Method for producing genetically modified T cells

Abstract

To provide methods for easily producing highly purified genetically modified T cells without the risk of contamination that can isolate and activate genetically modified T cells with high efficiency.SOLUTION: Provided is a method for producing genetically modified T cells comprising: (1) a capture step of contacting a liquid sample containing genetically modified T cells with a genetically modified T cell capturing agent to capture the genetically modified T cells with the genetically modified T cell capturing agent, the genetically modified T cell capturing agent comprising a carrier and a genetically modified T cell recognition / activation substance immobilized on the carrier, and the genetically modified T cell recognition / activation substance being capable of specifically binding to the genetically modified T cells via a modified T cell surface protein and being capable of activating the genetically modified T cells; and (2) an activation step of incubating the genetically modified T cells captured in the capture step and activating them with the genetically modified T cell recognition / activation substance.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing genetically modified T cells. [Background technology]

[0002] A new method of cancer treatment is known as cancer immunotherapy, in which T cells (unmodified T cells) collected from a patient are genetically modified to enhance their ability to attack cancer, and then these genetically modified T cells are returned to the original patient to treat the cancer. In order to produce the genetically modified T cells in this type of treatment, it is necessary to artificially genetically modify T cells derived from the patient, and then to activate and proliferate (cultivate) the resulting genetically modified T cells with high purity, and it is also necessary to perform these operations efficiently.

[0003] For example, JP 2017-513499 A (Patent Document 1) describes a technique for producing such genetically modified T cells. This method involves preparing a cell sample containing T cells (unmodified T cells) in a closed sterile cell culture system, magnetically separating the T cells, activating them, genetically modifying them, and then growing and washing the genetically modified T cells in a chamber. Patent Document 1 describes magnetic separation (enrichment) methods, including a positive selection method using magnetic beads immobilized with antigen-binding molecules (e.g., antibodies) specific to cell surface markers on the surface of T cells, and a negative selection method using magnetic beads immobilized with antibodies against surface antigens present on the surface of cells other than the genetically modified T cells of interest (contaminating cells). Patent Document 1 also describes activation methods using agonist antibodies (e.g., anti-CD3 antibodies, anti-CD28 antibodies) or cytokines bound to beads or nanostructures as regulators. Patent Document 1 also describes that after the genetically modified T cells are expanded, the genetically modified T cells are selected and separated (enriched) using magnetic beads onto which an antibody against a surface molecule (modified T cell surface protein) expressed by gene transfer is immobilized.

[0004] However, in conventional methods such as those described in Patent Document 1, cell separation and cell activation and proliferation must be performed in physically separate devices, which complicates the process and reduces the cell recovery rate. Furthermore, in such methods, there is a risk that the magnetic beads used in the separation of unmodified T cells and genetically modified T cells may become contaminated as impurities in the recovered genetically modified T cells. The presence of such magnetic beads may inhibit the proliferation of genetically modified T cells, and there is also a risk of administering them to patients. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2017-513499 Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention was made in consideration of the above-mentioned problems associated with the prior art, and aims to provide a method for producing genetically modified T cells, which enables highly efficient separation and activation of genetically modified T cells and easy production of highly pure genetically modified T cells that are free from the risk of contamination by impurities. [Means for solving the problem]

[0007] The present inventors conducted extensive research to solve the above-mentioned problems and found that the separation (capture) and activation of genetically modified T cells contained in a liquid sample can be easily and efficiently performed by using a genetically modified T cell capture agent containing a genetically modified T cell recognition and activation substance, which is capable of specifically binding to and activating genetically modified T cells via the T cell surface protein (modified T cell surface protein) of the genetically modified T cells, and a carrier to which the genetically modified T cell capture agent is immobilized. In particular, the inventors found that the genetically modified T cell capture agent can be packed into a column, thereby allowing all of the above steps to be easily performed in a single physical space within the column. Furthermore, the inventors found that incubating the genetically modified T cells captured by the genetically modified T cell capture agent results in internalization of the modified T cell surface protein and detachment of the genetically modified T cells from the capture agent, thereby easily recovering genetically modified T cells with high purity, especially when using the column.

[0008] Therefore, the present inventors discovered that it is possible to specifically separate, activate, and even recover the desired genetically modified T cells with high purity and efficiency without using magnetic beads, which have traditionally been used for cell separation, and thus completed the present invention.

[0009] That is, the aspects of the present invention are as follows. [1] A method for producing genetically modified T cells, (1) contacting a liquid sample containing genetically modified T cells with a genetically modified T cell capturing agent, and capturing the genetically modified T cells with the genetically modified T cell capturing agent; a capturing step in which the genetically modified T cell capturing agent comprises a carrier and a genetically modified T cell recognition / activation substance immobilized on the carrier, the genetically modified T cell recognition / activation substance being a substance capable of specifically binding to the genetically modified T cells via a modified T cell surface protein and activating the genetically modified T cells; and (2) an activation step in which the genetically modified T cells captured in the capture step are incubated and activated with the genetically modified T cell recognition / activation substance; A method for producing genetically modified T cells, comprising: [2] The method for producing genetically modified T cells according to [1], wherein the genetically modified T cells are CAR-T cells. [3] The method for producing genetically modified T cells described in [2], wherein the genetically modified T cell recognition / activation substance is a ligand for CAR. [4] The method for producing genetically modified T cells according to any one of [1] to [3], wherein the genetically modified T cell recognition / activation substance is at least one selected from the group consisting of Protein L and BCMA. [5] After the activation step, (3) a recovery step of recovering genetically modified T cells that have internalized the modified T cell surface protein and have detached from the genetically modified T cell capture agent; The method for producing the genetically modified T cell according to any one of [1] to [4], further comprising: [6] the capturing step is a step of adding the liquid sample to a column packed with the genetically modified T cell capturing agent to capture the genetically modified T cells; the activation step is a step of incubating and activating the genetically modified T cells in the column; and the recovery step is a step of recovering the genetically modified T cells that have flowed out of the column. [5] A method for producing genetically modified T cells. [7] After the recovery step, (4) a re-expression step in which the genetically modified T cells recovered in the recovery step are re-incubated to re-express the modified T cell surface protein on the cell surface. The method for producing genetically modified T cells according to [5] or [6], further comprising: [8] A composition comprising genetically modified T cells obtained by the method for producing genetically modified T cells according to any one of [1] to [7]. [9] The composition described in [8], which is a pharmaceutical composition. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a method for producing genetically modified T cells, which enables highly efficient isolation and activation of genetically modified T cells and easy production of highly pure genetically modified T cells that are free from the risk of contamination by impurities. Furthermore, according to the present invention, it is also possible to easily recover genetically modified T cells with high purity and high efficiency. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing one embodiment of the capture step of the method for producing genetically modified T cells. [Figure 2] FIG. 1 is a schematic diagram showing one embodiment of a method for producing genetically modified T cells. [Figure 3] 1 is a graph showing the number of CAR-Jurkat cells or Jurkat cells added to each column (added cells: vertical axis = number of added cells) and the number of cells captured in each column (vertical axis = number of captured cells) when a comparative carrier or a Protein L-immobilized carrier (large) was used, obtained in Test Example 1(1). [Figure 4] 1 is a graph obtained in Test Example 1(1) showing the capture rates of CAR-Jurkat cells or Jurkat cells when a comparative carrier or a Protein L-immobilized carrier (large) was used. [Figure 5] 1 is a graph showing the relationship between the fluorescence intensity derived from labeled CAR and the number of cells (count) when a comparative carrier was used (a) or when a Protein L-immobilized carrier (large) was used (b) (dotted line: apply, black filled area: flow-through 1), obtained in Test Example 1 (2). [Figure 6]1 is a graph showing the capture rate of T-CAR-T cells when a comparative carrier or a Protein L-immobilized carrier (large) was used, obtained in Test Example 2 (1). [Figure 7] 1 is a graph showing the capture rate of n-CAR-T cells or nT cells when Protein L-immobilized carrier (small) was used, obtained in Test Example 2 (2). [Figure 8] 1 is a graph showing the relationship between the fluorescence intensity and the cell number (count) derived from labeled CAR in each sample applied (a), flow-through 1 (b), flow-through 2 (c), or after 4 days of incubation (d) when Protein L immobilized carrier (large) was used, obtained in Test Example 3 (1) (dotted line: nT cells; black filled area: applied, flow-through 1, flow-through 2, and each sample after incubation). [Figure 9] 1 is a graph showing the relationship between the ratio of n-CAR-T cells and the rate of CAR gene retention (relative value) obtained in Test Example 3 (2). [Figure 10] 1 is a graph showing the CAR gene retention rate (relative value) of cells in the application, flow-through 1, and flow-through 2 when a comparative carrier or a Protein L immobilized carrier (large) was used, obtained in Test Example 3 (3). [Figure 11] 1 is a graph showing luciferase activity obtained in Test Example 4(1) when anti-BCMA CAR-Jurkat cells were added to B-CHO, CHO, BCMA-immobilized beads, or comparative beads. [Figure 12] 1 is a graph showing the capture rate of CAR-Jurkat cells when a comparative carrier or a BCMA-immobilized carrier was used, obtained in Test Example 4 (2). DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention will be described in detail below based on preferred embodiments thereof.

