Method for producing effector cell having desired specificity

Abstract

Provided is a method for producing an effector cell expressing a desired specificity site. In this method, material cells capable of differentiating into effector cells are proliferated, the material cells having a cassette deck structure that has cassette tape genes, which include a gene that encodes a marker protein in the genome of the material cells, so as to enable the expression of the marker protein in cells obtained by inducing differentiation of the material cells into effector cells or precursor cells of effector cells. The material cells are then induced to differentiate into effector cells or precursor cells thereof. The marker protein genes included in the resultant effector cells, etc., are replaced with genes encoding a protein contributing to a desired specificity, thereby making it possible to obtain effector cells expressing a desired specificity site.

Description

Method for producing effector cells with desired specificity

[0001] The present application relates to a method for producing effector cells expressing a desired specificity site. In particular, the present invention relates to a method for efficiently producing mature T cells or mature CAR-T cells expressing a T cell receptor (TCR) or chimeric antigen receptor (CAR) with desired specificity, or their precursor cells, by Recombinase-mediated Cassette Exchange (RMCE) or genome editing.

[0002] The presence of tumor-infiltrating T lymphocytes (TILs), highly specific T lymphocytes that recognize tumor tissue as foreign bodies and infiltrate tumors, has been reported in 50-80% of cases of many cancers and is considered evidence of an immune response against cancer. These TILs contain multiple types of T lymphocytes with different specificities, and if large amounts of TILs could be amplified and administered to patients, therapeutic effects could be expected. However, extracting and amplifying TILs themselves from each patient is difficult, and even if it were possible, it would be time-consuming and costly, and to date only limited effectiveness has been demonstrated.

[0003] Methods for introducing antigen receptor genes such as TCR and CAR into mature T cells are used clinically, and CAR in particular has already been approved. In these methods, antigen receptor genes are randomly introduced into mature T cells using retroviruses or lentiviruses. However, knocking TCR and CAR genes into the original TCR locus is expected to produce a more physiological expression pattern. Random gene introduction poses problems such as the inability to control the insertion site, risk of genome damage, and difficulty in controlling expression levels. Furthermore, application to therapies involving simultaneous administration of mature T cells expressing various TCRs, such as tumor-infiltrating T lymphocytes, requires introducing each type of TCR or CAR into the mature T cells one by one, which is not practical.

[0004] Some of the present inventors have proposed introducing a TCR at the level of pluripotent stem cells, inducing their differentiation into mature T cells, and using them in cellular immunotherapy (Patent Documents 1 to 4). Another group has also proposed introducing a CAR gene at the level of pluripotent stem cells (Patent Document 5). All of these methods are disclosed as feasible, in which a TCR or CAR gene is introduced at the iPS cell stage, followed by inducing differentiation into functional effector cells.

[0005] The present inventors have also proposed a method for introducing a desired TCR into the TCR locus of ES cells or iPS cells by inserting a cassette for introducing a TCR gene under the control of a promoter and endogenous enhancer (Patent Document 6). This method utilizes a method such as recombinase-mediated cassette exchange (RMCE), in which a structure capable of recombining specific portions of the cellular genome with an exogenous sequence (cassette tape) is first introduced into the target cells, similar to a cassette deck, and then the cassette is exchanged using recombinase enzymes such as Cre or Flippase (FLP). Specifically, pluripotent stem cells are constructed that include a cassette deck structure equipped with an empty cassette tape gene containing a drug resistance gene so that the gene is expressed under the control of the pluripotent stem cell's expression regulatory mechanism. By replacing the empty cassette tape gene with a cassette tape encoding the desired TCR gene, pluripotent stem cells expressing the exogenous TCR gene under the control of the endogenous TCR locus' expression regulatory mechanism can be produced. The resulting pluripotent stem cells can be proliferated as needed and induced to differentiate into mature T cells for use in cellular immunotherapy.

[0006] This method has made it possible to efficiently produce cells for cell therapy that stably express a desired TCR gene or CAR gene. However, while the technique of introducing a TCR into mature T cells or T cell precursors is widely used, inducing differentiation of pluripotent stem cells into mature T cells is not a common procedure and is not yet widely available in clinical settings. For antigens that are commonly expressed in many cancers, such as the WT1 antigen, mass production of mature T cells carrying a single TCR gene is useful for cellular immunotherapy. However, to regenerate TILs, it is necessary to isolate the TCR of TILs, then introduce it into iPS cells to generate multiple TCR-iPS cells, which then induce their differentiation into T cells, which is a time-consuming and labor-intensive process.

[0007] International Publication WO2016 / 010153 International Publication WO2016 / 010154 International Publication WO2016 / 010155 International Publication WO2017 / 179720 International Publication WO2014 / 165707 International Publication WO2020 / 022512

[0008] Themeli et al., Nat Biotechnol. (2013) 928-33Gong Ying et al., Journal of Cell Biology (2015) 1481-1489

[0009] An object of the present invention is to provide a method for easily producing effector cells having desired specificity-determining sites.A further object of the present invention is to provide effector cells having a so-called cassette deck structure that allows the specificity-determining sites to be easily exchanged.A further object of the present invention is to provide a method for simultaneously producing effector cells of the same species having different types of specificity-determining sites.

[0010] The present application provides a method for producing effector cells that express a desired protein, comprising the steps of: (1) providing a material cell that has the ability to differentiate into an effector cell, the material cell having a cassette deck structure in which the material cell has a cassette tape gene containing a gene encoding a marker protein in the genome of the material cell such that the marker protein can be expressed in cells obtained by inducing the material cell to differentiate into effector cells or precursor cells of effector cells; (2) growing the material cell; (3) inducing the material cell to differentiate into effector cells or precursor cells of effector cells; and (4) replacing the marker protein gene of the effector cells or precursor cells of effector cells with a gene encoding the desired protein.

[0011] Step (4) can be performed using RMCE or genome editing. When using RMCE as one embodiment of step (4), a pair of recombinase target sequences is provided upstream and downstream of the gene encoding the marker protein in step (1). The effector cells or their precursor cells obtained in (3) contain a gene encoding the marker protein sandwiched between a pair of recombinase target sequences. An example of this process is to culture the effector cells or their precursor cells in the presence of a recombinase together with a cassette tape exchange vector containing a pair of recombinase target sequences identical to the recombinase target sequences and a gene encoding a desired protein between the pair of recombinase target sequences, thereby exchanging the marker protein gene for a gene encoding the desired protein.

[0012] Another example of step (4) is a step of disrupting a marker protein gene contained in the effector cells or their precursor cells by genome editing, or replacing it with a gene encoding a desired protein, and introducing it into the source cells so that it is expressed therein.

[0013] In another aspect, the present application provides a method for producing effector cells that express a desired protein, the method comprising the steps of: (i) providing effector cells or their precursor cells that have a cassette deck structure in their genome that includes a cassette tape gene containing a gene encoding a marker protein so that the marker protein can be expressed, and that do not contain an exogenous drug resistance gene; and (ii) replacing the marker protein gene of the effector cells or their precursor cells with a gene encoding the desired protein.

[0014] In yet another aspect of the present application, there is provided a method for producing a material cell comprising a cassette deck structure containing a cassette tape gene, the method comprising the steps of: (a) preparing a cassette deck introduction vector comprising, from upstream to downstream, a first promoter sequence, a target sequence for a first recombinase, a gene encoding a marker protein linked so as to be expressed under the first promoter sequence, the target sequence for the first recombinase, the target sequence for a second recombinase, a second promoter sequence expressible in a material cell, a drug resistance gene linked so as to be expressed under the second promoter, and the target sequence for the second recombinase; (b) knocking in the cassette deck introduction vector prepared in (a) under the expression control mechanism of the material cell genome; (c) selecting cells into which the cassette deck introduction vector containing the gene encoding the marker protein has been successfully knocked in, the selection comprising the step of culturing the cells obtained in (b) in the presence of a drug to which the drug resistance gene is resistant; and (d) culturing the obtained cells in the presence of a second recombinase to remove the drug resistance gene. The material cell containing the cassette tape gene-containing cassette deck structure of this embodiment is preferably used for cassette tape exchange by the RMCE method.

[0015] In the method of the present application, the term "material cells" refers to cells used to introduce a gene encoding a marker protein, and includes cells having a cassette deck structure containing a cassette tape gene encoding the marker protein. The term "material cells" also refers to cells before they are induced to differentiate into effector cells. Pluripotent stem cells are preferably used as material cells, and ES cells and iPS cells are particularly preferably used.

[0016] Effector cells include, in particular, mature T cells or their precursor cells. In this case, examples of the marker protein and desired protein include a T cell receptor (TCR) or a chimeric antigen receptor (CAR). Alternatively, a fluorescent protein may be used as the marker protein.

