL-2 and anti-CD3 antibody. can More specifically, the radiation may be gamma radiation of 20 to 90 Gy, preferably gamma radiation of 50 to 70 Gy. The allo-PBMC may be included in 150 to 250 times the number of cells isolated in step 7), preferably 170 to 220 times. The concentration of IL-2 contained in the medium may be 800 to 1200 IU and the anti-CD3 antibody at a concentration of 10 to 50 ng/ml, and more preferably, the concentration of IL-2 is 900 IU to 1000 IU and the anti-CD3 antibody is 20 to 40 ng/ml. can be included

The expansion step of the present invention may be performed for 7 to 13 days, about 7 days when performed up to the semi-finished product expansion step, and additionally 11 to 13 days when performed up to the finished product expansion step. .

The T cells produced through the method for producing MR1-restricted T cells as described above may be characterized as CD8 + T and 4-1BB + cells, and CD8 +, 4-1BB +, and TCRva7.2 (-).

Accordingly, the present invention provides MR1-restricted T cells produced by the above production method, which are characterized in that they are CD8+T and 4-1BB+ cells.

The MR1-restricted T cells of the present invention may also be characterized as being TCRva7.2- and having CD69 ^high , CD45RO ⁺ , CD161 ^- , PD-1 ^low or CD57 ^low marker expression characteristics.

Therefore, the present invention provides for i) CD8+T and 4-1BB+; ii) TCRva7.2-; and iii) CD69 ^high , CD45RO ⁺ , CD161 ^- , PD-1 ^low or CD57 ^low marker expression characteristics, providing MR1-restricted T cells, preferably i) CD8 + T and 4-1BB +; ii) TCRva7.2-; and iii) MR1-restricted T cells having CD69 ^high , CD45RO ⁺ , CD161 ^- , PD-1 ^low and CD57 ^low marker expression characteristics.

Unlike conventional T cells, MR1-restricted T cells with marker expression characteristics as described above are limited in use depending on the expression of cancer antigens according to cancer type and HLA type, unlike conventional anti-cancer immune T cell therapeutics, regardless of HLA type. Since it is applicable to all types of cancer, it can be used as a novel T-cell therapeutic capable of binding to MR1.

T cells of the present invention are cultured T cells, such as all T cells such as primary T cells or T cells derived from cultured cell lines such as Jurkat, SupT1, etc., mammalian T cells, preferably human patient derived T cells or It may be a T cell precursor. In the case of mammalian origin, it can be obtained from blood, lymphocytes, bone marrow, thymus, or other tissues or fluids. Preferably, the T cells may be human T cells, CD4+ T cells, CD8 positive cytotoxic T lymphocytes, gamma delta T cells, tumor infiltrating lymphocytes or T cells isolated from peripheral blood mononuclear cells (PBMCs). .

In the present invention, MR1-restricted T cells prepared by the production method of the present invention or i) CD8 + T and 4-1BB +; ii) TCRva7.2-; and iii) CD69 ^high , CD45RO ⁺ , CD161 ⁻ , PD-1 ^low or CD57 ^low marker expression characteristics, and MR1-restricted T cells containing a pharmaceutical composition for preventing or treating cancer. In addition, the pharmaceutical composition of the present invention MR1-restricted T cells preferably i) CD8 + T and 4-1BB +; ii) TCRva7.2-; and iii) CD69 ^high , CD45RO ⁺ , CD161 ⁻ , PD-1 ^low and CD57 ^low marker expression characteristics, and MR1-restricted T cells.

In the present invention, "composition" is an active ingredient, together with the MR1-restricted T cell according to the present invention, inactive ingredients such as natural or artificial carriers, labels, or detection agents, or adjuvants, diluents, binders, stabilizers, buffers, salts , A lipophilic solvent, means a combination with an active ingredient such as a preservative, and includes a pharmaceutically acceptable carrier. Carriers may also include pharmaceutical excipients and additional proteins, peptides, amino acids, lipids, and carbohydrates (e.g., monosaccharides; disaccharides; trisaccharides; tetrasaccharides; oligosaccharides; sugars such as alditols, aldonic acids, esterified sugars); derivatives of, polysaccharides, sugar polymers, etc.) alone or in combination, and may be included in an amount of 1 to 99.99% by weight or volume%. Protein excipients include, but are not limited to, for example, human serum albumin, recombinant human albumin, gelatin, casein, and the like. Representative amino acid components that can act as buffers include, for example, alanine, arginine, glycine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. However, it is not limited thereto. Carbohydrate excipients may also include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose; disaccharides such as lactose, sucrose, trehalose, cellobiose, polysaccharides such as raffinose, maltodextrin, dextran, starch, and alditols such as mannitol, xylitol, maltitol, lactitol, sorbitol, and myoinositol. However, it is not limited thereto.