[0013] (T cells) In the present invention, unless otherwise specified, the term "T cells" includes "genetically modified T cells" that have undergone a desired artificial genetic modification, as well as other T cells. "Other T cells" includes unmodified T cells, i.e., T cells that have not undergone an artificial genetic modification, and T cells that have undergone an artificial genetic modification other than the desired genetic modification.

[0014] In the present invention, the origin of the T cells is not particularly limited, and they may be T cells derived from a healthy individual or from a patient (for example, a person with a weakened immune function, or a person suffering from a malignant tumor, an infectious disease, or an autoimmune disease). Furthermore, the T cells may be T cells induced to differentiate from pluripotent stem cells such as induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells), or somatic stem cells such as hematopoietic stem cells, or established T cell lines.

[0015] In the present invention, "artificial genetic modification" means modifying the function of a T cell by introducing a gene into or editing the gene of a T cell. The "artificial genetic modification" in the present invention may be a modification of a gene carried by a T cell, a deletion of a gene carried by a T cell, a modification in which an exogenous gene is introduced, or a combination of two or more of these.

[0016] The artificial gene modification method includes, but is not limited to, conventionally known methods and methods based thereon. Examples of methods for gene introduction into T cells include the introduction of a gene that modifies T cell function (DNA, mRNA, miRNA, antagomir, ODN, etc.) itself, or a vector into which the gene has been inserted (lentiviral vector, γ- or α-retroviral vector, adenoviral vector, adeno-associated viral vector, herpesvirus vector, equine encephalopathy viral vector, etc.). Examples of gene editing methods for T cells include editing of T cell genes (genome editing) using site-specific nucleases (meganuclease, zinc finger nuclease, TALEN, PPR, CRISPR-Cas, etc.). In the present invention, the artificial gene modification method may be one of these methods alone or a combination of two or more of these methods.

[0017] The gene that modifies the function of the T cell, i.e., the gene to be modified by the artificial genetic modification, is preferably at least one selected from the group consisting of genes encoding a fusion protein comprising a protein expressed on the surface of T cells (referred to herein as a "T cell surface protein") and at least one intracellular signaling domain, and genes encoding a fusion protein comprising a T cell surface protein, at least one costimulatory domain, and at least one intracellular signaling domain.

[0018] More specifically, the target genes to be modified by the artificial genetic modification include, for example, the following proteins: TSHR, CD19, CD20, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA , EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, ERBB2(Her2 / neu), MUC1, EG FR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6 K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPVE6,E7, MAGEA1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, and at least one protein selected from the group consisting of genotype receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1 These include genes encoding single-chain variable fragments (scFv) of antibodies against the above-mentioned proteins as antigens, and genes encoding proteins or peptides that have binding affinity to the above-mentioned proteins.

[0019] Furthermore, the target gene to be modified by the artificial genetic modification is preferably a gene encoding a protein that specifically exists on the surface of T cells, such as a T cell receptor (TCR), which is an antigen receptor. More specifically, such proteins include the following proteins: At least one selected from the group consisting of intracellular signaling domains of CD3ε, CD3γ, and CD3Δ; functional signaling domains of 4-1BB and CD3ζ; and transmembrane domains of α / β TCR and γ / δ TCR In this case, specific antigens for the antigen receptor (preferably TCR) include, for example, MART-1, P53, CEA, NY-ESO-1, MAGE-A3, MAGE-A4, and WT1.

[0020] Furthermore, the gene encoding the antigen receptor may be a gene encoding a chimeric antigen receptor (CAR), which is an artificial protein.

[0021] Among these, the target gene to be modified by the artificial genetic modification is preferably a gene encoding the antigen receptor, more preferably at least one selected from the group consisting of a gene encoding a T cell receptor (TCR) and a gene encoding a chimeric antigen receptor (CAR), and even more preferably a gene encoding a chimeric antigen receptor (CAR).

[0022] That is, the genetically modified T cells of the present invention preferably express, on their surface, a surface protein (herein sometimes referred to as a "modified T cell surface protein") encoded by the gene of interest modified by the artificial genetic modification. Such genetically modified T cells are more preferably at least one type selected from the group consisting of TCR-T cells that express a T cell receptor (TCR) encoded by the gene modified by the artificial genetic modification, and CAR-T cells that express a chimeric antigen receptor (CAR), and even more preferably CAR-T cells that express a chimeric antigen receptor (CAR) as the modified T cell surface protein.

[0023] Note that the genetically modified T cells to be subjected to the capture step described below (i.e., the genetically modified T cells contained in the liquid sample described below) must express the modified T cell surface protein on their cell surface. However, the genetically modified T cells obtained by the method for producing genetically modified T cells of the present invention need only be capable of expressing the modified T cell surface protein (i.e., possess a gene encoding the modified T cell surface protein in an expressible manner), and may also be T cells that possess a gene encoding the modified T cell surface protein but do not express it on their cell surface, i.e., genetically modified T cells in which the modified T cell surface protein has been internalized (sometimes referred to herein as "surface protein-internalizing genetically modified T cells").

[0024] (liquid sample) In the present invention, the "liquid sample" is not particularly limited as long as it is a liquid sample containing the genetically modified T cells, and examples include blood samples containing the T cells, such as blood (whole blood), diluted blood, serum, plasma, cerebrospinal fluid, umbilical cord blood, and apheresis; fractions containing the T cells obtained by separation from the blood samples; T cells induced from pluripotent stem cells, established T cell lines, and culture media or buffer solutions containing genetically modified T cells.

[0025] The medium for the culture solution is not particularly limited and may be any of the conventionally known media, such as Roswell Park Memorial Institute (RPMI) 1640 medium, minimal essential medium (α-MEM), Dulbecco's modified Eagle's medium (DMEM), F12 medium, feeder-free medium, and TexMACS GMP medium (Miltenyi Biotec). The medium may also contain cytokines such as IL-2 and fetal calf serum (FCS), as needed.

[0026] The buffer solution is not particularly limited and may be any known buffer solution, such as phosphate buffered saline (PBS), MACS buffer, HBSS buffer, etc. The buffer solution may contain albumin or fetal bovine serum (FCS) as needed.

[0027] (Genetically modified T cell capture agent) The genetically modified T cell capturing agent according to the present invention comprises a carrier and a genetically modified T cell recognition / activation substance immobilized on the carrier.