[0017] In the method of the present application, a starting cell is preferably produced that has, in its genome, only one cassette deck structure that contains a cassette tape gene containing a gene encoding a marker protein under an expression control mechanism. By simultaneously exchanging the cassette tape gene contained in each cell of an effector cell or a clone of a precursor cell that contains one cassette deck structure per cell with multiple types of cassette tape genes, it is possible to simultaneously produce multiple types of effector cells that express different proteins.

[0018] The present application also provides a cell preparation for immune cell therapy, which contains multiple types of effector cells expressing distinct proteins, produced by the method of the present invention.

[0019] FIG. 1 shows an outline of the TIL cocktail method when using the RMCE method provided by the method of the present application. When using genome editing, the recombinase target sequences lox2272 and loxP adjacent to the known TCR in the figure are unnecessary, and a genome editing vector is used instead of the recombinase Cre. This figure shows an outline of the TIL cocktail method provided by the method of the present application. This figure shows an outline of the procedure of Example 1. This figure shows the results of knocking in a cassette deck introduction vector into Jurkat cells in Example 1, followed by the removal of drug resistance factors by treatment with FLP and ganciclovir. The cells in FIG. 3 were further subjected to FACS Aria to obtain TCR-expressing cells. This is a schematic diagram of the procedure for treating the TCR-expressing cells in FIG. 4, i.e., cassette deck KI-Jurkat cells containing a WT1-specific TCR cassette tape, with a plasmid vector for cassette tape exchange of the NY-ESO1-specific TCR gene and a Cre recombinase expression vector. 5A , but a schematic diagram of the procedure using a linear DNA for NY-ESO1-specific TCR expression cassette tape replacement. FACS analysis of cells obtained by treating the TCR-expressing cells of FIG. 4, i.e., the WT1-specific TCR cassette tape KI-Jurkat cells, with an NY-ESO1-specific TCR expression cassette tape replacement plasmid vector, or a linear DNA for NY-ESO1-specific TCR cassette tape replacement and a Cre recombinase expression vector. A diagram showing an outline of the procedure in Example 2. A diagram showing an outline of the procedure in Example 2. A diagram showing a step of removing a drug resistance factor contained in the cassette deck introduction vector. A diagram showing the results of Example 2. A diagram showing the results of Example 3. A diagram showing the design concept of the guide RNA for cleaving the WT1-specific TCR (WT1-TCR) gene, which is a marker protein gene, and the 5' and 3' arms for the knock-in vector that introduces a desired sequence at the cleavage site in the genome editing of Example 4. 1 is a schematic diagram of a NYESO1-specific TCR (NYESO1-TCR) gene knock-in vector used in Example 4. FIG. 2 is a schematic diagram of a CRISPR-Cas9 vector used in Example 4.These are plasmid vectors containing a TCR cleavage guide RNA (gRNA#1) and a KI vector cleavage guide RNA (gRNA#5), each operably linked to a U6 promoter, and a gene encoding Cas9 operably linked to a CBh promoter. A schematic diagram showing the knock-in process of Example 4. A diagram showing the results of Example 4 (using gRNA#1). Top: Analysis of the expression of TCRβ with mouse Cβ and the expression of NYESO1 (NYESO1 tetramer: NYESO1). The numbers above indicate the expression in the upper right compartment (mTCRβ). ＋ NYES01 ＋ ) shows the percentage of total cells. Middle: Analysis of expression of TCRβ with mouse Cβ and expression of human TCR. Bottom: Analysis of expression of mouse TCRβ and human TCRαβ in both mouse TCRβ and NYESO1 tetramer positive cells confirmed in the top row. In the upper left section, the numbers of mouse TCRβ-positive, NYESO1 tetramer-positive, and human TCRαβ-negative cells are listed in the bottom row. Figure showing the results of Example 4 (using gRNA #4). Top: Analysis of expression of TCRβ with mouse Cβ and expression of NYESO1 (NYESO1 tetramer: NYESO1). The numbers at the top indicate the percentage of total cells in the upper right section (mTCRβ + NYESO1 +). Middle: Analysis of expression of TCRβ with mouse Cβ and expression of human TCR. Lower panel: Analysis of mouse TCRβ expression and human TCRαβ expression in mouse TCRβ and NYESO1 tetramer-positive cells confirmed in the upper panel. The numbers of mouse TCRβ-positive, NYESO1 tetramer-positive, and human TCRαβ-negative cells in the upper left section are shown in the bottom row.

[0020] In the present specification and claims, the term "effector cells" refers to cells that perform a specific function in the body after administration. Examples of effector cells include various mature T cells such as helper T cells, regulatory T cells, and cytotoxic T cells, immune cells such as natural killer (NK) cells, NKT cells, macrophages, and dendritic cells, as well as cells administered in a suspended state such as platelets, hormone-producing cells, immunosuppressive cells represented by mesenchymal stem cells, and nerve cells, as well as various tissues with two-dimensional structures such as the retina, retinal pigment epithelium, skin, and intestinal epithelium, and various organs with three-dimensional structures such as the heart, liver, and kidney.

[0021] The method of the present application is particularly suitable for use with immune cells, such as mature T cells, natural killer (NK) cells, NKT cells, macrophages, and dendritic cells, as effector cells. In particular, the method is suitable for use in producing mature T cells having a specificity-determining site such as a desired T cell receptor (TCR) or chimeric antigen receptor (CAR), or their precursor cells, as well as CAR-T cells or their precursor cells.

[0022] In the present application, the TCR gene used as the gene encoding the desired protein to be introduced into effector cells is not particularly limited, and may be a known rearranged TCR gene, or a TCR gene obtained by amplifying a TCR gene from a T cell specific for an antigen targeted by cell therapy using a known method. For example, a TCR specific to a cancer antigen is exemplified. Furthermore, in TIL therapy, TILs may be collected from a patient's cancer tissue and subjected to single-cell analysis to obtain and use a TCR of a frequently occurring killer T cell clone.

[0023] The term "chimeric antigen receptor" or "CAR" refers to a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a cytoplasmic sequence of a CD3ζ sequence sufficient to stimulate a T cell upon binding to an antigen, and optionally, cytoplasmic sequences of one or more (e.g., 2, 3, or 4) costimulatory proteins (e.g., one or more cytoplasmic sequences of a ligand that specifically binds to CD27, CD28, 4-1BB, OX40, CD30, CD40L, CD40, PD-1, PD-L1, ICOS, LFA-1, CD2, CD7, CD160, LIGHT, BTLA, TIM3, CD244, CD80, LAG3, NKG2C, B7-H3, and CD83) that provide costimulation of the T cell upon binding of the antigen binding domain to the antigen.

[0024] "Precursor cells" refer to cells that are in the process of differentiation but have the ability to differentiate into the cells in question. "Material cells capable of differentiating into effector cells" refer to cells that can be differentiated in vitro into effector cells or their precursor cells. Material cells are preferably cells that have the ability to differentiate into effector cells and can proliferate in vitro.

[0025] Examples of material cells capable of differentiating into effector cells include pluripotent stem cells and tissue stem cells.

[0026] Examples of tissue stem cells or somatic stem cells include neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.

[0027] Pluripotent stem cells are stem cells that have the pluripotency to differentiate into many cells present in the body and also have the ability to self-proliferate. Examples of pluripotent stem cells include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells). Pluripotent stem cells are preferably mammalian pluripotent stem cells, and more preferably human pluripotent stem cells. ES cells or iPS cells are preferably used as the source cells.

[0028] ES cells and iPS cells may be produced by known methods, or commercially available ones may be used. When using iPS cells, they may be produced from somatic cells obtained from the patient to be treated. In addition, a method has been proposed in which the HLA of ES cells or iPS cells is manipulated using genome editing technology to make pluripotent stem cells versatile (reference), and such versatile pluripotent stem cells may be used as the material cells of the present application.

[0029] As described above, the method of the present application is particularly suitable for use in obtaining mature T cells having a desired TCR or CAR as effector cells. Hereinafter, a method for obtaining mature T cells expressing a desired TCR by the RMCE method as effector cells expressing a desired protein will be described as an example.

[0030] First, we provide material cells that contain a cassette deck structure in which the genome of the material cell, a pluripotent stem cell, contains a set of recombinase target sequences and a cassette tape gene containing a gene encoding a marker protein between the target sequences, so that the marker protein can be expressed when the material cell is induced to differentiate into a T cell or a T cell precursor.