The pharmaceutical composition of the present invention can be formulated by a method known to those skilled in the art. For example, it can be used parenterally in the form of an injection of a sterile solution or suspension in water or other pharmaceutically acceptable liquids, if necessary. For example, in appropriate combination with a pharmaceutically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, excipient, vehicle, preservative, binder, etc., It can be formulated by mixing in a unit dosage form required for recognized pharmaceutical practice. The amount of the active ingredient in the formulation is such that an appropriate dose within the indicated range can be obtained.

In the present invention, the cancer may include both solid cancer and hematological cancer. For example, the cancers of the present invention include acute lymphocytic cancer, acute myelogenous leukemia, acinar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, rectal cancer, eye cancer, head and neck cancer, oral cancer, chronic lymphocytic leukemia, chronic bone marrow cancer, esophageal cancer, colon cancer, and cervical cancer. , glioma, Hodgkin's lymphoma, pharynx cancer, kidney cancer, larynx cancer, tendon cancer, lung cancer, melanoma, pancreatic cancer, peritoneal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, cervical cancer, ureter cancer, It may be one type of cancer selected from the group consisting of bladder cancer, colon cancer, and ovarian cancer. In the present invention, the cancer may preferably be melanoma, colon cancer, ovarian cancer or lung cancer.

The pharmaceutical composition of the present invention can be administered by oral administration, infusion, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intrarectal administration, topical administration, intranasal administration, etc., and the dosage depends on the route of administration, the patient It can be appropriately adjusted according to the age, gender, weight and severity of the patient and various factors. The pharmaceutical composition of the present invention may be administered in combination with other known anticancer agents for the purpose of preventing, improving or treating tumors or cancer symptoms.

In addition, the present invention provides a cancer treatment method comprising administering the MR1-restricted T cells to a subject in need thereof.

In addition, the present invention provides the use of the MR1-restricted T cells for cancer treatment and the manufacture of a cancer therapeutic agent.

The subject in need thereof refers to a subject suffering from cancer, or a subject likely to have cancer, or a subject suspected of having cancer, and may include mammals, including humans, without limitation.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1. Preparation of Panck T cells

The process of the present invention for preparing and priming new T cells capable of inducing cancer cell death and mass-producing them was performed as shown in FIG. 1 . The method of the present invention includes a stimulation phase of stimulating T cells with cancer cells and recombinant human IL-2 and an expansion phase of isolating stimulated T cells with high purity and culturing them in large quantities. It is carried out over 20 to 33 days.

More specifically, it is as follows.

1.1 Preparation of cancer cells for T cell stimulation

β ₂ -microglobulin (B2M), in addition to MR1, functions to present MHC I on the cell surface by binding to its ligand and forming a folding structure. Therefore, in order to inhibit the induction of HLA-T cells by MHC I by limiting the MHC I ligand, cancer cells in which B2M was knocked out were prepared. Cancer cells include A375 (melanoma cell line), SW480 (colon cancer cell line), SK-OV3 (ovarian cancer cell line), A549 (lung cancer cell line), A375 (malignant melanoma cell line), and K562 (chronic cancer cell line) in which MR1 is expressed at a low level. myeloid leukemia cells) and THP-1 (a human monocytic leukemia cell line) were used.

For the knockout of B2M in cancer cell lines, cancer cells were subjected to a mycoplasma test by PCR method to confirm that they were not contaminated with mycoplasma. For B2M knockout using the CRISPR/CAS9 system, the guide RNA targeting B2M was cloned into Guide-it CRISPR/CAS9 system plasmid (Clontech), including CRISPR/CAS9 and EGFP. . Cancer cells were cultured overnight in a 6-well plate, transfected with B2M gRNA CRISPR/CAS9/EGFP plasmid, and cultured for 72 hours. Transfection of the plasmid was confirmed by observing the level of intracellular EGFP expression under a fluorescence microscope. Cancer cells expressing EGFP were removed from the bottom, suspended in FACS Pre-sort buffer (BD), treated with APC-anti-human β (Biolegend) antibody, and reacted at 4° C. for 20 minutes. Using a FACS sorter, a cell group of EGFP(+)/B2M-APC(-) in which B2M expression was knocked out while expressing EGFP, a plasmid report, was isolated. The isolated cancer cells were seeded at 0.5 cell/well in a 96-well plate after measuring the cell number, and single cell cloning was performed. During cell culture, the formation of a cell group was confirmed in each well, and when the cell number increased, each clone was transferred to a 12-well plate to maintain the culture. Some cells of each clone were analyzed using the Guide-it™ Mutation Detection Kit (Clontech) to confirm the mutation of the B2M gene. The process is to amplify the B2M gene by PCR, enzymatically react with DNA hybridization, and conduct electrophoresis to detect the B2M gene. It involves identifying the truncated form of the gene.