[0028] [Genetically modified T cell recognition / activation substance] The "genetically modified T cell recognition / activation substance" of the present invention is a substance capable of specifically binding to the genetically modified T cells via the modified T cell surface protein and activating the genetically modified T cells. More specifically, the genetically modified T cell recognition / activation substance is a substance capable of specifically recognizing and binding to the modified T cell surface protein that is specifically present on the surface of the genetically modified T cells (i.e., the T cell surface protein modified by a desired artificial genetic modification or a T cell surface protein newly expressed on the T cell surface (e.g., CAR)), and is capable of activating only the genetically modified T cells via such specific binding.

[0029] In the present invention, "activation of genetically modified T cells" refers to at least one of an increase in the number of the genetically modified T cells (i.e., proliferation), an increase in the expression levels of cytokines expressed by the genetically modified T cells, such as IL-2, TGF-β, and IL-10, and internalization of the surface proteins of the genetically modified T cells described below.

[0030] Examples of the genetically modified T cell recognition / activation substance of the present invention include an antibody against the modified T cell surface protein, a fusion protein containing the antibody, or a conjugate of the antibody with a sugar chain; a protein, peptide, or aptamer that has binding affinity to the modified T cell surface protein; and if the modified T cell surface protein is an antibody or an antigen receptor (e.g., TCR, CAR, etc.), a substance that can activate the genetically modified T cells among proteins or peptides that serve as its antigen (e.g., the antigens exemplified above). One of these may be used alone, or two or more may be used in combination.

[0031] For example, when the genetically modified T cells of the present invention are CAR-T cells, the genetically modified T cell recognition / activation substance of the present invention is preferably a ligand for CAR (e.g., the specific antigen, anti-idiotype antibody, or antibody-binding substance (Protein L, Protein A, etc.)). As used herein, the term "antibody" is not limited to full-length antibodies (complete antibodies) but also includes antibody fragments and minibodies formed by binding antibody variable regions, so long as they have antigen-binding ability. Examples of antibody fragments include Fab, F(ab')2, Fab', diabodies, and single-chain antibodies (e.g., scFv, dsFv). Such antibodies can be produced by appropriately adapting and improving conventionally known methods, and commonly available antibodies can also be used as appropriate.

[0032] Among these, preferred substances for recognizing and activating genetically modified T cells according to the present invention are CD19, BCMA, CD20, HER2, protein L, protein A, anti-IgG antibodies, anti-Fab antibodies, anti-F(ab')2 antibodies, anti-Fab' antibodies, anti-diabody antibodies, anti-scFv antibodies, and anti-idiotype antibodies, with protein L and BCMA being particularly preferred. Protein L is a 35.8 kDa protein derived from Peptostreptococcus magnus, and is known to specifically bind to the κ light chain of immunoglobulins (particularly IgG).

[0033] [Carrier] In the present invention, the term "carrier" is not particularly limited as long as it is capable of immobilizing and supporting the genetically modified T cell recognition and activation substance and is insoluble in aqueous solutions such as the liquid sample, culture medium, and various buffers added as needed. The material of such a carrier is also not particularly limited, and examples include inorganic carriers such as zirconia, zeolite, silica, coated silica, silica gel, and glass vapor-deposited with a gold thin film; polysaccharide carriers insoluble in the aqueous solutions, such as agarose, cellulose, chitin, and chitosan; crosslinked polysaccharide carriers obtained by crosslinking the insoluble polysaccharide carriers with a crosslinking agent; crosslinked polysaccharide carriers obtained by crosslinking and insolubilizing water-soluble polysaccharides, such as dextran, pullulan, starch, alginate, and carrageenan; synthetic polymer carriers such as poly(meth)acrylate, polyvinyl alcohol, polyurethane, polystyrene, polyacrylamide, and polyglycidyl methacrylate; and crosslinked synthetic polymer carriers obtained by crosslinking the synthetic polymer carriers with a crosslinking agent. These may be used alone or in combination with one or more of these carriers.

[0034] The shape of the carrier is not particularly limited, but is preferably a shape that can be packed into the column described below, and examples thereof include particles, mesh, flat membrane, plate, and fiber, which may be porous or non-porous. Furthermore, the carrier according to the present invention may be in a shape integrated with the column container described below (monolith column), or may be in a shape fixed to the inner wall of the column container described below, with the genetically modified T cell recognizing and activating substance fixed to the innermost wall surface (i.e., the shape of the carrier is the same as the shape of the column container).

[0035] The size of the carrier is not particularly limited as long as it does not become a contaminant in the recovered genetically modified T cells. For example, it is preferable that the size of the carrier does not cause the genetically modified T cells to fall out of the column container described below during recovery. This can be adjusted appropriately depending on the size of the outlet of the column container. For example, if the carrier is particulate, the particle diameter is preferably 30 μm or more, and more preferably 100 to 250 μm.

[0036] The method for immobilizing the genetically modified T cell recognition and activation substance on the carrier can be any conventionally known method or a method similar thereto, and the genetically modified T cell recognition and activation substance can be immobilized directly or indirectly on the carrier.

[0037] Examples of methods for directly immobilizing the genetically modified T cell recognition and activation substance on the carrier include methods in which an active group such as an N-hydroxysuccinimide (NHS) activated ester group, an epoxy group, a vinyl group, a carboxy group, a maleimide group, a carbonylimidazole group, a haloacetyl group (a halogenated acetyl group), a tresyl group, a formyl group, or a haloacetamide is imparted to the surface of the carrier, or a carrier having such an active group on its surface is used, and the genetically modified T cell recognition and activation substance is immobilized on the carrier by covalent bonding between the active group and the genetically modified T cell recognition and activation substance.

[0038] The carrier to which the active group is attached may be a commercially available carrier as is, or may be prepared by introducing the active group onto the carrier surface under appropriate reaction conditions. For example, when the genetically modified T cell recognition / activation substance is covalently immobilized on the carrier, if a hydroxy group is present on the carrier surface, an active group-attaching agent can be used to form an active group capable of covalently binding to the genetically modified T cell recognition / activation substance from the hydroxy group. Examples of the active group-attaching agent include epichlorohydrin (forming an epoxy group as the active group), 1,4-butanediol diglycidyl ether (forming an epoxy group as the active group), tresyl chloride (forming a tresyl group as the active group), and vinyl bromide (forming a vinyl group as the active group). Alternatively, the hydroxy group can be converted to an amino group, a carboxy group, or the like, and then activated with an activator to form an active group capable of covalently binding to the genetically modified T cell recognition / activation substance. Examples of the activating agent include N-succinimidyl 3-maleimidopropionate (which forms a maleimide group as an active group), 1,1'-carbonyldiimidazole (which forms a carbonylimidazole group as an active group), and halogenated acetic acid (which forms a haloacetyl group as an active group).

[0039] Examples of methods for indirectly immobilizing the genetically modified T cell recognition / activation substance to the carrier include immobilization via a tag, such as polyhistidine, glutathione S-transferase, maltose-binding protein (MBP), cellulose-binding domain (CBD), myc tag, FLAG tag, cysteine-containing oligopeptide, lysine-containing oligopeptide, etc. The selection and length of the tag can be appropriately determined taking into consideration the binding strength of the genetically modified T cell recognition / activation substance to the carrier, steric hindrance caused by immobilizing the genetically modified T cell recognition / activation substance to the carrier, etc.

[0040] In the genetically modified T cell capturing agent of the present invention, the amount of the genetically modified T cell recognizing / activating substance immobilized on the carrier (the total amount if two or more types are used) can be adjusted appropriately depending on the binding affinity between the substance and the genetically modified T cells, and is not particularly limited, but may be, for example, in the range of 0.001 to 50 mg per mL of the genetically modified T cell capturing agent.