[0031] Examples of marker proteins include known ligands or receptors expressed on the surface of effector cells or their precursor cells. To produce mature T cells expressing a desired TCR, known TCRs or CARs for which tetramers are available are preferably used as marker proteins. Alternatively, examples of marker proteins include known fluorescent proteins. Many fluorescent proteins are known for use in genetic engineering, and many are commercially available. An appropriate fluorescent protein may be selected from these known fluorescent proteins.

[0032] In this specification and claims, the term "recombinase" refers to an enzyme that induces site-specific recombination, and a recombinase target sequence refers to a sequence that is recognized by the recombinase and can induce deletion, integration, or inversion between two target sequences. Examples of combinations of a recombinase and its target sequence include Cre recombinase and loxP and its derivatives, Flipperse (FLP) and frt, and clonase and attB / attP / attL / attR. As a combination of a recombinase target sequence and a recombinase contained in the cassette tape gene of the present application, it is preferable to use a recombinase system that can replace a cassette tape gene containing a desired protein gene flanked by a set of recombinase target sequences with a cassette tape gene containing a marker protein flanked by the same target sequences in the same orientation.

[0033] For example, when Cre recombinase is used as the recombinase, examples of target sequences include loxP, lox2272, lox511, and loxFas. In the presence of Cre recombinase, recombination between identical target sequences is promoted. Such recombination can replace, for example, a sequence flanked by lox2272 and loxP in the genome of a source cell with a sequence flanked by lox2272 and loxP on a vector.

[0034] The source cells used in the method of the present application have a cassette tape gene containing a gene encoding a marker protein in the genome of the source cells, such that the marker protein can be expressed in cells obtained by inducing the differentiation of the source cells into effector cells or precursor cells of effector cells. When obtaining mature T cells having a desired TCR or CAR as effector cells, the cassette tape gene may be introduced into the source cells so that the gene encoding the TCR is expressed under the control of the TCR expression control mechanism of the source cells, or may be introduced into the source cells together with an expression control function including a promoter and / or enhancer involved in TCR expression control. Preferably, the cassette tape gene is introduced so that it is expressed under the control of the TCR expression control mechanism of the source cells.

[0035] Hereinafter, we will refer to a structure including, from upstream, a promoter, a gene encoding a marker protein, and an enhancer as a "cassette deck structure," and a structure including a gene encoding a marker protein as a "cassette tape gene." First, we will explain step (1) of providing a material cell having a cassette deck structure including a cassette tape gene.

[0036] When the source cells are cells after TCR gene rearrangement, for example, iPS cells induced from T cells, a cassette tape gene containing a marker protein can be inserted under the physiological expression control of the rearranged TCR locus, i.e., a cassette deck structure containing the cassette tape gene can be constructed between the promoter and enhancer of the rearranged TCR locus.

[0037] When the source cells do not have a rearranged TCR locus, as disclosed in Patent Document 6 (International Publication No. WO2020 / 022512), there are three methods for constructing a cassette deck structure: (A) A cassette tape gene is introduced between the enhancer of the C region and the promoter of the V region of the TCR gene of the source cells so as to shorten the distance between them.

[0038] (B) In the material cell, the promoter of the V region of the TCR locus and the cassette tape gene are introduced upstream of the enhancer of the C region of the TCR locus, in that order from upstream to downstream, so that the promoter of the V region and the enhancer of the C region are close enough to exert their expression control function on the gene sandwiched between them.

[0039] (C) A cassette tape gene and a gene containing an enhancer of the C region of the TCR locus are introduced downstream of the promoter of the V region of the TCR locus in the material cell, in that order from upstream to downstream, so that the promoter of the V region and the enhancer of the C region are sufficiently close to each other to exert their expression control function on the gene sandwiched between them.

[0040] In the present application, the phrase "the promoter of the V region and the enhancer of the C region are close enough to exert their function of regulating the expression of the gene sandwiched between them" means that the distance between them is not particularly limited, as long as the promoter of the V region is under the control of the enhancer of the C region. For example, the distance between the promoter of the V region and the enhancer of the C region after introduction of the cassette tape gene may be about 8 to 50 kbp, about 10 to 40 kbp, about 12 to 32 kbp, or about 14 to 22 kbp.

[0041] The "enhancer of the C region of the TCR locus" and the "promoter of the V region of the TCR locus" may be sequences derived from the source cells, or may be sequences obtained from cells derived from another individual of the same species of animal as the source cells, or may be sequences obtained from cells derived from an animal of another species.

[0042] The cassette deck structure can be introduced into the source cells in a single operation or in multiple steps, and can also be carried out by conventionally known recombination techniques, such as homologous recombination, genome editing, and techniques that use a combination of recombinases such as Cre and Flippase.

[0043] When a rearranged TCR is used as the marker protein contained in the cassette tape gene, it is preferably a heterodimer of TCRα and TCRβ. When a rearranged TCRα and TCRβ heterodimer is expressed, the gene encoding the marker protein preferably has a sequence in which the rearranged TCRα gene and TCRβ gene are connected by a self-cleaving 2A peptide. By placing the self-cleaving 2A peptide between the α chain and the β chain, both genes can be expressed under the expression control system of a single gene.

[0044] Examples of 2A peptides that can be used include p2A, T2A, E2A, and F2A, with the p2A peptide being preferred because of its high cleavage efficiency. While either the TCRα gene or the TCRβ gene may be introduced upstream, a poly(A) sequence is preferably linked to the TCR gene introduced downstream.

[0045] An intron is preferably contained upstream of the TCR gene. The intron sequence may be any sequence that contains a splice donor sequence and a splice acceptor sequence in addition to a sequence that is removed by splicing, and examples thereof include the intron of the human polypeptide chain elongation factor α (EF1α) gene or the intron portion of the chicken β-actin (CAG) gene promoter.

[0046] It is preferable to introduce both the reconstructed TCRα gene and the TCRβ gene into effector cells after differentiation induction from the source cells so that they are under the control of a single gene expression control mechanism. The TCR gene locus into which the cassette deck structure containing the cassette tape gene on the source cell genome is introduced may be TCRα or TCRβ. It is preferable to delete the TCRα and β gene loci not used for gene introduction. Deletion of a specific gene locus may be performed using a known method as appropriate, such as a known genome editing technique such as CRISPR / Cas9 or Talen.

[0047] The method for creating a vector for expressing the reconstituted TCR α and β heterodimer and the method for introducing it into the material cells can be the method described in Patent Document 6 (WO2020 / 022512).

[0048] The following describes the above embodiment (B), i.e., a method for obtaining material cells for use in RMCE by introducing, in order from upstream, a promoter of the V region of the TCR locus and a cassette tape gene upstream of the enhancer of the C region of the TCR locus of a material cell, such that the promoter of the V region and the enhancer of the C region are sufficiently close to each other so as to exert their expression control function for the gene sandwiched between them. Note that the material cells may be cells in which the TCR locus has been rearranged, or cells in which the TCR locus has not been rearranged.

[0049] When the RMCE method is used, recombinase target sequences are inserted adjacent to the upstream and downstream of a gene encoding a marker protein into the cassette tape gene. In this aspect, there is provided a method for producing material cells having a cassette deck structure, comprising the steps of: (a) preparing a cassette deck transfer vector comprising, in order from upstream, a first promoter sequence expressible in effector cells after the material cells have differentiated into effector cells, a target sequence for a first recombinase, a cassette tape gene comprising a gene encoding a marker protein linked so as to be expressed under the first promoter sequence and the target sequence for the first recombinase, and a target sequence for a second recombinase, a second promoter sequence expressible in the material cells, a drug resistance gene linked so as to be expressed under the second promoter expressible in the material cells, and the target sequence for the second recombinase; (b) knocking in the cassette deck transfer vector prepared in (a) into the material cells; (c) culturing the cells obtained in (b) in the presence of a drug to which the drug resistance gene is resistant, and selecting cells in which the knock-in of the cassette deck transfer vector comprising the gene encoding the marker protein has been successful; and (d) treating the obtained cells with a second recombinase to remove the drug resistance gene.

[0050] In this embodiment, the first recombinase is specific to the target sequence of the recombinase contained in the effector cell having the cassette deck structure described above. The combination of the second recombinase and its target sequence is not particularly limited, as long as it does not cross-react with the first recombinase. For example, when a Cre / loxp system is used as the first recombinase, an example of the second recombinase is a FLP / frt system. The combination of the second recombinase and the target sequence is such that the site flanked by the target sequences is cross-reacted by the action of the second recombinase.

[0051] The first promoter is a promoter capable of inducing expression of a marker protein in cells induced to differentiate into effector cells. When a known TCR is used as the marker protein, the first promoter is preferably a promoter of the TCR locus of the source cells, such as a promoter of the V region of the TCR locus of the source cells.