In the analysis of each clone, the clones with the cut form by the enzymatic reaction were eliminated, and the DNA of the clones with the uncut form was separated from the agarose gel and subjected to gene sequence analysis. The gene sequence was translated into amino acids as well as DNA sequences, and clones having a mutation in the B2M sequence, especially a stop codon in the amino acid sequence, were selected. While maintaining the cell culture of the selected clone, 1.5-2x10 ⁶ cells were lysed with RIPA lysis buffer, protein was extracted, 50ug of protein was electrophoresed, transferred to a nitrocellulose membrane, and Western blotted with anti-B2M antibody (Abcam) to SW480 (ATCC, CCL-228) plus SK-OV3 (ATCC, HTB-79), A375 (KCLB, KCLB80003), A549 (ATCC, CCL185), THP-1 (ATCC, TIB-202) and K562 (KCLB, KCLB10243) ) was confirmed by knockout of the B2M protein, and the results are shown in FIG. 2 .

As shown in FIG. 2 , it was confirmed that the knockout of the B2M protein was effectively achieved in each cell line, and cancer cell lines in which the formation of MHC class I and endogenous MR1 were suppressed were prepared.

B2M knockout cancer cells were cultured by seeding 3-5x10 ⁶ cells in a 6-well plate, and the next day, a plasmid containing the MR1 gene linked to B2M and the G418 selection marker was transfected into the cells to induce overexpression. Cells overexpressing MR1 were first screened with gradually increasing concentrations of the G418 antibiotic, and when a sufficient number of cells was established, the cell group with strong MR1 expression was subjected to 1-2 FACS using anti-MR1-APC (Biolegend) antibody. Secondary separation was performed by sorting. Overexpression of MR1 was established in the SK-OV3-B2M ko-11, A375-B2M ko-4, and A549-B2M ko-28 cell lines in addition to the SW480-B2M ko-5 cell line.

After overexpression of B2M-linked MR1 was induced in the cancer cell line, after knocking out B2M, it was confirmed that cancer cells in which MR1 was overexpressed again were prepared. The results are shown in FIG. 3 .

As shown in FIG. 3 , it was confirmed that MR1 and B2M expressions were increased again by the induction of MR1 overexpression.

MHC I expression was confirmed in SW480 B2M KO/MR1 OV prepared by knocking out B2M and overexpressing MR1 and in SK-OV3/MR1 OV, a SK-OV3 cell line in which only MR1 was overexpressed without knocking out B2M. Binding with the anti-HLA-ABC antibody was confirmed to see if it was effectively inhibited. In addition, MHC I expression was confirmed in the SK-OV3-B2M KO/MR1 cell line, wild-type SK-OV3 (WtSK-OV3), and SK-OV3-B2M-ko cells in which only B2M was knocked out without inducing MRI overexpression.

As shown in FIG. 4a, the cell line SW480 B2M KO/MR1 OV overexpressing MR1 after B2M knockout produced in the present invention showed very low binding to anti-HLA-ABC antibodies, and the expression of MHC I was about 9.89%. confirmed to be Also, as shown in FIG. 4B , it was confirmed that MHC I expression disappeared in the SK-OV3 B2M KO/MR1 cell line prepared by knocking out B2M and overexpressing MR1. These results are in contrast to the strong MHC I expression of 99% or more in SK-OV3/MR1 OV cells prepared by overexpressing MR1 without knocking out B2M. In addition, it was confirmed that cancer cells exhibiting MR1-restricted expression were prepared through the present invention. These results indicate that it is effective to remove strong expression of MHC I by B2M knockout in order to induce MR1-restricted T cell activity.

The prepared B2M-knockout/MR1 overexpressing cancer cells were prepared by irradiation with 100 Gy gamma radiation one day before use in the following stimulation step.

In order to confirm the expression pattern of MR1 in cells of actual cancer patients, immune T cells were analyzed in cells isolated from ascites of cancer patients. First, cells were isolated from the ascites of 5 ovarian cancer patients provided by the IRB. Some of the isolated cells were stained with MR1 antibody (Biolegend), and stained with CD4 antibody (BD) and CD8 (Biolegend) antibody to confirm the presence of immune T cells in ascites-derived cells. The results of confirming the expression of MR1 in ascites-derived cells are shown in FIG. 5 .