[0041] Furthermore, the genetically modified T cell capturing agent of the present invention may further contain other components in addition to the genetically modified T cell recognizing / activating substance and the carrier, as long as the effects of the present invention are not impaired.

[0042] 〔column〕 In the present invention, the genetically modified T cell capturing agent is preferably packed in a column. In the present invention, a "column" refers to a single, physically unseparated space (column container) packed with a carrier capable of separating liquids, and allows for the addition and removal of liquids from the space. In this case, the carrier packed in the column is the genetically modified T cell capturing agent.

[0043] The shape of the column container is not particularly limited as long as it has an inlet through which the genetically modified T cells, the liquid sample, the medium, and other necessary components can be added to the column, and an outlet through which these can be discharged from the column (here, the inlet and outlet may be the same or different), and may be any shape, such as cylindrical, polygonal tubular, tubular, etc. Furthermore, the genetically modified T cell capture agent and the column container may be integrated into one shape (a monolithic column).

[0044] Furthermore, to prevent the genetically modified T cell capturing agent from falling out, the column may be provided with a mesh filter at the inlet and / or outlet, or the column container may have a convex structure facing inward, etc. The mesh filter is not particularly limited as long as it is large enough to prevent the genetically modified T cell capturing agent from passing through but allows cells to pass through, and examples include those with mesh openings of 30 μm or less.

[0045] The size of the column can be adjusted appropriately depending on the purpose and is not particularly limited. For example, the total volume in the column container (volume including the volume of the genetically modified T cell capturing agent) can be 0.1 to 1000 mL, and preferably 1 to 100 mL.

[0046] Furthermore, the density of the genetically modified T cell capturing agent in the column is not particularly limited, but may be, for example, 0.1 to 80% in terms of the ratio of the volume of the genetically modified T cell capturing agent to the total volume in the column container (volume including the volume of the genetically modified T cell capturing agent), and is preferably 1 to 50%.

[0047] (Genetically modified T cell manufacturing method) The method for producing genetically modified T cells of the present invention includes at least: (1) a capture step of contacting a liquid sample containing genetically modified T cells with the genetically modified T cell capture agent and capturing the genetically modified T cells with the genetically modified T cell capture agent; and (2) an activation step in which the genetically modified T cells captured in the capture step are incubated and activated with the genetically modified T cell recognition / activation substance; This method makes it possible to selectively capture, with high purity and efficiency, the genetically modified T cells of interest from a liquid sample containing contaminants other than the genetically modified T cells (for example, the above-mentioned "other T cells," cells other than T cells), separate the genetically modified T cells from the contaminants, and activate the genetically modified T cells.

[0048] The method for producing genetically modified T cells of the present invention will be described in more detail below by taking preferred embodiments as examples, with reference to the drawings where appropriate, but the present invention is not limited thereto. In the following description and drawings, identical or corresponding elements are designated by the same reference numerals, and redundant explanations will be omitted.

[0049] [Genetic modification process] The method for producing genetically modified T cells of the present invention may further include a preparatory step, prior to the capture step described below, of preparing genetically modified T cells to be subjected to the capture step described below. The preparatory step is not particularly limited, and genetically modified T cells that have already undergone the desired artificial genetic modification may be obtained and subjected to the capture step described below as they are, or genetically modified T cells obtained by subjecting the "other T cells (unmodified T cells, T cells that have undergone artificial genetic modification other than the desired genetic modification)" to the desired artificial genetic modification may be subjected to the capture step described below.

[0050] The genetically modified T cells to be subjected to the capture step described below, the method for artificial gene modification, and the target gene to be modified by the artificial gene modification are as described above, including preferred embodiments thereof.

[0051] [Capturing process] In the capture step according to the present invention, (1) A liquid sample containing the genetically modified T cells is contacted with the genetically modified T cell capturing agent, and the genetically modified T cells are captured by the genetically modified T cell capturing agent. Figure 1 shows a schematic diagram illustrating one embodiment of the capturing step according to the present invention, and Figure 2 shows a schematic diagram illustrating one embodiment of the method for producing genetically modified T cells including the capturing step according to the present invention.

[0052] As shown in Figures 1 and 2, for example, in the capture step, a column 3 is first prepared, packed with a genetically modified T cell capture agent 2 comprising a genetically modified T cell recognition / activation substance 21 and a carrier 22, and a liquid sample containing genetically modified T cells 1 and contaminating cells 13 is added to the column 3 (Figures 1 and 2(a)). Here, the genetically modified T cell recognition / activation substance 21 is a substance capable of specifically binding to the modified T cell surface protein 11 of the genetically modified T cells 1. As a result, the genetically modified T cells 1 are captured in the column 3 by the genetically modified T cell capture agent 2 via the binding between the modified T cell surface protein 11 and the genetically modified T cell recognition / activation substance 21 (Figures 1 and 2(b)). On the other hand, the contaminating contaminating cells 13 do not bind to the genetically modified T cell capture agent 2 and are not captured, and therefore flow out of the column 3 as a flow-through (effluent fraction; the same applies below) (Figure 1(c)). Therefore, the capture step makes it possible to easily separate the genetically modified T cells 1 from the contaminating cells 13 with high purity and high efficiency.

[0053] In Figures 1 and 2, for the sake of illustration, the genetically modified T cell 1 is shown bound to one genetically modified T cell recognition and activation substance 21 via one modified T cell surface protein 11 (Figures 1 and 2 (b)), but in the capture step and the activation step described below, the genetically modified T cell 1 may be bound to multiple nearby genetically modified T cell recognition and activation substances 21.

[0054] [Cleaning process] The method for producing genetically modified T cells of the present invention may further include a washing step, if necessary, after the capture step to further thoroughly wash away and remove contaminants other than genetically modified T cells (e.g., contaminant cells 13). An example of the washing step is washing the genetically modified T cell capture agent that has captured genetically modified T cells with a washing solution. Examples of the washing solution include the medium and the buffer solution (e.g., neutral buffered saline, MACS buffer (manufactured by Miltenyi Biotec), etc.). More specifically, an example of the washing step is passing the washing solution through column 3.

[0055] [Activation process] In the activation step according to the present invention, (2) The genetically modified T cells captured in the capturing step are incubated while still captured with the genetically modified T cell recognition and activation substance, and then activated with the genetically modified T cell recognition and activation substance. Figure 2 shows a schematic diagram illustrating one embodiment of the method for producing genetically modified T cells according to the present invention, which includes an activation step.

[0056] As shown in Figure 2, in the activation step, after the capture step (Figure 2 (a) and (b)), genetically modified T cells 1 are incubated while bound to a genetically modified T cell capture agent 2, for example, in a column 3. In this case, the genetically modified T cells 1 can be incubated by closing the outlet of the column 3 or by adjusting the medium flow rate as needed. Furthermore, when a column is used for the incubation as shown in Figure 2, the column 3 may be left stationary or shaken or rotated. The medium may be passed through the column 3 at a constant flow rate. The medium may be caused to flow within the column using a shaker, a rotor, a propeller, or the like, or two or more of these may be combined.

[0057] In the present invention, "incubation" refers to cultivating cells at a constant temperature, but does not necessarily require cell proliferation. The incubation conditions are not particularly limited and can be set appropriately depending on the genetically modified T cells. 1 Examples of incubation conditions include incubation in the above-mentioned medium at 37°C in a 5% CO atmosphere for 1 hour to 2 days.

[0058] Here, the genetically modified T cell recognition / activation substance 21 contained in the genetically modified T cell capture agent 2 is a substance capable of activating the modified T cell surface protein 11. Therefore, only the genetically modified T cells 1 captured and separated in the capture step are specifically activated (i.e., by internalizing the modified T cell surface protein 11, inducing proliferation of the genetically modified T cells 1, and / or inducing an increase in the amount of cytokines expressed by the genetically modified T cells 1), and the activated genetically modified T cells of interest can be obtained with high purity and high efficiency.

