JP7738826B2 - Method for producing genetically modified T cells - Google Patents

Description

The present invention relates to a method for producing genetically modified T cells.

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. To produce the genetically modified T cells in this type of treatment, a technology is required to artificially genetically modify patient-derived T cells, and then activate and proliferate (cultivate) the resulting genetically modified T cells with high purity, and there is also a demand for this technology to be carried out efficiently.

As a technique for producing such genetically modified T cells, for example, JP 2017-513499 A (Patent Document 1) describes a method for 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, as the magnetic separation method (enrichment method), a method using magnetic beads to which antigen-binding molecules (antibodies, etc.) specific to cell surface markers on the surface of T cells are immobilized (positive selection method), and a method using magnetic beads to which antibodies against surface antigens present on the surface of cells other than the genetically modified T cells of interest (contaminating cells) are immobilized (negative selection method). The activation method describes a method in which an agonist antibody (anti-CD3 antibody, anti-CD28 antibody, etc.) or cytokine bound to beads or nanostructures acts as a modulator. Patent Document 1 also describes how, after the genetically modified T cells have been 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.

However, conventional methods such as those described in Patent Document 1 require cell separation and cell activation and proliferation to be carried out in physically separate devices, which makes the process complicated and leads to a decrease in cell recovery rate. Furthermore, with 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 mixed into the recovered genetically modified T cells as impurities. 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.

Special Publication No. 2017-513499

The present invention was made in consideration of the problems associated with the prior art described above, and aims to provide a method for producing genetically modified T cells that enables highly efficient isolation and activation of genetically modified T cells and facilitates the production of highly pure genetically modified T cells that are free from the risk of contamination by impurities.

As a result of extensive research aimed at solving the above-mentioned problems, the inventors have 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 as a capture body, which includes a genetically modified T cell recognition and activation substance that can specifically bind to and activate 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 recognition and activation substance is immobilized. In particular, the inventors have found that the genetically modified T cell capture agent can be packed into a column, which allows all of the above steps to be easily performed in a single physical space within the column. Furthermore, the inventors have found that by incubating the genetically modified T cells while they are captured by the genetically modified T cell capture agent, the modified T cell surface protein is internalized and the genetically modified T cells are detached from the capture agent, making it possible to easily recover genetically modified T cells with high purity, particularly when using a column.

As a result, the inventors discovered that it is possible to specifically isolate, 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 isolation, and thus completed the present invention.

That is, the aspects of the present invention are as follows.
[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; 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 gene-modified T cells according to [1], wherein the gene-modified T cells are CAR-T cells.
[3]
The method for producing genetically modified T cells according to [2], wherein the genetically modified T cell recognizing and activating 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 and activation substance is at least one selected from the group consisting of Protein L and BCMA.
[5]
After the activation step,
(3) The method for producing genetically modified T cells according to any one of [1] to [4], further comprising a recovery step of recovering 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.
[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 a genetically modified T cell according to [5].
[7]
After the recovery step,
(4) The method for producing genetically modified T cells according to [5] or [6], further comprising a re-expression step of re-incubating the genetically modified T cells recovered in the recovery step to re-express the modified T cell surface protein on the cell surface.
[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 according to [8], which is a pharmaceutical composition.

The present invention makes it possible to provide a method for producing genetically modified T cells that allows for highly efficient isolation and activation of genetically modified T cells and facilitates the production of highly pure genetically modified T cells that are free from the risk of contamination by impurities. Furthermore, the present invention also makes it possible to easily recover genetically modified T cells with high purity and high efficiency.