As shown in FIG. 5, it was confirmed that MR1 was strongly expressed in ascites-derived cells ascites 6 and 13, moderately expressed in ascites 5 and ascites 10, and weakly expressed in ascites 9. However, it was confirmed that MR1 expression was relatively increased in cancer patient-derived cells compared to weak expression in normal cells or cancer cell lines. In addition, as a result of confirming the binding to the CD4 and CD8 antibodies, it was confirmed that a certain percentage of immune T cells were present in the cells derived from the ascites of the cancer patient. These immune T cells were expected to have better induction of stimulation to MR1.

1.2 stimulation phase

The stimulation step is a step in which T cells are stimulated by cancer cells and treated with an appropriate combination of cytokines to induce MR1 receptor-expressing T cells.

Stimulating T cells with the SW480 B2M KO/MR1 OV cell line or the SK-OV3/MR1 OV cell line prepared in 1.1 above can induce the proliferation of T cells (Panck T cells) expressing MR1 receptors having specificity limited to MR1. can

As the first step to prepare MRI-restricted T cells, PBMCs are isolated from blood of healthy donors or ovarian cancer patients by density gradient using Ficoll-paque (Cytiva) or pan T cells are isolated (Pan T cell isolation Kit, Miltenyi Biotec). As in Example 1.1, the B2M-knockout/MR1 overexpressing cancer cells irradiated with 100 Gy gamma radiation the day before were prepared by attaching and culturing 5x10 ^5/ well cells to a 12-well plate. Attached irradiated B2M-knockout/MR1 overexpressing cancer cells and isolated PBMC-derived T cells were co-cultured at a T cell ratio of 3 to 4 times that of cancer cells to induce T cell stimulation (Day 1).

Thereafter, human rIL-2 (50 IU) was added only twice on days 2 and 5 of culture using a CTL medium (Wellgene) supplemented with 3% auto-plasma.

On Day 7, the newly attached B2M-knockout/MR1 overexpressing cancer cells irradiated with 100 Gy gamma irradiation and the T cells stimulated in the previous step were co-cultured and a second stimulation was applied. On Day 9 and Day 11, the medium was replaced with fresh CTL+3% auto-plasma medium supplemented with rhIL-2 (50 IU). On Day 11, newly attached 100Gy gamma irradiated B2M-knockout/MR1 overexpressing cancer cells were prepared. Thereafter, on Day 12, the newly attached B2M-knockout/MR1 overexpressing cancer cells irradiated with 100 Gy gamma radiation were co-cultured with the previously stimulated T cells, re-stimulated overnight, and then the stimulation step was terminated.

Expression of 4-1BB was not confirmed in CD8+ T cells before stimulation with irradiated B2M-knockout/MR1 overexpressing cancer cells, but expression of 4-1BB was confirmed in CD8+ T cells subjected to the stimulation method.

1.3 expansion phase

The expansion step is a step for mass proliferation of CD8 + T cells specifically expressing the MR1 receptor prepared by stimulation induction in 1.2. Since it was confirmed that 4-1BB was expressed in the CD8+ T cells that had undergone the induction step, T cells expressing the MR1 receptor were isolated using this. In addition, to exclude the presence of MAIT cells, TCRva7.2+ cells were sorted out.

In other words, T cells induced to obtain desired cells were collected as single cell suspensions in FACS Pre-sort buffer (BD), anti-hCD8-V450 (Biolegend), anti-h4-BB-PeCy7 (BD), and anti-TCRva7 After treating with .2-APC (Biolegend) antibody and reacting at 4℃ for 20 minutes, CD8(+) and TCRva7.2(-) T cells were gated using FACs sorting, and then only CD8+ 4-1BB+ T cells separated. The results of analyzing the isolated cells are shown in FIG. 6 .

As shown in Figure 6, the purity of CD8+, 4-1BB+ cells was confirmed to be 97.1%, and CD8+TCRva7.2+ cells were found to be 0.05%, indicating that the T cells prepared in the present invention were highly pure CD8+, 4- It was confirmed that it was prepared with 1BB+, TCRva7.2(-) cells.