[0059] [Recovery process] In the method for producing genetically modified T cells of the present invention, after the capturing step, the genetically modified T cells 1 may be detached from the genetically modified T cell capturing agent 2 and collected by an appropriate known method (physical method, chemical method, etc.). However, in the method for producing genetically modified T cells of the present invention, (3) a recovery step in which the genetically modified T cells that have been detached from the genetically modified T cell capturing agent due to internalization of the modified T cell surface protein by the activation step are recovered. 2 is a schematic diagram showing one embodiment of the method for producing genetically modified T cells according to the present invention, which includes a recovery step.

[0060] As shown in FIG. 2, in the method for producing genetically modified T cells of the present invention, the activation step of the present invention causes the modified T cell surface protein 11 of the genetically modified T cell 1 to be internalized within the genetically modified T cell 10 (FIG. 2(c)). The surface protein-internalized genetically modified T cells 12, which have internalized the modified T cell surface protein 11, can no longer bind to the genetically modified T cell recognition and activation substance 21 and are therefore detached from the genetically modified T cell capture agent 2 (FIG. 2(c)). The detached surface protein-internalized genetically modified T cells 12 flow through the column 3 as flow-through (FIG. 2(d)). Therefore, the recovery step makes it possible to easily recover the genetically modified T cells of interest (in this case, the surface protein-internalized genetically modified T cells 12) with high purity.

[0061] Furthermore, in the activation step, some of the cells proliferated from the genetically modified T cells 1 are unable to bind to the genetically modified T cell recognition / activation substance 21 in the column and are released into the culture medium, and therefore the cells recovered in the recovery step may include such genetically modified T cells 1 (genetically modified T cells expressing the modified T cell surface protein 11).

[0062] The outflow method may be a method in which the column 3 is closed in the activation step, and the medium is then discharged all at once after incubation for a certain period of time, or a method in which the medium is passed through the column 3 while incubation is performed, and the flow-through fraction is collected.

[0063] Furthermore, the recovery step may further include, if necessary, a step of passing a solution such as the washing solution through column 3 and recovering the flow-through fraction in order to recover surface protein-internalized gene-modified T cells 12 with even higher efficiency.

[0064] In the method for producing genetically modified T cells of the present invention, as in the example above, by using a column, genetically modified T cells 1 can be selectively separated (captured), activated (incubated), and recovered easily and with high purity and efficiency from a liquid sample containing contaminant cells 13 other than the desired genetically modified T cells 1 and other contaminants using a single column 3.

[0065] [Re-expression step] The genetically modified T cells obtained by the method for producing genetically modified T cells of the present invention may be used as a composition containing the genetically modified T cells of interest. However, when the method for producing genetically modified T cells of the present invention includes the recovery step according to the present invention, the genetically modified T cells obtained are genetically modified T cells that have internalized a surface protein, as described above. Therefore, the method for producing genetically modified T cells of the present invention can be carried out by: (4) a re-expression step in which the genetically modified T cells recovered in the recovery step are re-incubated to re-express the modified T cell surface protein on the cell surface. 2 is a schematic diagram showing one embodiment of the method for producing gene-modified T cells according to the present invention, which includes a re-expression step.

[0066] As shown in Figure 2, according to the method for producing genetically modified T cells of the present invention, the genetically modified T cells 12 with internalized surface proteins recovered in the recovery step (Figure 2(d)) can be re-incubated to re-express the modified T cell surface protein 11 at a high expression rate (Figure 2(e)). Therefore, the re-expression step allows the genetically modified T cells 1 of interest that express the modified T cell surface protein 11 on their cell surface to be easily obtained with high purity and high efficiency.

[0067] The incubation conditions in the re-expression step are not particularly limited and can be set appropriately depending on the genetically modified T cells 1. Examples of the incubation conditions include incubation in a culture vessel such as a plate, dish, flask, bioreactor, culture bag, or culture tank in the above-mentioned medium at 37°C in a 5% CO atmosphere for 2 to 14 days.

[0068] (composition) The genetically modified T cells obtained by the method for producing genetically modified T cells of the present invention (i.e., the genetically modified T cells obtained in the activation step, the recovery step, or the re-expression step; more preferably, the genetically modified T cells obtained in the recovery step or the re-expression step; even more preferably, the genetically modified T cells obtained in the re-expression step) may be used as a composition containing the genetically modified T cells of interest, or a medium containing the genetically modified T cells may be used as a composition containing the genetically modified T cells of interest after further culturing and expanding the cells, or concentrating the cells by centrifugation or the like, or by an appropriate combination of these methods. This allows for the production of a composition containing highly pure genetically modified T cells that are free from the risk of contamination by impurities such as magnetic beads.

[0069] The composition may contain components derived from the culture medium, the washing solution, etc., in addition to the genetically modified T cells of interest. The resulting composition can be used as a pharmaceutical composition for cell therapy, disease prevention, or disease treatment. The pharmaceutical composition can be transplanted into humans or non-human animals, and may further contain conventionally known excipients or additives in addition to the composition, depending on the purpose.

[0070] (Genetically modified T cell manufacturing equipment) In the method of producing genetically modified T cells of the present invention, an apparatus equipped with the column packed with the genetically modified T cell capturing agent can be suitably used as an apparatus for producing genetically modified T cells.

[0071] In addition to the column, the apparatus for producing genetically modified T cells may further include a supply means (sensor, valve, pump, tank, etc.) for supplying the culture medium and the washing solution to the column; a discharge means (sensor, valve, pump, tank, etc.) for discharging the culture medium and the washing solution from the column; and a detection means (sensor, etc.) for detecting contaminating cells and genetically modified T cells that have flowed out after the capture step or activation step.

[0072] Furthermore, when the method for producing genetically modified T cells of the present invention further comprises the recovery step or the re-expression step, the apparatus for producing genetically modified T cells may further include a recovery means (sensor, valve, tank, centrifuge, etc.) for recovering the genetically modified T cells that have flowed out in the recovery step; an incubation means (sensor, culture vessel, shaking device, rotating device, propulsion device, etc.) for further incubating the recovered genetically modified T cells in the re-expression step; etc.

[0073] The configuration of the apparatus for producing genetically modified T cells is not limited to the above, and may be a combination of the above means as appropriate, or may further include a control means for controlling each of the above means. [Example]

[0074] The present invention will be explained in more detail below based on test examples, but the present invention is not limited to the following embodiments.

[0075] <Genetically modified T cell capture agent> In each of the following test examples, Protein L-immobilized Toyopearl (TOYOPEARL AF-rProtein L-650F, manufactured by Tosoh Corporation) was used as a Protein L-immobilized carrier (small) (genetically modified T cell capture agent). As a control, Toyopearl (TOYOPEARL HW65, manufactured by Tosoh Corporation) without immobilized Protein L was used as a comparative carrier for the Protein L-immobilized carrier (small).

[0076] Furthermore, Protein L-immobilized Toyopearl (TOYOPEARL AF-rProtein L-650F, manufactured by Tosoh Corporation) was classified into particle sizes ranging from 100 to 250 μm and used as a Protein L-immobilized carrier (large) (genetically modified T cell capture agent). As a control, Protein L-unimmobilized Toyopearl (TOYOPEARL HW65, manufactured by Tosoh Corporation) was classified into particle sizes ranging from 100 to 250 μm and used as a comparative carrier for Protein L-immobilized carrier (large).