[0052] The V region of the TCR locus has one promoter for each V gene. The V region promoter of the TCR locus is not particularly limited and may be selected appropriately. For example, when the TCRβ locus is used, the Vβ20-1 promoter can be used. The promoter sequence can be obtained by designing primers and amplifying them by PCR so as to obtain a DNA fragment of the promoter sequence immediately upstream from the translation initiation point in the first exon of the V gene.

[0053] The promoter that can be expressed in the source cells is not particularly limited as long as it can induce the expression of a linked drug resistance gene in the cells. Examples include, but are not limited to, the cytomegalovirus (CMV) promoter, the simian virus 40 (SV40) promoter, and the phosphoglycerate kinase (PGK) promoter. An example is the mouse phosphoglycerate kinase (PGK) gene promoter (pPGK).

[0054] As the drug resistance gene, any known drug resistance gene that can function as a marker in the source cells may be used, and examples thereof include resistance genes to hygromycin, puromycin, neomycin, etc.

[0055] The drug resistance gene is preferably a fusion gene having a drug sensitivity gene downstream thereof. A drug sensitivity gene means a gene that, when expressed, can induce apoptosis of cells in response to an exogenously added substance. Such a drug sensitivity gene is not particularly limited and may be appropriately selected from known gene types. Examples include the thymidine kinase gene of herpes simplex virus and varicella-zoster virus. An example of a drug that induces apoptosis in cells into which such a gene has been incorporated is ganciclovir. A polyA sequence is preferably connected downstream of the drug resistance gene.

[0056] The vector used in the present application may be appropriately selected from vectors used in genetic recombination, and examples thereof include vectors such as viruses, plasmids, and artificial chromosomes. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and Sendai viral vectors. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC). Plasmids for mammalian cells may be used as the plasmid. Commercially available vectors may be appropriately selected and used depending on the purpose.

[0057] The site for introducing the cassette deck structure into the TCR locus of the source cell genome is determined, and a targeting vector for cassette deck introduction is constructed. The site for introduction may be any site where the C enhancer derived from the source cell can activate the V promoter when a sequence containing the V promoter and cassette tape gene is introduced in this order from upstream.

[0058] For example, when a cassette deck structure is introduced into the TCRβ locus of a source cell, it can be introduced when rearrangement of the TCRβ locus has not occurred, and a region close to the enhancer is preferred, for example, upstream of Dβ2 and downstream of Cβ1. When a cassette deck structure is introduced into the TCRα locus of a source cell, it can be introduced when rearrangement of the TCRα locus has not occurred, and a region close to the enhancer is preferred, for example, upstream of the most upstream Jα gene and downstream of the most downstream Vα gene.

[0059] Once the site of introduction in the TCR locus of the genome of the source cell has been determined, sequences homologous to the sequences upstream and downstream of the introduction site are introduced as the 5' arm and 3' arm, respectively, to enable homologous recombination. In this context, the term "homologous sequences" refers to the 5' arm and 3' arm sequences, each of which may have sufficient homology to allow homologous recombination to occur.

[0060] For example, when introducing a cassette deck structure into the unrearranged TCRβ locus of the genome of a source cell, a DNA fragment extending from about 110 bp upstream of the Dβ2 gene to about 1.6 kbp further upstream can be used as the 5'-arm sequence, and a DNA fragment extending from about 50 bp upstream of the Dβ2 gene to about 1.6 kbp downstream can be used as the 3'-arm sequence. The DNA fragments of the 5'-arm and 3'-arm can be obtained by PCR amplification using primers capable of specifically amplifying the respective sequences and the genomic DNA of the source cell as a template.

[0061] Specifically, examples of targeting vectors for cassette deck introduction include vectors containing, from upstream to downstream, a sequence (5' arm) homologous to the 5' side of the introduction site within the TCR locus of the source cell genome, a promoter sequence for the TCR locus V region, a target sequence for a first recombinase, a cassette tape gene containing a gene encoding a marker protein linked to be expressed under the first promoter sequence and the target sequence for the first recombinase, a target sequence for a second recombinase, a second promoter sequence expressible in the source cell, a drug resistance gene linked to be expressed under the second promoter expressible in the source cell, and the target sequence for the second recombinase, and a sequence (3' arm) homologous to the 3' side of the introduction site in the source cell. When amplifying each sequence by PCR, primers are designed to add a DNA sequence for introducing the resulting PCR product into the drug resistance vector. Using the resulting PCR product, a vector can be constructed using known methods, such as Gibson assembly, or commercially available kits.

[0062] The targeting vector for cassette deck introduction may further comprise a promoter and a marker gene that can be expressed in the source cells downstream of the 3' arm. An example of such a promoter-marker combination is the combination of the MC1 promoter and the diphtheria toxin gene (DTA).

[0063] Step (b): The targeting vector for introducing the cassette deck is knocked into the TCR locus of the source cell by homologous recombination. Knock-in can be performed by a known method, for example, electroporation.

[0064] To increase the efficiency of knock-in by homologous recombination, it is preferable to introduce two single-strand breaks (nicks) at the knock-in site, for example, near the initiation site (upstream) of the 3' arm, before knocking in the targeting vector for introducing the cassette deck. Nicks can be introduced by known methods, such as using the CRISPR / Cas9n system.

[0065] Step (c): In source cells where homologous recombination has been successful, the drug resistance gene is expressed due to the action of the introduced promoter. Therefore, source cells where homologous recombination has been successful can be selected in the presence of a drug to which the drug resistance gene is resistant. Furthermore, when a marker gene is incorporated downstream of the 3' arm of the targeting vector for cassette deck introduction, source cells into which the so-called outer portions of the 5' arm sequence and 3' arm sequence, excluding the cassette tape gene and drug resistance gene, are introduced express a marker, such as a cytotoxin, and the cells do not survive. Therefore, by selecting surviving cells, cells in which the cassette deck introduction vector has been successfully knocked in can be selected. The selected source cells may be further confirmed by PCR to select only cells in which the V region promoter and cassette tape gene have been knocked in.

[0066] In step (d), a second recombinase is applied to the cells into which the cassette deck introduction vector has been knocked in. To apply the second recombinase, for example, a second recombinase expression vector is introduced into the cells. Expression of the second recombinase expression vector removes the drug resistance gene, resulting in the production of material cells containing a cassette deck structure having a promoter, a set of recombinase target sequences, and a gene encoding a marker protein between the target sequences, and an enhancer in the genome of the material cells, such that the marker protein can be expressed in cells induced to differentiate from the material cells into effector cells or precursor cells of effector cells.

[0067] If the drug resistance gene is a fusion gene having a drug sensitivity gene downstream thereof, after step (d), cells in which removal of the drug resistance gene has failed may be removed by further culturing the cells in the presence of a substance that can induce apoptosis by the drug sensitivity gene.

[0068] The above is one embodiment of step (1) of the method of the present application when using the RMCE method: a step of providing a source cell that has the ability to differentiate into an effector cell, and that has, within the genome of the source cell, a cassette tape gene that includes a set of recombinase target sequences and a gene encoding a marker protein between the target sequences, such that the marker protein can be expressed in cells that have been induced to differentiate into effector cells or precursor cells of effector cells.

[0069] For example, in the above-mentioned case of "(A) introducing a cassette tape gene between the enhancer of the C region of the TCR gene of the material cell and the promoter of the V region so as to shorten the distance between them," a cassette deck transfer vector that does not have a first promoter sequence can be prepared in step (a). Also, in the case of "(C) introducing, downstream of the promoter of the V region of the TCR gene locus of the material cell, the cassette tape gene and a gene including an enhancer of the C region of the TCR gene locus, in that order from upstream to downstream, so that the promoter of the V region and the enhancer of the C region are sufficiently close to each other to exert their expression control function for the gene sandwiched between them," a cassette deck transfer vector that does not have a first promoter sequence and includes an enhancer of the C region of the TCR gene agent of the material cell downstream of the drug resistance gene and the target sequence of the second recombinase can be used.

[0070] Step (2): Proliferating the provided material cells. The method for proliferating the material cells may be appropriately selected depending on the type of material cells.

[0071] Step (3): The expanded source cells are differentiated into effector cells. When the source cells are pluripotent stem cells and the effector cells are T cells, known methods can be used to induce differentiation of pluripotent stem cells into T cells, such as those described in Non-Patent Documents 1 and 2 and Patent Documents 1 to 5. By using such methods, when the source cells are pluripotent stem cells, differentiation into mature T cells is possible without disrupting the cassette deck structure constructed at the TCR locus. TCR rearrangement may be suppressed by deleting the Rag1 gene or the Rag2 gene. It is sufficient to delete either the Rag1 gene or the Rag2 gene.