Thereafter, the isolated T cells were treated with ALys505N serum free medium (CSTI), 3% allo-plasma, allo-PBMC irradiated with 60Gy gamma radiation (more than 200 times the number of isolated Panck T cells), rhIL-2 (1000 IU) , and anti-hCD3 antibody (BD, 30ng / ml) to start the culture in an optimized medium containing rhIL-2 (1000IU) and 3% allo-plasma every 2 to 3 days thereafter ALys505N ( CSTI), cultured for 7 days while adding a medium, and then frozen as a semi-finished product or cultured for 11 to 13 days to obtain a large amount of Panck T cells having cancer cell killing efficacy.

The frozen semi-finished Panck T cells were thawed again for expansion culture, and allo-PBMC irradiated with ALys505N serum free medium (CSTI), 3% allo-plasma, and 60 Gy gamma radiation (more than 200 times the number of isolated Panck T cells), rhIL -2 (1000 IU), and an anti-hCD3 antibody (BD, 30 ng / ml) in an optimized medium containing culture proceeds for 11 to 13 days, Panck T cells can be prepared.

The cells prepared in this way can be defined as MR1-restricted T cells as T cells that specifically express the MR1 receptor, and MRI-restricted T cells prepared through the stimulation and proliferation steps of the present invention It was defined as 'Panck T cells'.

Example 2. Comparison of Panck T cell recovery effect by adding cytokines

In order to compare the production efficiency of Panck T cells by adding rIL-2, rIL-7 and rIL-15 performed in the stimulation step of the Panck T cell production process of the present invention, Cytokine was divided into i) IL-2 alone treatment, ii) IL-7 and IL-15 treatment, and iii) IL-2, IL-7 and IL-15 treatment groups, and the effects were compared.

experimental group density IL-2 alone i) IL-2; 50IU IL-7 and IL-15 i) IL-7; 5ng/mlii) IL-15; 5ng/mL IL-2, IL-7 and IL-15 i) IL-2; 50 IUii) IL-7; 5ng/mL
iii) IL-15; 5ng/mL

In order to exclude the effect of expansion, the total number of T cells obtained the next day after stimulation with irradiated B2M-knockout/MR1 overexpressing cancer cells in the last stimulation step on day 12 of culture was compared, and the results are shown in FIG. 7 shown in

As shown in Figure 7, IL-7 and IL-15, IL-2, IL-7 and IL in both the SW480 B2M KO/MR1 OV cell line and the SK-OV3/MR1 OV cell line compared to IL-2 treatment alone. It was confirmed that the total number of activated T cells obtained in the -15 treatment group increased.

However, the ratio of CD8+/4-1BB+ Panck T cells obtained from actual Panck T cell isolation was not significantly different among the three treatment groups, and the ratio of CD8+/CD62L+ memory T cells (boxed) was rather IL-2 alone. It was confirmed that the treatment was high.

That is, although the total number of T cells obtained in the induction phase was increased in the IL-2, IL-7, and IL-15 treatment groups than in the IL-2 treatment alone, the desired isolated CD8+/4-1BB+ Panck T cell ratio was substantially increased. showed a slight difference from IL-2 alone treatment, so if necessary, in order to increase the cell yield in the stimulation step, similar effects can be achieved even if IL-2 is treated alone or IL-7 and IL-15 are treated in combination confirmed. In the following examples, treatment with IL-2 alone was preferentially used for the preparation of CD8+4-1BB+ Panck T cells.

Example 3. Panck T cell isolation and phenotypic analysis

In order to confirm the phenotype of Panck T cells prepared and expanded by the method of Example 1, various T cell markers were analyzed. Specifically, Panck T cells are anti-hCD8-V450 (Biolegend), anti-hCD69-PeCy7 (Biolegend), anti-hCD161-BV605 (Biolegend), 5-OP-RU tetrmer-APC (Creative Biolabs ), anti-TCRva7.2-APC (Biolegend), anti-hCD62L-BV510 (BD), anti-hCD45RO (BD), anti-hCD57-FITC (BD), anti-hPD-1-FITC (BD) antibodies was treated and reacted at 4℃ for 20 minutes, and CD69 (involved in T cell activation), CD161, tetramer, TCRva7.2 (MAIT cell marker), CD62L (memory T cell marker), CD45RO (T cell activation/ proliferation), CD57 (T cell senescence marker), and PD-1 (T cell exhaustion marker) were analyzed. The cell marker confirmation results are shown in FIG. 8 .

As shown in Figure 8, it was confirmed that Panck T cells did not express MAIT cell markers tetramer and TCRva7.2, but expressed CD69 ^high , CD45RO ⁺ , CD161 ^- , PD-1 ^low , and CD57 ^low phenotypes. did Through the above results, it was confirmed that the Pank T cells of the present invention exhibit the properties of effector memory T cells.