FIG. 1 is a schematic diagram showing one embodiment of the capture step of the method for producing genetically modified T cells. FIG. 1 is a schematic diagram showing one embodiment of a method for producing genetically modified T cells. FIG. 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). FIG. 1 is a graph showing the capture rates of CAR-Jurkat cells or Jurkat cells when a comparative carrier or a Protein L-immobilized carrier (large) was used, obtained in Test Example 1 (1). FIG. 1 is a graph showing the relationship between the fluorescence intensity derived from the 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). 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). 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). FIG. 10 is a graph showing the relationship between the fluorescence intensity derived from the labeled CAR and the number of cells (count) in each sample of application (a), flow-through 1 (b), flow-through 2 (c), or after 4 days of incubation (d) when a Protein L-immobilized carrier (large) was used, obtained in Test Example 3 (1) (dotted line: n-T cells; black filled areas: each sample of application, flow-through 1, flow-through 2, and after incubation). FIG. 1 is a graph showing the relationship between the ratio of n-CAR-T cells and the CAR gene retention rate (relative value), obtained in Test Example 3 (2). FIG. 1 is a graph obtained in Test Example 3(3) showing the CAR gene retention rates (relative values) of cells in the apply, flow-through 1, and flow-through 2 when a comparative carrier or a Protein L-immobilized carrier (large) was used. 1 is a graph obtained in Test Example 4(1) showing luciferase activity when anti-BCMA CAR-Jurkat cells were added to B-CHO, CHO, BCMA-immobilized beads, or comparative beads. 1 is a graph obtained in Test Example 4(2) showing the capture rates of CAR-Jurkat cells when a comparative carrier or a BCMA-immobilized carrier was used.

The present invention will now be described in detail with reference to preferred embodiments.

(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" include unmodified T cells, i.e., T cells that have not undergone any artificial genetic modification, and T cells that have undergone an artificial genetic modification other than the desired genetic modification.

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 system, or a person suffering from a malignant tumor, an infection, 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.

In the present invention, "artificial genetic modification" means modifying the function of T cells by introducing a gene into or editing the gene of T cells. "Artificial genetic modification" in the present invention may be modification of the gene itself possessed by T cells, deletion of the gene itself possessed by T cells, modification by introduction of an exogenous gene, or a combination of two or more of these.

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.), or a vector into which such a gene has been inserted (lentiviral vector, gamma- or alpha-retroviral vector, adenoviral vector, adeno-associated viral vector, herpesvirus vector, equine encephalopathy viral vector, etc.). Examples of methods for gene editing of 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.

The gene that alters the function of the T cell, i.e., the gene to be altered 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.

More specifically, the target genes to be modified by the artificial gene 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, B7H 3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-β , SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, prostase, PAP, E LF2M, ephrinB2, 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, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE- A1, legumain, HPV E6, 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, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TE S1, 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 as an antigen, and genes encoding proteins or peptides that have binding affinity to the above proteins.

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 an antigen receptor, a T cell receptor (TCR). More specifically, such proteins include, for example, the following proteins:
Examples of the specific antigen for the antigen receptor (preferably TCR) include at least one protein 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.

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

Among these, the 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).

In other words, the genetically modified T cells of the present invention preferably express on their surface a surface protein (sometimes referred to herein 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 expressing a T cell receptor (TCR) encoded by the gene modified by the artificial genetic modification, and CAR-T cells expressing a chimeric antigen receptor (CAR), and even more preferably CAR-T cells expressing a chimeric antigen receptor (CAR) as the modified T cell surface protein.

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 "genetically modified T cells with surface protein internalization").

(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 solutions and buffer solutions containing genetically modified T cells.

The medium for the culture solution is not particularly limited and may be any suitable conventionally known medium, 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, or TexMACS GMP medium (Miltenyi Biotec). The medium may also contain cytokines such as IL-2 or fetal bovine serum (FCS), as needed.

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

(Genetically modified T cell capture agent)
The genetically modified T cell capturing agent of the present invention comprises a carrier and a genetically modified T cell recognizing and activating substance immobilized on the carrier.

[Genetically modified T cell recognition/activation substance]
The "genetically modified T cell recognition and activation substance" according to 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 and 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 (e.g., CAR) newly expressed on the surface of the T cells), and capable of activating only the genetically modified T cells via such specific binding.

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

Examples of substances that recognize and activate genetically modified T cells according to the present invention include antibodies against the modified T cell surface protein, fusion proteins containing the antibodies, and conjugates of the antibodies with sugar chains; proteins, peptides, or aptamers that have binding affinity to the modified T cell surface protein; and, when the modified T cell surface protein is an antibody or an antigen receptor (e.g., TCR, CAR, etc.), proteins or peptides that serve as its antigens (e.g., the antigens exemplified above) that can activate the genetically modified T cells. One of these may be used alone, or two or more may be used in combination.