[0072] T cells are cells that express CD3 and at least one of CD4 and CD8. Depending on the therapeutic purpose, they may be differentiated into either killer T cells that express CD8 or helper T cells that express CD4. They may also be precursor cells thereof.

[0073] The resulting T cells have a cassette deck structure in their genome that includes, in order from upstream, a promoter, a set of recombinase target sequences, and a cassette tape gene containing a gene encoding a known TCR (marker protein) between the target sequences, and an enhancer, and express the known TCR (marker protein).

[0074] The presence of a known TCR marker protein in the obtained T cells can be confirmed using a specific antibody or tetramer, thereby selecting cells having a cassette deck structure.

[0075] The method of the present invention makes it possible to produce a large number of mature T cells that have a cassette deck structure containing a cassette tape gene containing the same marker protein and express the marker protein.

[0076] Step (4): The marker protein gene of the effector cells or their precursor cells is replaced with a gene encoding a desired protein. A specific example of this step when using the RMCE method is described below. The effector cells or their precursor cells are cultured in the presence of a recombinase together with a cassette tape exchange vector containing a pair of recombinase target sequences identical to the recombinase target sequence and a gene encoding a desired protein between the pair of recombinase target sequences, thereby exchanging the cassette tape gene containing the marker protein gene for a cassette tape gene containing a gene encoding the desired protein.

[0077] The cassette tape gene containing a gene encoding a known TCR between the target sequences of a pair of recombinases is replaced with a cassette tape gene containing a desired rearranged TCR gene between the target sequences of the same recombinases.

[0078] The rearranged TCR gene to be introduced into T cells can be prepared by amplifying and isolating the TCR gene from T cells specific to the antigen targeted by cell therapy using a known method. For example, a TCR specific to a cancer antigen can be used. Furthermore, in TIL therapy, TILs can be collected from a patient's cancer tissue and subjected to single-cell analysis to obtain and use TCRs from frequently occurring killer T cell clones.

[0079] A cassette tape exchange vector for introducing a TCR is created. The TCR is preferably a heterodimer of TCRα and TCRβ, similar to the TCR used as a marker protein. When expressing a heterodimer of rearranged TCRα and TCRβ, the gene encoding the marker protein preferably has a sequence in which the rearranged TCRα gene and TCRβ gene are connected by a self-cleaving 2A peptide. Furthermore, it is preferable for the structure to have an intron upstream of the TCRα and TCRβ, which are sandwiched between recombinase target sequences and contain the 2A peptide between them, and a polyA downstream.

[0080] The vector for cassette tape exchange may be appropriately selected from the vectors used for genetic recombination described above, such as viruses, plasmids, artificial chromosomes, etc. Alternatively, a linear DNA vector may be used.

[0081] By introducing a cassette tape exchange vector into a mature T cell having a cassette deck structure in the presence of a recombinase, the TCR gene contained in the cassette tape exchange vector can be exchanged with the TCR gene contained as a marker protein.

[0082] Specifically, the cassette tape exchange vector and the recombinant enzyme expression vector may be knocked into mature T cells by a known method, such as electroporation.

[0083] The cassette exchange can be confirmed by confirming the presence of the TCR used as a marker protein in the resulting cells. The identification of a known TCR can be carried out using a tetramer or a specific antibody.

[0084] The method of the present application makes it possible to produce a large number of effector cells of the same type, each having a cassette deck structure containing one identical cassette tape gene, and by exchanging the cassette tape gene, it is possible to express a desired protein.

[0085] The present method can also be performed using genome editing. When genome editing is used, the source cells having a cassette deck structure provided in step (1) are cells that have a cassette tape gene containing a gene encoding a marker protein in the genome of the source cells, such that the marker protein can be expressed in cells induced to differentiate from the source cells into effector cells or effector cell precursor cells. Here, the "cassette deck structure" refers to a structure that includes, from upstream, a promoter, a gene encoding the marker protein, and an enhancer, with the gene encoding the marker protein serving as the cassette tape. When genome editing is used, the marker protein does not need to be sandwiched between target genes of a recombinase. Steps (2) and (3) are the same as when using the RMCE method. In the effector cells containing the cassette tape gene containing the marker protein obtained in step (3), genome editing is used to disrupt or replace the marker protein contained in the effector cells or their precursor cells, thereby introducing a gene encoding the desired protein so that it is expressed in the source cells. Below, we will explain a method that uses the CRISPR / Cas9 system, a genome editing method, to destroy a marker protein in effector cells that contain a cassette tape gene containing a rearranged TCR as a marker protein and introduce another rearranged TCR.

[0086] i) Design of guide RNA for cleaving marker protein gene (see Figure 10) The guide RNA is designed to cleave the portion responsible for the specificity of the marker protein gene. For example, if the marker protein is a TCR gene in which TCRα and TCRβ are connected by p2A, as shown in Figure 5A, the guide RNA can be designed to cleave one of CDR1 to CDR3, which contributes to the specificity of either TCR chain. The following describes the cleavage of CDR3 within the TCRβ chain variable region. ii) Design of arms for rearranged TCR gene knock-in vector (see Figure 10) A guide RNA recognition site for cleaving the marker protein is determined within the CDR3 region, and approximately 20 bp upstream from the CRISPR / Cas9 cleavage site is designated as the homologous sequence for the 5' arm, and approximately 20 bp downstream is designated as the homologous sequence for the 3' arm. iii) Design of rearranged TCR gene knock-in vector (see Figure 11) A knock-in vector for introducing the TCR is created. When knocking in a heterodimer of rearranged TCRα and TCRβ, a gene encoding an amino acid sequence in which the rearranged TCRα gene and TCRβ gene are linked by a self-cleaving 2A peptide is preferred. The sequence to be knocked in is sandwiched between the homologous sequences of the 5' arm and 3' arm determined in step ii), and recognition sequences for a guide RNA for cleavage of the KI vector are installed at both ends. A gene encoding a self-cleaving 2A peptide is placed at the most upstream of the sequence to be knocked in sandwiched between the 5' arm and 3' arm, and a stop codon is placed at the most downstream of the gene encoding the desired protein. Specific examples of rearranged TCR gene KI vectors include those having, from upstream, a guide RNA recognition sequence for cleavage of the KI vector, a 5' arm sequence, a gene encoding the 2A peptide, a gene encoding TCRβ, a gene encoding the 2A peptide, a gene encoding TCRα, a stop codon, a 3' arm sequence, and a guide RNA recognition sequence for cleavage of the KI vector. iv) Preparation of a vector for CRISPR / Cas9 A vector is prepared in which the guide RNA sequence for cleaving the marker gene and the guide RNA sequence for cleaving the reconstructed TCR gene knock-in vector are expressed individually or simultaneously with the Cas9 gene.The vector can be created by inserting two guide RNA sequences into a commercially available CRISPR / Cas9 vector so that they are operable downstream of the promoter. A schematic diagram of a vector with two guide RNAs inserted is shown in Figure 12. Commercially available Cas9 gene expression vectors include pX330, pCAS-Guide, and pGuide-it. v) Knock-in of a reconstructed TCR gene knock-in vector and a CRISPR / Cas9 vector into source cells. Knock-in of the vector into source cells can be performed using known methods, such as electroporation. The reconstructed TCR gene is integrated into the specific region of the marker TCR gene by knocking it in (Figure 13). In Figure 13, the sequence from the start codon of the gene encoding the marker protein of the source cell to the stop codon of the knocked-in reconstructed TCR gene is translated. The 5' fragment of the marker TCR β chain is cleaved by self-cleavage of the p2A peptide, losing specificity. The remaining portion of the marker TCR is located 3' from the termination codon at the end of the knocked-in rearranged TCR gene and is not translated. The translated rearranged TCR α and β chains are generated as two proteins by self-cleavage of the p2A peptide between them. Thus, the TCR protein used as the marker is replaced by the desired rearranged TCR protein.

[0087] The present method allows for the mass production of mature T cells or their progenitors that express a desired TCR by producing a large number of mature T cells or their progenitors with a cassette deck structure containing a cassette deck gene containing a known rearranged TCR as a marker protein and then replacing the cassette deck gene with a cassette deck gene containing a desired TCR gene. By introducing only one cassette deck structure into a single mature T cell or its progenitor cell, it is possible to simultaneously exchange cassette deck genes for multiple TCRs in multiple mature T cells or their progenitor cells ( Figure 1A ). For example, disrupting the TCR expression mechanism without a marker protein can yield effector cells that have only one cassette deck structure introduced into each cell. This method of exchanging cassette tapes for multiple TCRs into a cell clone with only one cassette deck structure is suitable for use in TIL therapy.