Example 4. Confirmation of efficacy of Panck T cells in vitro

In order to confirm the ability of Panck T cells prepared by the method of the present invention to kill cancer cells, an in vitro experiment was performed. As target cancer cells, A375 (KCLB, KCLB80003), SW480 (ATCC, CCL-228), and MR1 ko-10-40-5 established by knocking out MR1 from SW480 were prepared. Panck T cells were obtained from healthy human blood. Prepared S0006-T cells and #11-210518-T cells and #12-210526-T cells prepared from ovarian cancer patients were used (donor blood was provided by the IRB).

The experimental groups were the group treated with only the culture medium, the target cancer cell line treated with lysis buffer to kill 100% SW480 only, the positive control group of MR1 ko-10-40-5 only, and the negative control group of Panck T only treated with each Panck T cell. Compared to the cancer cell line, the cancer cell killing effect was analyzed in the Panck T cell treatment group.

In a 96U-well plate, 1x10 ⁴ cells of the target cancer cell line were added to each well, and Effector Panck T cells were added in 1x10 ⁴ T cells in a 1:1 ratio of the cancer cell line and 5x10 ⁴ T cells in a 1:5 ratio. and then incubated for 16 hours. Cancer cell killing efficacy was analyzed through LDH (CytoTox96 Non-radioactive Cytotoxicity assay, Promega) analysis method. In order to compare whether the cancer cell killing effect is MR1-binding toxicity by the MR1-specific receptor of Panck T cells, SW480 and A375 were used as target cancer cell lines, and SW480-MR1 KO 10-40 in which MR1 expression was knocked down as a control group -5 cells were compared as a negative control, and also by blocking the MHC I protein of the cancer cell line using an anti-MHC I antibody (BD), it was confirmed whether it affects the cancer cell killing effect of Panck T cells. The results of confirming the cytotoxicity to cancer cells are shown in FIG. 9 .

As shown in Figure 9, the cancer cell killing effect was confirmed in all three types of Panck T cell treatment groups. In particular, the apoptotic effect was significantly reduced in KO 10-40-5 cells in which MR1 expression was knocked down in all experimental groups, and the apoptotic effect was not significantly affected in the anti-MHC I antibody blocking experimental group. It was confirmed that the cancer cell killing effect of cells was the MR1-limited cancer cell killing effect.

In addition, in Example 4, the amount of cytokine produced by Panck T cells secreted during the cancer cell death experiment by Panck T cells was analyzed by CBA analysis (BD, Cytometric Bead Array) method. The experimental groups were the group treated with only the culture medium, the target cancer cell line treated with lysis buffer to kill 100% SW480 only, the positive control group of MR1 ko-10-40-5 only, and the negative control group of Panck T only treated with each Panck T cell. In comparison with the Panck T cell treatment group of cancer cell lines, cytokines produced from the cancer cell killing effect were analyzed.

The culture medium of each experimental group was treated with the cytokine capture bead mix of each IL-2, IL-4, IL-6, IL-10, TNF, IFN-g, and IL-17, reacted for 3 hours at room temperature, and then each cytokine The cytokine binding strength included in the culture medium of the beads was confirmed by the mean value of flow cytometry, and the results of IFN-g and TNF, in which significant changes were observed, are shown in FIG. 10 .

As shown in FIG. 10 , IFN-g and TNF cytokines were increased in the experimental group treated with Panck T cells, but such cytokines were not increased in KO 10-40-5 cells in which MR1 expression was knocked down. In addition, since cytokines increased without being affected by anti-MHC I antibody blocking, it was confirmed that Panck T cells were MR1-restricted T cells.

The cancer cell killing efficacy of Panck T cells can be confirmed through the marked increase in IFN-g production in T cells of all experimental groups, whereas the production of TNF showed a significant increase in killing cancer cell lines, but blocking anti-MHC I did not observe a consistent effect.

Example 5. In vivo Panck T anticancer activity assay

To confirm the in vivo anticancer activity of the Panck T cells of the present invention, a blood tumor model was used. Tumors were formed by subcutaneously administering 4x10 ⁶ cells of LCL (human lymphoblastoid cell line) cells to immunodeficient NSG mice, and tumor sizes were measured. Before Panck T cell administration, groups were divided based on the average tumor size of all mice, and each individual mouse was labeled with a number. In the first experiment, the number of Panck T cells was administered at 10x10 ⁶ cells/mouse, and in the second experiment, the number of cells used in the first experiment was lower than that of 6.6x10 ⁶ cells/mouse and 3.3x10 ⁶ cells/mouse. administered. As a negative control, 1xDPBS was used, and CD8+/4-1BB- T cells (Normal T) isolated as normal T cells were used. The experiment was repeated twice, and the results confirming the LCL tumor removal efficacy are shown in FIGS. 11 and 12.