For example, when the genetically modified T cell of the present invention is a CAR-T cell, the genetically modified T cell recognizing and activating substance of the present invention is preferably a ligand for CAR (e.g., the specific antigen, an anti-idiotype antibody, or an antibody-binding substance (Protein L, Protein A, etc.)). In this specification, the term "antibody" is not limited to a full-length antibody (complete antibody), but also includes antibody fragments and minibodies formed by binding antibody variable regions, so long as they have the ability to bind to an antigen. Examples of the antibody fragment include Fab, F(ab') ₂ , Fab', diabody, and single-chain antibody (e.g., scFv, dsFv). Such antibodies can be produced by appropriately adopting and improving conventionally known methods, and commonly available antibodies can also be used as appropriate.

Among these, the genetically modified T cell recognizing and activating substance according to the present invention is preferably CD19, BCMA, CD20, HER2, protein L, protein A, anti-IgG antibody, anti-Fab antibody, anti-F(ab') ₂ antibody, anti-Fab' antibody, anti-diabody antibody, anti-scFv antibody, or anti-idiotype antibody, 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 immunoglobulin (particularly IgG).

[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/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 thin gold 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.

The shape of the carrier is not particularly limited, but is preferably one that can be packed into the column described below. Examples include particulate, mesh, flat membrane, plate, and fiber shapes, which may be porous or non-porous. Furthermore, the carrier of the present invention may be integrated with the column container described below (monolith column), or may be fixed to the inner wall of the column container described below, with the genetically modified T cell recognition/activation substance fixed to the innermost wall surface (i.e., the shape of the carrier is the same as the shape of the column container).

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 when being recovered. This can be adjusted appropriately depending on the size of the column container outlet. For example, if the carrier is particulate, the particle diameter is preferably 30 μm or more, and more preferably 100 to 250 μm.

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

Examples of methods for directly immobilizing the genetically modified T cell recognizing and activating substance on the carrier include providing the surface of the carrier with an active group such as an N-hydroxysuccinimide (NHS) activated ester group, epoxy group, vinyl group, carboxy group, maleimide group, carbonylimidazole group, haloacetyl group (halogenated acetyl group), tresyl group, formyl group, or haloacetamide, or using a carrier having such an active group on its surface and immobilizing the genetically modified T cell recognizing and activating substance on the carrier via a covalent bond between the active group and the genetically modified T cell recognizing and activating substance.

As the carrier to which the active group is attached, commercially available carriers may be used as is, or the carrier may be prepared by introducing active groups 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 hydroxy groups are present on the carrier surface, an active group-attaching agent can be used to form active groups from the hydroxy groups that can be covalently bonded to the genetically modified T cell recognition/activation substance. 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, active groups that can be covalently bonded to the genetically modified T cell recognition/activation substance can be formed by converting the hydroxy groups to amino groups, carboxy groups, or the like, and then activating them with an activator. Examples of the activating agent include N-succinimidyl 3-maleimidopropionate (which forms a maleimide group as the active group), 1,1'-carbonyldiimidazole (which forms a carbonylimidazole group as the active group), and halogenated acetic acid (which forms a haloacetyl group as the active group).

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, or lysine-containing oligopeptide. The selection and length of the tag can be determined appropriately, taking into consideration the binding strength of the genetically modified T cell recognition/activation substance to the carrier and steric hindrance caused by immobilizing the genetically modified T cell recognition/activation substance to the carrier.

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 examples include an amount in the range of 0.001 to 50 mg per mL of the genetically modified T cell capturing agent.

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 and activating substance and the carrier, as long as the effects of the present invention are not impaired.

〔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 to an extent that liquid can be further added to and discharged from the space. In this case, the carrier packed in the column is the genetically modified T cell capturing agent.

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 culture 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). It may be cylindrical, polygonal, tubular, or any other shape. The genetically modified T cell capture agent and column container may also be integrated into one shape (a monolithic column).

Furthermore, the column may be equipped with a mesh filter at the inlet and/or outlet, or the column container may have a convex structure on the inside, in order to prevent the genetically modified T cell capturing agent from falling out. The mesh filter may be of any size, as long as it is impermeable to the genetically modified T cell capturing agent but permeable to cells; for example, one with a mesh size of 30 μm or less may be used.

The size of the column can be adjusted appropriately depending on the purpose and is not particularly limited, but examples include a total volume in the column container (volume including the volume of the genetically modified T cell capture agent) of 0.1 to 1000 mL, preferably 1 to 100 mL.