[0088] An example of the present method for TIL therapy is the TIL cocktail method, in which multiple TCR cassette tapes are exchanged for a cell clone with only one cassette deck structure. First, TILs are collected from a patient's cancer tissue, and single-cell analysis of the TILs can provide information on the TCRs of multiple frequently occurring killer T cell clones. The resulting multiple TCRs are inserted into a cassette tape exchange vector (Figure 1B). Meanwhile, using pluripotent stem cells that have been generalized through HLA manipulation as source cells, regenerative T cell clones with a cassette deck structure are produced using the present method, and a TCR is introduced into these regenerative T cells. The present invention is used in the step of introducing a TCR into these regenerative T cells with a cassette deck structure. In principle, this strategy can be applied to all patients from whom TILs can be collected. Because T cells themselves are generalized, while TCR genes are personalized, this strategy can be considered a combination of personalized medicine and generalized medicine.

[0089] A method has been proposed in which the HLA of ES cells or iPS cells is manipulated using genome editing technology to make pluripotent stem cells versatile, and such versatile pluripotent stem cells may be used as the material cells of the present application.

[0090] When using the RMCE method, the TIL cocktail method involves: 1) inserting a rearranged TCR gene into the TCR locus of the base ES / iPS cells in a position where expression is controlled under the influence of an endogenous enhancer (Figure 1A). 2) By placing lox2272 and loxp upstream and downstream of the inserted TCR gene, respectively, it creates a cassette tape structure inside a cassette deck. 3) The TCR gene to be introduced is a known TCR for which a tetramer for staining is available. 4) To limit the expression of the TCR locus in the cells to a single locus, the TCR locus without the rearranged TCR gene is destroyed. 5) T cells are regenerated from the ES / iPS cells thus created. 6) Meanwhile, lox2272 and loxp are placed upstream and downstream of the TCR gene to be introduced (cassette tape exchange vector). 7) The mixture of multiple TCR genes is introduced into the cells together with Cre.

[0091] When genome editing is used, the TIL cocktail method involves: 1) inserting a rearranged TCR gene into the TCR locus of the base ES / iPS cell in a position where expression is controlled under the influence of an endogenous enhancer. 2) At this time, the TCR locus without the rearranged TCR gene is destroyed so that only one TCR locus is expressed in the cell. 3) The TCR gene to be introduced is a known TCR for which a tetramer for staining is available. 4) Regenerating T cells from the ES / iPS cells thus created. 5) Meanwhile, a genome editing vector is prepared to introduce a desired sequence into the sequence site specific to the introduced TCR gene, and a knock-in vector (a vector for cassette tape replacement) to introduce the rearranged TCR gene. 6) The genome editing vector is then applied to the cells along with the knock-in vectors for introducing multiple types of rearranged TCR genes.

[0092] This method has the following advantages: 1) it allows gene transfer without the risk of mispairing while mixing multiple types of TCR vectors, and 2) it allows the replaced cells to be sorted as tetramer-negative cells. As a result, it is possible to produce a large number of T cells that express the same TCR as TIL. Meanwhile, the present invention is applicable not only to TIL-derived TCR genes, but also to the introduction of one or more TCR genes or CAR genes against common cancer antigens into regenerated T cells.

[0093] The present application will be described in further detail below with reference to examples. Hereinafter, a cassette tape gene containing a specific gene will be referred to as "the cassette tape of the specific gene." In this example, cells containing a cassette deck containing a cassette tape of a WT1-specific TCR gene under the control of the TCR expression control mechanism of Jurkat cells, which are mature T cells, were prepared. By applying a cassette tape exchange vector for the NY-ESO1-specific TCR gene to the cassette tape of the obtained cassette deck, the cassette tape of the WT1-specific TCR gene was successfully exchanged for a cassette tape of the NY-ESO1-specific TCR gene. A series of procedures is shown in Figure 2. 1) Knock-in of a cassette deck containing a TCR gene cassette tape into the TCRβ locus Dβ2 region (hereinafter also referred to as "TCRDβ2 gene region") of Jurkat cells by homologous recombination:

[0094] Reagents and antibodies used: KOD-FX (Toyobo, KFX-101), Amaxa R Cell Line Nucleofector RKit V (Lonza, VACA-1003), puromycin dihydrochloride (Wako, 160-23151), ganciclovir (Wako, 078-04481), PE / Cy7 anti-human TCRα / β monoclonal antibody (BioLegend, 306719), APC anti-human CD3 monoclonal antibody (BioLegend, 300439), APC / Cyanine7 anti-human CD3 antibody (BioLegend, 300426), APC mouse IgG2bκ isotype control antibody (BioLegend, 400322), HLA-A*24:02 modified WT1 Tetramer-CYTWNQMNL-PE (MBL, TS-M014-1), HLA-A*02:01 NY-ESO-1 Tetramer-SLLMWITQC-PE (MBL, TB-M011-1)

[0095] In all experiments, the cells were cultured in a culture medium with the following composition. When selecting with a drug, the cells were cultured with the following composition and each drug added. *The composition of the penicillin / streptomycin / L-glutamine solution is 10,000 U / mL penicillin, 10,000 μg / mL streptomycin, and 29.2 mg / mL L-glutamine, resulting in final concentrations of 100 U / mL, 100 μg / mL, and 292 μg / mL, respectively.

[0096] In this example, the vector was introduced into the cells by electroporation. Ｒ Cell Line Nucleofector Ｒ We followed the instructions in the Kit V kit manual. After suspending the cells and vector in the reagent provided with the kit, we transferred them to the cuvette provided with the kit and placed them in an Amaxa Nucleofector II (Lonza). We then transfected the cells with the vector using the built-in program X001.

[0097] The cell source used was the J.RT3-T3.5 Jurkat cell line (hereinafter referred to as Jurkat β mutant), a subspecies of the Jurkat cell line in which the rearranged TCR β gene is no longer expressed due to a mutation (Ohashi et al., Science 316, 606-609, 1985).

[0098] A targeting vector for cassette deck introduction was prepared, which contained the cassette tape gene in which the reconstructed TCR (corresponding to the marker protein) shown in Figure 2A was flanked by lox2272 and loxP. Although not shown in Figure 2A, the vector also contained the DCT gene and a promoter for its expression downstream of the 3' arm homologous region.

[0099] A cassette tape containing the Vβ20-1 promoter and TCR gene was inserted into the TCRβ locus of Jurkat cells, a T cell line, approximately 50 bp upstream of the Dβ2 gene (Fig. 2B) using a targeting vector for cassette deck introduction (KI targeting vector) (Fig. 2A). A schematic diagram of the inserted cassette is shown in Fig. 2C.

[0100] To increase the efficiency of knock-in by homologous recombination, two single-strand breaks (nicks) were introduced approximately 50 bp upstream of the Dβ2 gene using the CRISPR / Cas9n system. The targeting vector for cassette deck introduction was introduced into Jurkat β mutant along with the two CRISPR / Cas9n vectors shown below.

[0101] In cells that have integrated the cassette deck introduction targeting vector into their genomic DNA by homologous recombination, the EF1α promoter acts to express a fusion gene (PurorΔTK) consisting of a puromycin resistance gene and the kinase domain of herpesvirus thymidine kinase (ΔTK) (Figure 2C). Therefore, clones that had integrated the drug resistance gene in the cassette deck introduction targeting vector into their genome were selected using 0.25 μg / mL puromycin in culture medium (positive selection). On the other hand, cells that had integrated the vector portion outside the 5' and 3' arm sequences of the cassette deck introduction targeting vector into their genomic DNA by random integration were eliminated due to the production of diphtheria toxin (DTA) within the cells (negative selection).

[0102] Next, from the cells (clones) selected in this manner, clones in which the TCR gene and the drug resistance gene portion from the Vβ20-1 promoter to frt were integrated into the TCRDβ2 gene region were identified by PCR. From these, cells in which the TCR gene cassette tape was inserted into only one allele of the TCRDβ2 gene region and the vector had not been integrated into other regions of the genomic DNA by random integration were selected by PCR.

[0103] The CRISPR / Cas9n vectors used were the following: A: 5'-CACCGAGGTTAGTCTGACTGTGTG-3' (SEQ ID NO: 1) B: 5'-AAACCACACAGTCAGACTAACCTC-3' (SEQ ID NO: 2) C: 5'-CACCCTGCCGCTGCCCAGTGGTTG-3' (SEQ ID NO: 3) D: 5'-AAACCAACCACTGGGCAGCGGCAG-3' (SEQ ID NO: 4) The oligonucleotides A and B (vector 1) and C and D (vector 2) were annealed and inserted into the plasmid pX460 cleaved with the restriction enzyme BbsI.