As shown in FIG. 11 , the reduction in tumor volume in all experimental groups in which Panck T cells were administered once, 10x10 ⁶ cells/mouse was confirmed by average tumor growth by group (left) and tumor growth by individual (right).

In addition, as shown in FIG. 12, LCL tumor growth was significantly inhibited even in the experimental groups administered with Panck T cells at a cell number of ^{6.6x10 6} cells/mouse and 3.3x10 ⁶ cells/mouse, which were lower than the cell number of 10x10 6 cells/mouse. Average tumor growth by group (left) and individual tumor growth (right) were confirmed. On the other hand, in the experimental group (Neg group) using CD8+/4-1BB- T cells, which are normal T cells, the growth of early cancer cells increased rapidly, showing a difference from the Panck T cell-administered group. The above results show that unlike normal T cells, Panck T cells prepared by the method of the present invention can inhibit tumor growth more quickly and effectively even at a lower concentration.

Example 6. In vivo Panck T anticancer activity assay in SW480 solid cancer

In order to confirm the anticancer effect in the solid carcinoma SW480 tumor model, Panck T cells isolated from ovarian cancer patients and prepared by the method of Example 1 (group 1), isolated from healthy normal donors and prepared by the method of Example 1 Panck T cells (2 groups) were administered to a solid carcinoma SW480 tumor model at a dose of 5x10 ⁷ cells/mouse. After that, the change in tumor size was confirmed in each experimental group, and the results are shown in FIG. 13 .

In addition, in order to confirm the anticancer effect in the solid carcinoma SW480 tumor model, Panck T cells isolated from another ovarian cancer patient and prepared by the method of Example 1 were used in the solid carcinoma SW480 tumor model at a dose of 5x10 ⁷ cells/mouse. Administered (Panck-1_#14). After that, the change in tumor size in each experimental group was confirmed. After separating PBMCs from blood, they were stained with anti-hCD45-PE/Cy7 (BD) and anti-hCD8-V450 (Biolegend) antibodies to analyze the percentage and number of human hCD8+ cells. The PBS administration group was used as a control group, and the results of tumor growth confirmation, CD8+ T cell ratio and cell number in each experimental group are shown in FIG. 14 .

As shown in FIG. 13 , it was confirmed that Panck T cell group 1 isolated from an ovarian cancer patient exhibited stronger anticancer efficacy compared to Panck T cell group 2 isolated from a normal person.

In addition, as shown in FIG. 14, it was repeatedly confirmed that Panck T cells isolated from other ovarian cancer patients also showed strong anticancer efficacy, and the ratio and number of human CD8+ T cells were increased compared to the control group (PBS). Confirmed.

Example 7. Confirmation of TCR Diversity in Panck T Cells

The T cell receptor (TCR, T cell receptor) expressed in Panck T cells was analyzed for each donor isolated from normal people and ovarian cancer patients and prepared by the method of Example 1. Each Panck T cell was subjected to single cell RNA sequencing at the single cell level, and the ratio of TCR alpa/beta clones expressed in about 10,000 single cells in each sample was analyzed, and the results are shown in FIG. 15 .

TCR clonotypes of Panck T cells prepared from each of 5 ovarian cancer patients (#9, 10, 11, 12, 13) and 1 normal person (S0006) were analyzed, and various TCR clonotypes were shown for each individual and each donor. . This showed the diversity of activated T cell receptors derived from cancer cell lines regardless of the mis-match or half-match of HLA-A type between the stimulator cancer cell line and the donor.

As shown in FIG. 15, it was confirmed that the prepared Panck T cells were not limited to the HLA type and had various T cell receptor sequences, and representative Top 10 clonotype TCRs were compared in Panck T cells prepared from each donor blood. It was confirmed that TCR diversity was shown when.

Each TCR sequence analyzed from Panck T cells was cloned into a lentiviral vector and then produced as a virus. The endogenous TCR expression is knocked out and NFAT, a transcription factor, is activated by the newly injected external TCR signal stimulation, and the virus produced is introduced into Jurkat cells (BPS, TCR KO Jurkat-NFAT-luciferase) capable of expressing luciferase, and TCR Signal activity was analyzed to select positive TCR clones. The method for analyzing TCR signal activity is shown in FIG. 16, and the analysis results of clones showing positive TCR signal activity in the selection of TCR clones are shown in FIG. 17.