The density of the genetically modified T cell capture agent within 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 capture agent to the total volume within the column container (volume including the volume of the genetically modified T cell capture agent), and preferably 1 to 50%.

(Method for producing genetically modified T cells)
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" or cells other than T cells), separate the genetically modified T cells from the contaminants, and activate the genetically modified T cells.

The following provides a more detailed explanation of preferred embodiments of the method for producing genetically modified T cells of the present invention, with reference to the drawings where appropriate, but the present invention is not limited to these. In the following explanation and drawings, identical or corresponding elements are designated by the same reference numerals, and redundant explanations will be omitted.

[Genetic modification process]
The method for producing genetically modified T cells of the present invention may further include a preparatory step of preparing genetically modified T cells to be subjected to the capture step described below, prior 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.

The genetically modified T cells to be subjected to the capture step described below, the method of artificial genetic modification, and the target gene to be modified by the artificial genetic modification are as described above, including their preferred embodiments.

[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.

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 ((a) of Figures 1 and 2). 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 ((b) of Figures 1 and 2). 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) ((c) of Figure 1). Therefore, the capture step makes it possible to easily separate genetically modified T cells 1 from contaminating cells 13 with high purity and efficiency.

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.

[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). Examples of the washing step include 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, examples of the washing step include passing the washing solution through column 3.

[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.

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 in a column 3 while still bound to a genetically modified T cell capture agent 2. In this case, the genetically modified T cells 1 can be incubated by closing the outlet of the column 3 or adjusting the flow rate of the medium as needed. Furthermore, when a column is used for the incubation as shown in Figure 2, the column 3 may be left stationary, 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, rotor, propeller, or the like, or two or more of these may be combined.

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% _CO2 atmosphere for 1 hour to 2 days.

Here, the genetically modified T cell recognition and 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), making it possible to obtain activated genetically modified T cells of interest with high purity and high efficiency.

[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) It is preferable that the method further comprises a recovery step of recovering 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. 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 a recovery step.

As shown in Figure 2, according to 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 (Figure 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/activation substance 21 and are therefore detached from the genetically modified T cell capture agent 2 (Figure 2(c)). The detached surface protein-internalized genetically modified T cells 12 flow through the column 3 as flow-through (Figure 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.

Furthermore, as a result of 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 and activation substance 21 in the column and are released into the 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).

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

If necessary, the recovery step may further include 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.

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, highly pure, and efficiently 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.

[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 with surface proteins internalized, as described above. Therefore, the method for producing genetically modified T cells of the present invention includes the following steps:
(4) It is preferable that the method further comprises a re-expression step of re-incubating the genetically modified T cells recovered in the recovery step to re-express the modified T cell surface protein on the cell surface. 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 a re-expression step.

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 makes it possible to easily obtain genetically modified T cells 1 of interest that express the modified T cell surface protein 11 on their cell surface with high purity and efficiency.

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% _CO2 atmosphere for 2 to 14 days.

(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 is free from the risk of contamination by contaminants such as magnetic beads.

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

(Genetically modified T cell manufacturing device)
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.

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.

Furthermore, when the method for producing genetically modified T cells of the present invention further includes the recovery step and/or re-expression step, the apparatus for producing genetically modified T cells may further include recovery means (sensor, valve, tank, centrifuge, etc.) for recovering the genetically modified T cells that have flowed out in the recovery step; and 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.

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 control means for controlling each of the above means.

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

<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).

Furthermore, Protein L-immobilized Toyopearl (TOYOPEARL AF-rProtein L-650F, manufactured by Tosoh Corporation) was classified so that the particle size was between 100 and 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 so that the particle size was between 100 and 250 μm, and used as a comparative carrier for the Protein L-immobilized carrier (large).

His-tagged BCMA (extracellular domain, amino acid sequence: SEQ ID NO: 4) was also prepared in E. coli and immobilized on TOYOPEARL HW40-EC (Tosoh Corporation) carriers classified to have a particle size of 100-200 μm, and used as a BCMA-immobilized carrier (genetically modified T cell capture agent). As a control, TOYOPEARL HW40-EC (Tosoh Corporation, non-BMCA-carrying) carriers classified to have a particle size of 100-200 μm were used as a comparative carrier for the BCMA-immobilized carrier.