[0104] Integration of the TCR gene into the TCRDβ2 gene region was confirmed by amplifying the 5′ arm region and 3′ arm region using the following primers and PCR method.

[0105] 5' arm region: Primer 1, 5'-ACGGCTGAAATCTCCCTAACCC-3' (SEQ ID NO: 5) Primer 2, 5'-ATACGAAGTTATAGCTAGTCTTCCGTGATGGCCTCACACCA-3' (SEQ ID NO: 6) PCR was performed using a KOD-FX (Toyobo, KFX-101) PCR kit, with 35 cycles of 94°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 4 minutes.

[0106] 3' arm region: Primer 3, 5'-GTCCAGACCCACGTCACC-3' (SEQ ID NO: 7) Primer 4, 5'-GGGGACCGAGGGGCTGGAAG-3' (SEQ ID NO: 8) PCR was performed using KOD-FX (Toyobo, KFX-101) with 35 cycles of 94°C for 2 minutes, followed by 98°C for 10 seconds and 68°C for 2 minutes and 30 seconds.

[0107] Integration of the TCR gene into only one allele of the TCRDβ2 gene region was confirmed using the following primers and PCR reaction conditions: Primer 5, 5'-CCTCCTGTCATAAGGTGCCAT-3' (SEQ ID NO: 9) Primer 6, 5'-CCACTTTGCTGTCTTGGCCTT-3' (SEQ ID NO: 10) PCR was performed using a KOD-FX (Toyobo, KFX-101) with 35 cycles of 94°C for 2 minutes, followed by 98°C for 10 seconds, 60°C for 30 seconds, and 68°C for 8 minutes.

[0108] Whether the vector had been integrated into the genomic DNA by random integration was confirmed by amplifying the diphtheria toxin (DTA) gene using the following primers and PCR reaction conditions: Diphtheria toxin (DTA) gene Primer 7, 5'-AGCCAAAATCTGGTACACAAGG-3' (SEQ ID NO: 11) Primer 8, 5'-CTGAGCACTACACGCGAAGCA-3' (SEQ ID NO: 12) PCR was performed using a KOD-FX (Toyobo, KFX-101) PCR system, with 35 cycles of 94°C for 2 minutes, 98°C for 10 seconds, 58°C for 30 seconds, and 68°C for 25 seconds.

[0109] Diphtheria toxin-free cells were used below as "targeting vector KI-Jurkat cells for cassette deck introduction."

[0110] 2) Introduction of FLP vector into KI-Jurkat cells, a targeting vector for cassette deck introduction (preparation of Jurkat cells expressing exogenous TCR) Materials

[0111] The Flipperse expression vector (pCAGGS-FLPe) was introduced into KI-Jurkat cells by electroporation, using a targeting vector for cassette deck introduction. FLP recognizes frt sequences and deletes the region flanked by them. Figures 2C and 2D show the Dβ2 region of the TCRβ locus after knock-in (Figure 2C) and the TCRβ locus with deletion of the portion flanked by frt sequences (Figure 2D).

[0112] Transient FLP expression by electroporation results in a mixture of cells in which the region flanked by frt is deleted and cells in which the deletion did not occur. Therefore, ganciclovir was used to remove cells in which the deletion failed (cells in which FLP-mediated recombination failed). Ganciclovir does not normally work on human cells such as Jurkat cells. In contrast, in cells containing PurorΔTK, ganciclovir becomes a nucleic acid analog when phosphorylated by ΔTK, inhibiting DNA replication and resulting in the arrest of cell growth or cell death.

[0113] After culturing for 9 days with 12 μM ganciclovir in culture medium, the expression of TCR and CD3 on the cell membrane was analyzed by FACS. The results are shown in Figure 3. Ganciclovir selection increased the number of cells expressing TCR. However, many cells still did not express TCR, so TCR-expressing cells were isolated using FACA Aria (BD) and used below (Figure 4).

[0114] After culturing the isolated cells, deletion of the region flanked by the frt sequences was confirmed by PCR, and reanalysis was performed by FACS. It was confirmed that the isolated cells had deleted the region flanked by the frt sequences and maintained TCR expression (Figure 4). The obtained cells were used in the following experiment as "TCR expression cassette deck KI-Jurkat cells." 3) TCR cassette tape replacement in TCR expression cassette deck KI-Jurkat cells using plasmid DNA

[0115] This is the process shown in Figures 2D and 2E of Figure 2. An outline of this process is shown again in Figure 5A. From the top, the TCR expression cassette KI-Jurkat cell TCR β locus Dβ2 region, the TCR cassette tape exchange vector (plasmid vector), and the TCR β locus after cassette tape exchange are shown schematically. Downstream of the Vβ20-1 promoter in the TCR β locus Dβ2 region is an intron-TCR β-p2A-TCR α-polyA flanked by Lox2272 and Loxp.

[0116] The vector used for TCR cassette tape exchange was a plasmid vector having intron-TCRβ-p2A-TCRα-polyA flanked by Lox2272 and Lox. TCRs against the NY-ESO1 antigen were used as TCRα and β.

[0117] To perform cassette tape replacement, a TCR cassette tape replacement vector and a Cre expression vector (pCAG-nls-Cre) were introduced into TCR expression cassette deck KI-Jurkat cells by electroporation. After electroporation, the cells were cultured for 7 days. To confirm that the region flanked by lox2272 and loxP had been replaced by the cassette replacement method, the presence or absence of NY-ESO1 tetramer-positive cells was confirmed using FACS. The results are shown in Figure 6. NY-ESO1 tetramer-positive cells were not detected in cells that had not undergone cassette tape replacement, but NY-ESO1 tetramer-positive cells were detected in cells transfected with the TCR cassette tape replacement vector and Cre expression vector.

[0118] 4) TCR cassette replacement in KI-Jurkat cells using linear DNA. To improve the efficiency of the TCR cassette replacement method, we attempted to use linear DNA, which is smaller than plasmid DNA. A schematic diagram is shown in Figure 5B. The linear DNA was generated by PCR amplification of the region flanked by lox2272 and loxP in the TCR cassette replacement vector (plasmid vector) described above (see Materials below). The linear DNA for TCR cassette replacement and a Cre expression vector (pCAG-nls-Cre) were introduced into TCR expression cassette KI-Jurkat cells by electroporation. In this example, the TCR introduced was directed against the NY-ESO1 antigen. After 14 days of culture, the presence or absence of NY-ESO1 tetramer-positive cells was confirmed using FACS to confirm that the region flanked by lox2272 and loxP had been replaced by cassette tape exchange. The results are shown in Figure 6. NY-ESO1 tetramer-positive cells were detected, as in the case of TCR cassette tape exchange using plasmid DNA.

[0119] Materials: Linear DNA for TCR cassette tape replacement: Primer 9, 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO: 13) Primer 10, 5'-CAGGAAACAGCTATGACCATG-3' (SEQ ID NO: 14) PCR was performed using a KOD-FX (Toyobo, KFX-101) reactor, with 35 cycles of 94°C for 2 minutes, followed by 98°C for 10 seconds, 59.1°C for 30 seconds, and 68°C for 4 minutes.

[0120]

[0121] 1) Knock-in of a cassette deck containing a WT-1-specific TCR gene (cassette tape gene) flanked by lox sites into the Dβ2 region of the TCRβ locus of human iPS cells The following iPS cell lines were used: HKM1 line: derived from human monocytes (established by the Kawamoto Hiroshi Laboratory, Institute for Virus Research and Frontier Medical Sciences, Kyoto University) I14s04 line: derived from human monocytes (established by the Center for iPS Cell Research and Application, Kyoto University)

[0122] 2) TCR knock-in (gene editing using CRISPR / Cas9) A targeting vector for cassette deck introduction was prepared in the same manner as in Example 1. The structure of the vector is shown in Figure 7A. The TCR gene used was a TCR (KM#3-3) obtained from a WT1-specific TCR. The plasmids of CRISPR / Cas9 and guide RNA expression vector 1 and cassette deck introduction targeting vector 2, mixed in the ratio described above, were transfected into iPS cells using electroporation. Two days later, the cells were harvested and 5 x 10 4 / well (6-well plate), and the next day drug selection was performed using puromycin at the final concentrations shown below.

[0123] Electroporation was performed using 1 x 10 iPS cells per session. ６ A total of 10 μg of plasmid DNA was used, and NEPA21 was used under the following conditions: Day 0: Electroporation Day 2: Cell reseeding 5×10 ４cells / well Day 3: Puromycin 180 ng / ml Day 4: Puromycin 150 ng / ml Days 5-9: Normal culture Day 12: iPSC colony pick-up

[0124] PCR was performed on the genomic DNA of each of the established iPS cell clones to confirm and select strains in which the desired knock-in had been achieved.