[0077] His-tagged BCMA (extracellular domain, amino acid sequence: SEQ ID NO: 4) was prepared in E. coli and immobilized onto TOYOPEARL HW40-EC (Tosoh Corporation) carriers classified to a particle size of 100-200 μm for use as BCMA-immobilized carriers (genetically modified T cell capture agents). As a control, TOYOPEARL HW40-EC (Tosoh Corporation, non-BMCA-loaded) was classified to a particle size of 100-200 μm for use as a comparative carrier for BCMA-immobilized carriers.

[0078] Furthermore, His-tagged BCMA (extracellular domain, amino acid sequence: SEQ ID NO: 4) was prepared using Escherichia coli and bound to Anti-His-tag mAb-Magnetic Beads (manufactured by MBL Life Science) according to its protocol, and used as BCMA-immobilized beads (gene-modified T cell stimulant). Also, as a control, Anti-His-tag mAb-Magnetic Beads (manufactured by MBL Life Science) were used as-is as comparative beads for the BCMA-immobilized beads.

[0079] <T cell> In each of the following test examples, the following T cells were used as each T cell.

[0080] (Naive T cells (n-T cells)) Naive T cells (n-T cells) were isolated from hPBMC (human peripheral blood mononuclear cells, manufactured by Lonza) by negative selection using a Naive Pan T Cell Isolation Kit (manufactured by Miltenyi). The ratio of n-T cells in the isolated cell fraction was confirmed by staining with anti-CCR7 antibody-Alexa488 (manufactured by BioLegend) and anti-CD45RA antibody-PE (manufactured by BioLegend) and analyzing by flow cytometry. As a result, the ratio of n-T cells, that is, CCR7 and CD45RA double-positive cells, was 78.52%.

[0081] (CAR-T cells) The nT cells or T cells (Promab) isolated from hPBMCs as described above were suspended in CAR-T cell medium (CAR-T Cell Medium (Fetal Bovine Serum (Promab)) supplemented with 10 ng / mL recombinant human IL7 (Peprotech), 10 ng / mL recombinant human IL15 (Peprotech), 20 ng / mL recombinant human IL21 (Peprotech), and 10 μM Pan Caspase Fmk inhibitor Z-VAD (z-VAD-fmk (Fujifilm)); the culture method using these supplements is described in WO 2018 / 135646). The suspension was placed in a U-bottom 96-well plate (Corning) at a density of 1 x 10 nT cells. 5 cells / well, T cells were 5 × 10 4 Next, Dynabeads Human T-Activator CD3 / CD28 (Gibco) were added so that the number of beads was three times the number of cells, and the cells were incubated for one day.

[0082] [n-CAR-T cells] After one day of incubation, nT cells were plated at 5.0 × 10 cells per well onto a 96-well plate coated with 30 μg / ml Retronectin (Takara Bio). 4Cells were seeded at 1.5 cells / well, and anti-CD19 CAR lentivirus (CAR structure: anti-CD19scFv-4-1BB-CD3ζ (Creative BioLabs)) was added to an MOI of 12.5 or 25, followed by centrifugation at 1500g, 32°C, for 90 minutes for lentiviral infection. After appropriate passage and medium changes, the cell population on day 7 postinfection was incubated with 2.0 μg of CD19-biotin (BPS Biosciences) and stained with 0.2 μg of streptavidin-PE (BioLegend). The fluorescent-positive population was isolated using a cell sorter (FACSaria, BD). The cell population separated above was stimulated again by adding Dynabeads Human T-Activator CD3 / CD28 (Gibco) in an amount of three times the number of beads relative to the number of cells, and the cells obtained by expansion and culture were designated n-CAR-T cells.

[0083] [T-CAR-T cells] After one day of incubation, T cells were cultured in 200 μL of T cell suspension (cell count: 5.0 × 10 4 cells) and Anti-CD19 CAR Lentivirus (Creative BioLabs) 2.5 × 10 6 TU and Polybrene (final concentration: 5 μg / ml (Sigma-Aldrich)) were mixed in a 1.5 mL tube and then centrifuged (800 g, 25°C, 90 minutes) for lentiviral infection. After appropriate passage and medium changes, the cell population on day 17 post-infection was reacted with 2.0 μg of CD19-biotin (BPS Bioscience), stained with 0.2 μg of streptavidin-PE (BioLegend), and the fluorescent-positive population was isolated using a cell sorter (FACSaria, BD). The isolated cell population was incubated in the CAR-T cell medium for 5 days, and the resulting cells were used as T-CAR-T cells.

[0084] (Test Example 1) Capture of CAR-presenting cells by Protein L immobilized carrier (1) First, 1 mL of the Protein L-immobilized carrier (large) or its comparative carrier was packed into a column (Muromac Mini Column M, Muromachi Chemical Co., Ltd.) equipped with a 90 μm polyester mesh filter (MobiTec Co., Ltd.) and washed with 5 mL of CAR-Jurkat medium (Growth Medium 2H, BPS Bioscience Co., Ltd.). Next, 1 × 10 Anti-BCMA CAR-Jurkat cells (CAR-Jurkat cells, CAR structure: anti-BCMAscFv-CD28-4-1BB-CD3ζ, CAR expression rate: 96.04%, BPS Bioscience Co., Ltd.) were added. 6 cells, or Jurkat cells (BPS Bioscience) at 1 × 10 6 The cells were suspended in 1 mL of the CAR-Jurkat medium and then added to the washed column. A portion of the sample before addition to the column was collected as an application.

[0085] Furthermore, each column was washed by passing 1 mL of the CAR-Jurkat medium five times, and the flow-through (Flow-through 1) was collected. The number of cells in the applied solution (number of added cells) and the number of cells collected in Flow-through 1 were counted using a cell counter (ViCELL, Beckman Coulter), and the number of cells adsorbed (captured) on each column (number of captured cells) was calculated from the difference between these numbers. The number of added cells and the number of captured cells are shown in Figure 3. The ratio of the number of captured cells to the number of cells in each applied solution (capture rate) was also calculated. The results are shown in Figure 4. As shown in Figures 3 and 4, it was confirmed that CAR-presenting cells (CAR-Jurkat cells) could be specifically captured (separated) using the Protein L-immobilized carrier (large).

[0086] (2) First, 1 mL of the Protein L-immobilized carrier (large) or its comparative carrier was packed into a column container (Muromac Mini Column M, Muromachi Chemical Co., Ltd.) equipped with a 90 μm polyester mesh filter (MobiTec), and washed with 5 mL of CAR-Jurkat medium (Growth Medium 2H, BPS Bioscience). Next, equal amounts of the CAR-Jurkat cells and Jurkat cells were mixed to obtain a total cell count of 1 × 10 6 The cells were suspended in 1 mL of the CAR-Jurkat medium and then added to the washed column. A portion of the sample before addition to the column was collected as an application.

[0087] Each column was washed with 1 mL of the CAR-Jurkat medium five times, and the flow-through (Flow-through 1) was collected. The cells in the application and those collected in Flow-through 1 were stained with 0.3 μg of BCMA-biotin (BPS Biosciences) and then with 0.2 μg of streptavidin-PE to label CAR on the cell surface. Analysis was performed using flow cytometry (Guava easyCyte, Luminex). The percentage of CAR-expressing cells, i.e., CAR-positive cells, was calculated from the ratio of CAR-labeled cells to the total cells collected at each time point (application or Flow-through 1) for each column. The results for the Protein L-immobilized carrier (large) column showed a CAR-positive cell percentage of 55.1% at the time of column application (application) and 2.2% at the time of washing (Flow-through 1). On the other hand, in the comparative carrier-packed column, the percentage of CAR-positive cells present at the time of column loading (apply) was 55.1%, and the percentage of CAR-positive cells present at the time of washing (flow-through 1) was 55.7%.