Furthermore, His-tagged BCMA (extracellular domain, amino acid sequence: SEQ ID NO: 4) was prepared in E. coli and bound to Anti-His-tag mAb-Magnetic Beads (MBL Life Sciences) according to the manufacturer's protocol, and used as BCMA-immobilized beads (genetically modified T cell stimulator). As a control, Anti-His-tag mAb-Magnetic Beads (MBL Life Sciences) were used as they were to compare the BCMA-immobilized beads.

<T cells>
In each of the following test examples, the following T cells were used as the respective T cells.

(Naive T cells (n-T cells))
Naive T cells (n-T cells) were isolated from hPBMCs (human peripheral blood mononuclear cells, Lonza) by negative selection using a naive T isolation kit (Naive Pan T Cell Isolation Kit, Miltinyi). The proportion of n-T cells in the isolated cell fraction was confirmed by staining with anti-CCR7 antibody-Alexa488 (BioLegend) and anti-CD45RA antibody-PE (BioLegend) and analyzing by flow cytometry. As a result, the proportion of n-T cells, i.e., CCR7 and CD45RA co-positive cells, was 78.52%.

(CAR-T cells)
The n-T cells or T cells (Promab) isolated from hPBMCs as described above were cultured in CAR-T cell medium (CAR-T Cell Medium (Fetal Bovine Serum (Promab))) supplemented with 10 ng/mL recombinant human IL-7 (Peprotech), 10 ng/mL recombinant human IL-15 (Peprotech), 20 ng/mL recombinant human IL-21 (Peprotech), and 10 μM Pan Caspase FMK inhibitor. The cells were suspended in a medium supplemented with Z-VAD (z-VAD-fmk (Fujifilm)); the culture method using the supplemented factors is described in WO 2018/135646). This was seeded into a U-bottom 96-well plate (Corning) at 1 x 10 ⁵ cells/well for n-T cells and 5 x 10 ⁴ cells/well for T cells. 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 plate was incubated for one day.

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

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

(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 container (Muromac Mini Column M, Muromachi Chemical Co., Ltd.) equipped with a 90 μm polyester mesh filter (MobiTec), and the column was washed with 5 mL of CAR-Jurkat medium (Growth Medium 2H, BPS Bioscience). Next, 1 x 106 Anti-BCMA CAR-Jurkat cells (CAR-Jurkat cells, CAR structure: anti-BCMAscFv-CD28-4-1BB-CD3ζ, CAR expression rate: 96.04%, manufactured by BPS Bioscience) or 1 x ^{106 Jurkat} cells (manufactured by BPS Bioscience) were suspended in 1 mL of the CAR-Jurkat medium and then applied to the washed column. In addition, a portion of the sample before application to the column was collected as an application.

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 application (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 application (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) can be specifically captured (separated) using the Protein L-immobilized carrier (large).

(2)
First, 1 mL of the above 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 above CAR-Jurkat cells and Jurkat cells were mixed, and a total of 1 × ¹⁰ cells was suspended in 1 mL of the above CAR-Jurkat medium and then added to the washed column. In addition, a portion of the sample before addition to the column was collected as an application.

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 cells in the applied column and the cells collected in Flow-through 1 were each stained with 0.3 μg of BCMA-biotin (BPS Bioscience), followed by 0.2 μg of streptavidin-PE to label CAR on the cell surface. Analysis was performed by flow cytometry (Guava easyCyte, Luminex), and the proportion of CAR-expressing cells, i.e., CAR-positive cells, was determined from the ratio of CAR-labeled cells to the total cells collected in each column at each time point (applied or Flow-through 1). As a result, in the column packed with Protein L immobilized carrier (large), 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 2.2%. On the other hand, in the column packed with comparative carrier, 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%.

The flow cytometry results are shown in Figure 5. In Figure 5(a) and (b), the vertical axis represents the cell count, and the horizontal axis represents the fluorescence intensity derived from the labeled CAR, with the more labeled CAR (CAR-positive) the cells are, the further to the right they are. In Figure 5(a) and (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) allows for the specific capture (separation) of cells expressing CAR (CAR-positive cells: CAR-Jurkat cells) via CAR, and that contaminating cells not expressing CAR (Jurkat cells) can be more effectively removed by washing.