As shown in FIG. 17, as a result of individual individual analysis according to each blood donor, not only the clones selected in the cancer cell line (stimulator) and TCR signal activity analysis used in the manufacture of each Panck T cell, but also the same as the selected clone Clones (PC-like) having the same clonotype but different CDR3 sequences were also selected. Most of the selected TCR clones were selected from TCR sequences analyzed from Panck T cells, which showed strong anticancer activity when analyzed for in vivo anticancer activity.

Example 8. Confirmation of anticancer effect of Panck T cells derived from ovarian cancer patients

Panck T cells were prepared from several ovarian cancer patients by the method of Example 1, and their killing activity against various cancer cells was confirmed. SW480, A375, and SK-OV3 were used as cancer cells, and the results of confirming cancer cell killing activity are shown in FIG. 18 .

As shown in FIG. 18, about 83% in SW480, about 74% in A375, and about 77% in SK-OV3 showed cancer cell killing activity. Regardless, these results show that Panck T cells can universally kill various types of cancer cells.

Although specific parts of the present invention have been described in detail above, it is clear that these specific techniques are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (21)

1) preparing cancer cells in which B2M (β ₂ -microglobulin) was knocked out;
2) inducing overexpression of B2M-linked MR1 in the cancer cells of step 1);
3) irradiating radiation to the cancer cells in step 2);
4) co-cultivating the cancer cells prepared in step 3) and the T cells;
5) culturing the T cells after culturing in step 4) in a medium containing one or more cytokines selected from the group consisting of IL-2, IL-7 and IL-15; and
6) co-culturing the cancer cells prepared in step 3) and the T cells cultured in step 5); A method for producing MR1-restricted T cells comprising a.
The method of claim 1, wherein the radiation in step 3) is gamma rays of 70 to 130 Gy.
The method of claim 1, wherein the co-cultivation of step 4) involves culturing cancer cells and T cells at a cell ratio of 1:1 to 1:5.
The method of claim 1, after step 5)
5-1) co-cultivating the cancer cells prepared in step 3) and the T cells cultured in step 5); and
5-2) culturing the T cells after culturing in step 5-1) in a medium containing at least one cytokine selected from the group consisting of IL-2, IL-7 and IL-15; Repeating 1 to 5 times; Including, method.
The method of claim 1, wherein the cytokine in step 5) is IL-2 alone; IL-7 and IL-15; or IL2, IL-7 and IL-15.
The method of claim 5, wherein the IL-2 is contained in a concentration of 20 to 80 IU, and the IL-7 and IL-15 are contained in a concentration of 1 to 10 ng/ml.
The method according to claim 1, further comprising culturing T cells in a medium containing IL-2 after step 5).
The method of claim 1, wherein steps 4) to 6) are performed for 8 to 20 days.
According to claim 1,
7) isolating CD8+, 4-1BB+ and TCRva7.2(-) cells from among the cells prepared in step 5); and
8) culturing the cells separated in step 7); A method comprising an extension step comprising a.
The method of claim 9, wherein step 8)
The method of culturing the cells isolated in step 7) in a medium containing irradiated allo-PBMC, IL-2 and anti-CD3 antibody.
11. The method of claim 10, wherein the radiation is 20 to 90 Gy gamma radiation.
11. The method of claim 10, wherein the allo-PBMC is contained in 150 to 250 folds of the cells isolated in step 7).
The method of claim 10, wherein the IL-2 is 800 to 1200 IU and the anti-CD3 antibody is contained at 10 to 50 ng/ml.
10. The method of claim 9, wherein the expanding step is performed for 7 to 13 days.
The method of claim 1 , wherein the MR1-restricted T cells are CD8+ T and 4-1BB+ cells.
MR1-restricted T cells produced by the method of any one of claims 1 to 15.
17. The MR1-restricted T cell of claim 16, which is CD8+T and 4-1BB+.
The MR1-restricted T cell according to claim 16, wherein the cell is TCRva7.2- and has the characteristics of CD69 ^high , CD45RO ⁺ , CD161 ^- , PD-1 ^low or CD57 ^low marker expression.
i) CD8+T and 4-1BB+;
ii) TCRva7.2-; and
iii) MR1-restricted T cells with CD69 ^high , CD45RO ⁺ , CD161 ^- , PD-1 ^low or CD57 ^low marker expression characteristics.
A pharmaceutical composition for preventing or treating cancer comprising the MR1-restricted T cells of claim 16.
A pharmaceutical composition for preventing or treating cancer comprising the MR1-restricted T cells of claim 19.

Images