[0088] The flow cytometry results are shown in Figure 5. In Figure 5(a)-(b), the vertical axis represents cell counts, and the horizontal axis represents fluorescence intensity derived from labeled CAR, with the more labeled CAR (CAR-positive) the cells are, the further to the right they are. In Figure 5(a)-(b), the dotted lines represent the results at the time of column application (apply), the black areas represent the results at the time of washing (flow-through 1), and the area indicated by "R2" represents CAR-positive cells. As shown in Figure 5, when the comparative carrier was used (a), the proportion of CAR-positive cells did not change between apply and flow-through 1. However, when the Protein L-immobilized carrier (large) was used (b), almost no CAR-positive cells were found in flow-through 1, confirming that they were captured by the Protein L-immobilized carrier (large). This confirmed that the Protein L immobilized carrier (large) can specifically capture (separate) cells expressing CAR (CAR-positive cells: CAR-Jurkat cells) via CAR, and can more effectively remove contaminating cells (Jurkat cells) that do not express CAR by washing.

[0089] (Test Example 2) Capture of CAR-T cells by Protein L immobilized carrier (1) First, 1 mL of the Protein L-immobilized carrier (large) or its comparative carrier was loaded into a column container (Muromac Mini Column M, Muromachi Chemical Co., Ltd.) equipped with a 90 μm polyester mesh filter (MobiTec), and washed with 5 mL of the CAR-T cell medium. Next, 1 × 10 T-CAR-T cells (CAR positive rate at time of loading: 40%) were added. 6 The cells were suspended in 1 mL of the CAR-T cell medium and then added to the washed column. A portion of the sample before addition to the column was collected as an application.

[0090] Furthermore, each column was washed by passing 1 mL of the CAR-T cell medium five times, and the flow-through (Flow-through 1) was collected. The number of cells in the applied solution and the number of cells collected in Flow-through 1 were counted using a cell counter (ViCELL, Beckman Coulter), and the difference between these counts was used to calculate the number of cells adsorbed (captured) on each column (the number of captured cells) and the ratio of the number of captured cells to the number of cells in the applied solution (the capture rate). Figure 6 shows the T-CAR-T cell capture rate when using the Protein L-immobilized carrier (large) or a comparative carrier. As shown in Figure 6, it was confirmed that T-CAR-T cells could be captured using the Protein L-immobilized carrier (large).

[0091] (2) In addition, 0.1 ml of the Protein L-immobilized carrier (small) was filled into a column container (Mobicol F column, MobiTec) equipped with a 35 μm polyester mesh filter (MobiTec), washed three times with 200 μL of the CAR-T cell medium, and then filled with 200 μL of the CAR-T cell medium. Next, 5 × 10 n-CAR-T cells (CAR positive rate at time of addition: 58.91%) were added. 5 cells, or 5 × 10 nT cells 5 The cells were each suspended in 200 μL of the CAR-T cell medium and added to a column packed with the medium. Next, 500 μL of the CAR-T cell medium was added and aspirated from the bottom of the column, which was then washed five times, and the flow-through (Flow-through 1) was collected.

[0092] The number of cells added to the column and the number of cells recovered in Flow-Through 1 were each counted using a hemocytometer, and the difference between these counts was used to calculate the number of cells adsorbed (captured) on each column (number of captured cells) and the ratio of the number of captured cells to the number of cells added to the column (capture rate). Figure 7 shows the capture rate of n-CAR-T cells or nT cells when using the Protein L-immobilized carrier (small). As shown in Figure 7, it was confirmed that the Protein L-immobilized carrier (small) could also specifically capture (separate) CAR-T cells (n-CAR-T cells).

[0093] (Test Example 3) Detachment and recovery of CAR-T cells from Protein L-immobilized carrier (1) First, 1 mL of the Protein L-immobilized carrier (large) was packed into a column container (Muromac Mini Column M, Muromachi Chemical Co., Ltd.) equipped with a 90 μm polyester mesh filter (MobiTec), and washed with 5 mL of the CAR-T cell medium. Next, 5 × 10 n-CAR-T cells were added. 6 cells, or 5 × 10 nT cells 6 The cells were each suspended in 1 mL of the CAR-T cell medium and then added to the washed column. A portion of the sample before addition to the column was collected as an application. Each column was washed by passing 1 mL of the CAR-T cell medium through it five times, and the flow-through (flow-through 1) was collected.

[0094] Next, the top and bottom of the column were capped and incubated for 3 hours (37°C, 5% CO2), after which 5 mL of the CAR-T cell medium was passed through the column and the flow-through (flow-through 2) was collected. The collected flow-through 2 was transferred to a 24-well plate (Falcon) at 2 × 10 5Cells were seeded at 0.2 μg / ml and incubated for 4 days. Cells from the column application (apply), wash flow-through (flow-through 1), and recovery flow-through (flow-through 2), as well as cells after 4 days of incubation, were stained with 2.0 μg of CD19-biotin (BPS Bioscience) and then 0.2 μg of streptavidin-PE (BioLegend) to label CAR on the cell surface. Analysis was performed using a flow cytometer (Guava easyCyte, Luminex) to determine the percentage of CAR-expressing T cells, i.e., CAR-T cells, based on the ratio of CAR-labeled cells to the total cells recovered at each time point.

[0095] The flow cytometry results are shown in Figure 8. In Figure 8(a) to (d), the vertical axis represents the cell count, and the horizontal axis represents the fluorescence intensity derived from the labeled CAR, with the amount of labeled CAR increasing to the right. In Figure 8(a) to (d), the dotted lines represent the results for nT cells (negative control), and the black areas represent the results for the applied, flow-through 1, flow-through 2, and post-incubation samples. The area marked "CAR" represents CAR-positive cells, i.e., CAR-T cells. Figure 8(a) to (d) also show the percentage of CAR-T cells present at each time point when using n-CAR-T cells.

[0096] As shown in Figure 8, when 38% CAR-positive n-CAR-T cells were added, the CAR expression rate in the flow-through at the time of collection (Flow-through 2, (c)) was 7%, while the CAR expression rate in the cells after 4 days of incubation (d) was 63%. This confirmed that incubation on the Protein L-immobilized support (large) resulted in the internalization of CAR on the cell surface, and the captured cells were detached and collected. Furthermore, it was confirmed that the CAR gene expression rate of the collected cells increased when they were re-incubated, thereby enabling the high purity of CAR-T cells.

[0097] (2) The above n-CAR-T cells (CAR positive rate at time of addition: 58.91%) and the above nT cells were mixed so that the n-CAR-T cell number ratio (n-CAR-T cells / (n-CAR-T cells + nT cells)) was 1, 0.5, 0.25, 0.125, 0.0625, or 0.03125. Genomic DNA was extracted from each mixed cell population, and the CAR gene retention rate of n-CAR-T cells was determined. That is, first, the mixed cell population (total cell number 1 × 10 5 The cells were suspended in 100 μL of Reagent A from the Kaneka Simple DNA Extraction Kit Version 2 (Kaneka Corporation), heated at 98 °C for 8 minutes, and then 14 μL of Reagent B was added to extract genomic DNA. Next, 4 μL of the extracted genomic DNA solution was added with 10 μL of TagPath qPCR Master Mix, GC (Thermo Corporation), 0.18 μL of 100 μM forward primer (nucleotide sequence: SEQ ID NO: 1), 0.18 μL of 100 μM reverse primer (nucleotide sequence: SEQ ID NO: 2), 0.5 μL of 10.1 μM probe (nucleotide sequence: SEQ ID NO: 3), 1 μL of TaqMan Copy Number Reference Assay, human, RNase P (Thermo Corporation), and 4.14 μL of sterile water, and real-time PCR was performed using QuantStadio 3 (Thermo Corporation). The RNase P gene was used as the reference gene, and the retention rate of the CAR gene was calculated using the ΔΔCT method.