(Test Example 2) Capture of CAR-T cells by Protein L-immobilized carrier (1)
First, 1 mL of the above 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 the CAR-T cell medium. Next, 1 x 10 ⁶ cells of the above T-CAR-T cells (CAR-positive rate at time of loading: 40%) were suspended in 1 mL of the CAR-T cell medium, and then loaded onto the washed column. In addition, a portion of the sample before loading onto the column was recovered as an application.

Furthermore, 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 number of cells in the applied solution and the number of cells collected in flow-through 1 were each 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 (number of captured cells) and the ratio of the number of captured cells to the number of cells in the applied solution (capture rate). Figure 6 shows the capture rate of T-CAR-T cells 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).

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

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 n-T cells when using Protein L-immobilized carrier (small). As shown in Figure 7, it was confirmed that CAR-T cells (n-CAR-T cells) can also be specifically captured (separated) using Protein L-immobilized carrier (small).

(Test Example 3) Detachment and recovery of CAR-T cells from Protein L-immobilized carrier (1)
First, 1 mL of the above 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 the column was washed with 5 mL of the above CAR-T cell medium. Next, 5 × 10 ⁶ cells of the above n-CAR-T cells or 5 × 10 ⁶ cells of the above n-T cells were suspended in 1 mL of the above CAR-T cell medium, and then added to the washed column. In addition, a portion of the sample before addition to the column was collected as an application. Furthermore, each column was washed by passing 1 mL of the above CAR-T cell medium five times, and the flow-through (flow-through 1) was collected.

Next, the top and bottom of the column were capped and the column was incubated for 3 hours (37°C, 5% _CO ), 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 seeded into a 24-well plate (manufactured by FALCON) at 2 x 10 ⁵ cells/ml and incubated for 4 days. Each of the cells in the flow-through upon column loading (apply), the flow-through upon washing (flow-through 1), and the flow-through upon recovery (flow-through 2), as well as the cells after 4 days of incubation, were each stained with 2.0 μg of CD19-biotin (manufactured by BPS Bioscience) and then with 0.2 μg of streptavidin-PE (manufactured by BioLegend) to label CAR on the cell surface. Analysis was performed by flow cytometry (Guava easyCyte, manufactured by Luminex), and the proportion of CAR-expressing T cells, i.e., CAR-T cells, was calculated from the ratio of the number of CAR-labeled cells to the total cells collected at each time point.

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 more labeled CAR present further to the right. In Figure 8(a) to (d), the dotted lines represent the results for n-T cells (negative control), and the black filled areas represent the results for the applied, flow-through 1, flow-through 2, and post-incubation samples. The areas marked "CAR" represent 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 n-CAR-T cells were used.

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 recovery (flow-through 2, (c)) was 7%, whereas the CAR expression rate in the cells after 4 days of incubation (d) was 63%, confirming that incubation on the Protein L-immobilized carrier (large) resulted in the internalization of CAR on the cell surface, and the captured cells were detached and recovered. Furthermore, it was confirmed that the CAR gene expression rate of the recovered cells increased when they were re-incubated, thereby enabling the CAR-T cells to be highly purified.

(2)
The above n-CAR-T cells (CAR positive rate at time of addition: 58.91%) and the above n-T cells were mixed so that the n-CAR-T cell number ratio (n-CAR-T cells/(n-CAR-T cells + n-T 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 number of cells: 1 x 10 ⁵ cells) was suspended in 100 μL of Reagent A of Kaneka Easy DNA Extraction Kit Version 2 (manufactured by Kaneka Corporation) and heated at 98°C for 8 minutes. After that, 14 μL of Reagent B was added to extract genomic DNA. Next, 4 μL of the extracted genomic DNA solution was mixed with 10 μL of TagPath qPCR Master Mix, GC (manufactured by Thermo), 0.18 μL of 100 μM forward primer (base sequence: SEQ ID NO: 1), 0.18 μL of 100 μM reverse primer (base sequence: SEQ ID NO: 2), 0.5 μL of 10.1 μM probe (base sequence: SEQ ID NO: 3), 1 μL of TaqMan Copy Number Reference Assay, human, RNase P (manufactured by Thermo), and 4.14 μL of sterile water, and real-time PCR was performed using QuantStudio 3 (manufactured by Thermo). Using the RNase P gene as a reference gene, the retention rate of the CAR gene was calculated by the ΔΔCT method.

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.

(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 above 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 above n-CAR-T cells and the above n-T cells were mixed, and a total cell count of 1 × 10 ⁶ cells was suspended in 1 mL of the CAR-T cell medium and then added to the washed column. In addition, a portion of the sample before addition to the column was recovered as an application. Furthermore, 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 recovered. Next, the top and bottom of the column were capped and incubated for 3 hours (37°C, 5% CO ₂ ), 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 CAR gene retention rates in cells in the column loading (apply), wash flow-through (flow-through 1), and recovery flow-through (flow-through 2) were calculated using the ΔΔCT method with the same real-time PCR method as in (2) above, using the RNase P gene as the reference gene. 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 during recovery, so real-time PCR was not performed on the recovery flow-through (flow-through 2).

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).

(Test Example 4) Stimulation and capture of CAR-presenting cells by BCMA-immobilized carrier (1)
BCMA-highly expressing CHO cells (B-CHO, BPS Bioscience) were suspended in Thaw Medium 3 (BPS Bioscience) at 3 x 10 ⁴ cells and seeded onto a 96-well plate (Corning). CHO cell line K1 (CHO, negative control) was suspended in Ham's F12 medium (Fujifilm) at 3 x 10 ⁴ cells and seeded onto a 96-well plate (Corning). Both were incubated for 6 to 12 hours (5% CO ₂ , 37°C) to allow the cells to adhere.

The above-mentioned BCMA-immobilized beads were added to wells of the 96-well plate other than the B-CHO or CHO wells at 12.5, 25, 50, 100, or 200 ng/well, or the above-mentioned comparison beads were added to wells at 200 ng/well. 1 x 105 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 seeded into wells containing ^each cell or bead, and the wells were incubated at 37°C and 5% _CO2 for 6 hours.

Next, using a luciferase assay kit (ONE-Step ^™ Luciferase Assay System, manufactured by BPS Bioscience), the luciferase activity of the anti-BCMA CAR-Jurkat cells was measured with a plate reader (manufactured by Tecan) according to the protocol. The results are shown in Figure 11. As shown in Figure 11, the BCMA-immobilized beads exhibited stimulatory activity against the anti-BCMA CAR-Jurkat cells.

(2)
First, 0.1 mL of the above-mentioned BCMA-immobilized carrier or its comparative carrier was filled 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 ⁵ cells of anti-BCMA CAR-Jurkat cells (CAR-Jurkat cells, CAR structure: anti-BCMAscFv-CD28-4-1BB-CD3ζ, CAR expression rate: 96.04%, BPS Bioscience) were suspended in 100 μL of the above-mentioned CAR-Jurkat medium, and 100 μL of this suspension was added to the washed column. In addition, a portion of the sample before addition to the column was collected as an application sample.

Next, the top and bottom of the column were capped and sealed, and the column was incubated for 35 minutes (37°C, ₅ % CO ), after which 100 μL of PBS(-) was added to the top of the column and 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 each counted using a hemocytometer, and the difference between these numbers 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 in the applied column (capture rate). The capture rates of CAR-Jurkat cells when using the BCMA-immobilized carrier or a comparative carrier are shown in Figure 12. As shown in Figure 12, it was confirmed that CAR-presenting cells (CAR-Jurkat cells) could be specifically captured (separated) by the BCMA-immobilized carrier.

The present invention makes it possible to provide a method for producing genetically modified T cells that allows for highly efficient isolation and activation of genetically modified T cells and facilitates the production of highly pure genetically modified T cells that are free from the risk of contamination by impurities. Furthermore, the present invention also makes it possible to easily recover genetically modified T cells with high purity and high efficiency.

1, 10...genetically modified T cells, 11...modified T cell surface protein, 12...genetically modified T cells internalizing surface protein, 13...contaminating cells, 2...genetically modified T cell capture agent, 21...genetically modified T cell recognition/activation substance, 22...carrier, 3...column.

SEQ ID NO: 1
<223> Forward primer SEQ ID NO: 2
<223> Reverse primer SEQ ID NO: 3
<223> Probe

Claims (3)

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