[0098] The relative value of the CAR gene retention rate in each mixed cell population was calculated, assuming that the CAR gene retention rate when the n-CAR-T cell number ratio was 1. The results are shown in Figure 9. As shown in Figure 9, there was a high correlation between the n-CAR-T cell number ratio and the CAR gene retention rate.

[0099] (3) A column container (Muromac Mini Column M, Muromachi Chemical Co., Ltd.) equipped with a 90 μm polyester mesh filter (MobiTec) was filled with 1 mL of the Protein L-immobilized carrier (large) or its comparative carrier, and washed with 5 mL of the CAR-T cell medium. Next, equal amounts of the n-CAR-T cells and the nT cells were mixed, resulting in a total cell count of 1 × 10 6 The cells were suspended in 1 mL of the CAR-T cell medium and then applied to the washed column. A portion of the sample before application to the column was collected as an application. Each column was washed by passing 1 mL of the CAR-T cell medium through it five times, and the flow-through (flow-through 1) was collected. The top and bottom of the column were then capped and incubated for 3 hours (37°C, 5% CO2), after which 5 mL of the CAR-T cell medium was passed through the column and the flow-through (flow-through 2) was collected.

[0100] The CAR gene retention rate in cells in the column application (apply), wash flow-through (flow-through 1), and recovery flow-through (flow-through 2) was calculated by real-time PCR using the RNase P gene as the reference gene and the ΔΔCT method, as described in (2) above. Note that when the comparative carrier was used, most of the cells were lost in the wash flow-through (flow-through 1) and no cells were recovered at recovery, so real-time PCR was not performed on the recovery flow-through (flow-through 2).

[0101] The CAR gene retention rate of cells at the time of column loading (apply) was set to 1, and the relative value of the CAR gene retention rate of cells in each flow-through was calculated. The results are shown in Figure 10. As shown in Figure 10, CAR-T cells carrying the CAR gene were specifically captured (separated) by the Protein L-immobilized carrier (large). Furthermore, it was confirmed that the cells recovered as flow-through after incubation on the Protein L-immobilized carrier (large) carried the CAR gene but did not express it on the cell surface, i.e., they were CAR-T cells with internalized CAR (surface protein-internalized gene-modified T cells).

[0102] (Test Example 4) Stimulation and capture of CAR-presenting cells by BCMA-immobilized carriers (1) BCMA-highly expressing CHO cells (B-CHO, manufactured by BPS Bioscience) were used at a concentration of 3 × 10 4 The cells were suspended in Thaw Medium 3 (BPS Bioscience) to give 3 × 10 cells, and seeded on a 96-well plate (Corning). CHO cell line K1 (CHO, negative control) was used. 4 The cells were suspended in Ham's F12 medium (Fujifilm) and seeded onto a 96-well plate (Corning). Both were incubated for 6 to 12 hours (5% CO2, 37°C) to allow the cells to adhere.

[0103] The BCMA-immobilized beads were added to wells of the 96-well plate other than those containing B-CHO or CHO at 12.5, 25, 50, 100, or 200 ng / well, or the control beads were added to wells at 200 ng / well. 1 x 10 anti-BCMA CAR-Jurkat cells (Anti-BCMA CAR / NFAT (Luciferase) Reporter Jurkat cells, CAR structure: anti-BCMAscFv-CD28-4-1BB-CD3ζ, manufactured by BPS Bioscience) were added to each well containing the cells or beads. 5 The cells were seeded and incubated at 37°C in 5% CO2 for 6 hours.

[0104] Then, luciferase assay kit (ONE-Step TM Luciferase Assay System (BPS Bioscience) was used to measure the luciferase activity of anti-BCMA CAR-Jurkat cells using a plate reader (Tecan) according to the protocol. The results are shown in Figure 11. As shown in Figure 11, BCMA-immobilized beads exhibited stimulatory activity against anti-BCMA CAR-Jurkat cells.

[0105] (2) First, 0.1 mL of the BCMA-immobilized carrier or its comparative carrier was loaded into a column container (Mobicol F, MoBiTec) equipped with a 90 μm polyester mesh filter and washed with 1 mL of PBS(-). Next, 1.3 × 10 Anti-BCMA CAR-Jurkat cells (CAR-Jurkat cells, CAR structure: anti-BCMAscFv-CD28-4-1BB-CD3ζ, CAR expression rate: 96.04%, BPS Bioscience) were added. 5 The cells were suspended in 100 μL of the CAR-Jurkat medium, and 100 μL of the suspension was added to the washed column. A portion of the sample before addition to the column was collected as an application.

[0106] The columns were then capped and sealed. After incubation for 35 minutes (37°C, 5% CO), 100 μL of PBS(-) was added to the top of the column. The column was centrifuged at 8,000 rpm for 1 minute, and the flow-through (Flow-through 1) was collected. The number of cells in the applied column and the number of cells collected in Flow-through 1 were counted using a hemocytometer. The difference between these counts was used to calculate the number of cells adsorbed (captured) by each column (the number of captured cells) and the ratio of the number of captured cells to the number of cells in the applied column (the capture rate). Figure 12 shows the capture rates of CAR-Jurkat cells using the BCMA-immobilized carrier and the comparative carrier. As shown in Figure 12, it was confirmed that CAR-presenting cells (CAR-Jurkat cells) could be specifically captured (separated) using the BCMA-immobilized carrier. [Industrial Applicability]

[0107] According to the present invention, it is possible to provide a method for producing genetically modified T cells, which enables highly efficient isolation and activation of genetically modified T cells and easy production of highly pure genetically modified T cells that are free from the risk of contamination by impurities. Furthermore, according to the present invention, it is also possible to easily recover genetically modified T cells with high purity and high efficiency. [Explanation of symbols]

[0108] 1, 10...genetically modified T cells, 11...modified T cell surface protein, 12...genetically modified T cells internalizing surface proteins, 13...contaminating cells, 2...genetically modified T cell capture agent, 21...genetically modified T cell recognition and activation substance, 22...carrier, 3...column. [Sequence List Free Text]

[0109] SEQ ID NO:1 <223> Forward primer SEQ ID NO:2 <223> Reverse primer SEQ ID NO:3 <223> probe

Claims

1. 1. A method for producing genetically modified T cells, comprising: (1) contacting a liquid sample containing genetically modified T cells with a genetically modified T cell capturing agent, and capturing the genetically modified T cells with the genetically modified T cell capturing agent; a capturing step, in which the genetically modified T cell capturing agent comprises a carrier and a genetically modified T cell recognition / activation substance immobilized on the carrier, the genetically modified T cell recognition / activation substance being a substance capable of specifically binding to the genetically modified T cells via a modified T cell surface protein and activating the genetically modified T cells; (2) an activation step in which the genetically modified T cells captured in the capture step are incubated and activated with the genetically modified T cell recognition / activation substance; and (3) a recovery step in which, after the activation step, the genetically modified T cells that have internalized the modified T cell surface protein and have detached from the genetically modified T cell capturing agent are recovered; Including, the genetically modified T cells are CAR-T cells; the substance for recognizing and activating genetically modified T cells is Protein L; the modified T cell surface protein is a CAR; A method for producing genetically modified T cells.

2. the capturing step is a step of adding the liquid sample to a column packed with the genetically modified T cell capturing agent to capture the genetically modified T cells; the activation step is a step of incubating and activating the genetically modified T cells in the column; and the recovery step is a step of recovering the genetically modified T cells that have flowed out of the column. The method for producing the genetically modified T cells according to claim 1.

3. After the recovery step, (4) a re-expression step in which the genetically modified T cells recovered in the recovery step are re-incubated to re-express the modified T cell surface protein on the cell surface. The method for producing genetically modified T cells according to claim 1 or 2, further comprising:

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