[0125] 3) Removal of the drug selection mechanism (site-specific recombination using FLP) Thymidine kinase is expressed downstream of the targeting vector for cassette deck introduction shown in Figure 7A, which toxicifies ganciclovir (GCV) within the cells, resulting in cell death when co-cultured with GCV. Using this mechanism, FLP expression vector 3 was electroporated, FLP was transiently expressed, and then drug selection was performed using the GCV drug at the final concentration shown below. An outline of this process is shown in Figure 7B.

[0126] Day 0: Electroporation Day 2: Cell reseeding 2-4 x 10 ３ cells / well Day 3-6: GCV 5ug / ml Day 7-12: Regular culture Day 12: iPSC colony pick-up

[0127] PCR was performed on the genomic DNA of each of the established iPS cell clones to identify strains in which the target PurorΔTK site was deleted.

[0128] The obtained iPS cells having a cassette deck structure containing a cassette tape of a TCR gene having a lox2272-TCR-loxP structure containing WT1-specific TCR were induced to differentiate into CD8 single positive T cells (CTL).

[0129] CTL induction was performed using the method described in Patent Document 4 (WO 2017 / 179720). Specifically, iPS cells were induced to develop T cell precursors, which were CD4CD8 double-positive cells, and then the CD4CD8 double-positive cells were isolated. The isolated CD4CD8 double-positive cells were further induced to develop CD8 single-positive cells. The results are shown in Figure 8. WT1-specific CD8 single-positive T cells were obtained, in which the CD8 antigen was heterozygous for the CD8 α chain and CD8 β chain. In other words, the obtained cells were CD8 single-positive T cells that had a cassette deck containing a cassette tape of a WT1-specific TCR gene.

[0130] Cassette tape exchange of CD8 single-positive T cells was carried out using a cassette deck containing the cassette tape of the WT1-specific TCR gene obtained in Example 2. 1) TCR cassette tape exchange of CD8 SPT cells using plasmid DNA

[0131] Cassette tape exchange was performed according to the outline shown in Figure 5A. The cassette tape exchange vector was a circular plasmid vector containing intron-TCRβ-p2A-TCRα-polyA flanked by Lox2272 and Lox. TCRs against the NY-ESO1 antigen were used as TCRα and β. A plasmid vector encoding an expressible GFP gene flanked by Lox2272 and Lox was used as the mock vector.

[0132] To replace the cassette tape, a TCR cassette tape replacement vector or a mock vector and a Cre expression vector (pCAG-nls-Cre) were introduced by electroporation into CD8S P T cells containing a cassette deck containing a WT1-specific TCR gene cassette tape. After electroporation, the cells were cultured for 13 days. To confirm replacement by the cassette tape replacement method, the presence or absence of NY-ESO1 tetramer-positive cells was confirmed using FACS. Cells in which the TCR was replaced by cassette tape replacement were detected as NY-ESO1 tetramer-positive cells. The results are shown in Figure 9. As a negative control, 10 μg of a mock vector was used instead of the TCR cassette tape replacement vector (NY-ESO1).

[0133] When the mock vector was introduced, no NY-ESO1 tetramer-positive cells were generated. Cassette exchange using a cassette vector containing the NY-ESO1 TCR was performed twice, changing the electroporation conditions. Although the efficiency was low in both cases, NY-ESO1 tetramer-expressing cells were generated, confirming that cassette exchange was successful.

[0134] The marker protein, TCR, was replaced with another TCR using genome editing. Cells (TCR expression cassette deck KI-Jurkat cells) were used in which the reconstructed WT1-specific TCR prepared in Example 1 was knocked into the Dβ2 region of the TCRβ locus of Jurkat cells. The TCRβ locus of these cells has the structure shown in Figure 5A, WT1-TCR KI Human TCRβ locus. In Example 4, these cells are referred to as "Jurkat WT1-TCR cells." In this example, genome editing was used to express NYESO1-TCR in place of WT1-TCR in Jurkat WT1-TCR cells.

[0135] Two guide RNAs were created (gRNA#1 and gRNA#4) targeting the gene portion encoding the CDR3 region of the TCR β chain of WT1-TCR. The 20 bp 5'-arm and 20 bp 3'-arm regions from the cleavage site of each guide RNA were designated the 5'-arm and 3'-arm, respectively. A conceptual diagram of the homologous sequence and 5'-arm and 3'-arm design of the two guide RNAs is shown in Figure 10. The vertical lines separating the base sequences in the figure correspond to the reading frame of protein translation. Additionally, a different guide RNA (gRNA#5) was used for cleavage of the NYESO-1-TCR knock-in vector. The recognition sequence of gRNA#5 is underlined below: 5'-GCATCGTACGCGTACGTGTTTGG-3' (SEQ ID NO: 15). The DNA sequence of the gRNA portion of the gRNA#5 vector was a previously reported sequence (Nature Protocol 11, 118-133, 2016).

[0136] The NYESO1-TCR genes used for replacement with WT1-TCR were a chimeric TCR β chain in which the variable region (Vβ) of the TCR β chain of a known human TCR that recognizes the NYESO1 antigen was linked to a mouse TCR constant region (mCβ), and a chimeric gene in which a chimeric TCR α chain with a similar structure was linked via the p2A peptide gene (Figure 11).

[0137] A schematic diagram of the NYESO1-TCR gene knock-in vector is shown in Figure 11. The NYESO1-TCR knock-in vector is a plasmid DNA vector that contains, from upstream, the recognition sequence for gRNA#5, a 5' arm sequence, a p2A sequence, the NYESO1-TCR gene in which the TCR β chain and the TCR β chain are linked by the p2A sequence, a 3' arm sequence, and the recognition sequence for gRNA#5. It also contains a stop codon at the 3' end of the NYESO1-TCR gene.

[0138] Preparation of guide RNA expression vector for CRISPR / Cas9 gRNA # 1, gRNA # 4, gRNA # 5 were introduced into the plasmid vector pX330, which can express both guide RNA and Cas9, and vectors for CRISPR / Cas9 were created by introducing gRNA # 1 & gRNA # 5, gRNA # 4 & gRNA # 5. As an example, a schematic diagram of a vector expressing gRNA # 1, gRNA # 5 and Cas9 is shown in Figure 12.

[0139] Introduction of NYESO1-TCR KI vector and CRISPR / Cas9 vector into Jurkat WT1-TCR cells. Electroporation was used to introduce the vectors into the cells. Electroporation was performed according to the Amaxa® Cell Line Nucleofector® Kit V kit manual. 2 x 10 cells were added to 100 μL of the reagent provided with the kit. ６ After suspending the cells and the vector, the cells were transferred to a cuvette provided with the kit and set in an Amaxa Nucleofector II (Lonza), and the vector was introduced into the cells using the built-in program X001.

[0140] Conditions for gene transfer by electroporation Condition 1: WT1-TCR gene is cleaved by gRNA#1

[0141] Condition 2: WT1-TCR gene cleaved by gRNA#4

[0142] Six days after gene transfection, TCR expression was analyzed using a flow cytometer for the eight types of cells (conditions 1-1 to 2-4) and non-transfected Jurkat WT1-TCR cells. The following two antibodies and one tetramer were used for the analysis. Antibodies: Human TCRαβ antibody-PECy7 (BioLegend, 306720) Mouse TCRβ antibody-APC (BioLegend, 109212) Tetramers: NYESO1-tetramer-PE (MBL, TB-M011-1)

[0143] The human TCRαβ antibody recognizes TCRs with human C regions, but not TCRs with mouse C regions. On the other hand, the mouse TCRβ antibody recognizes TCRs with mouse C regions, but not TCRs with human C regions. Furthermore, the NYESO1-tetramer recognizes only the NYESO1-TCR. Under these experimental conditions, the human TCRαβ antibody recognizes WT1-TCR (KM#3-3), while the mouse TCRβ antibody and the NYESO1-tetrame are thought to recognize the NYESO1-TCR. The results are shown in Figures 14A and 14B.

[0144] Non-transfected Jurkat WT1-TCR cells were confirmed to express a TCR (KM#3-3) recognized by the human TCRαβ antibody, but no expression of a TCR recognized by the mouse TCRβ antibody or NYESO1-tetramer was observed (upper and middle rows of the Jurkat WT1-TCR cell column on the right in Figures 14A and 14B). In contrast, in cell groups co-transfected with the NYESO1-TCR vector and a gRNA / Cas9 expression vector for knocking the NYESO1-TCR vector into the CDR3 region of WT1-TCR, cells expressing NYESO1-TCR were observed, albeit in very small numbers (Figure 14A, upper and middle rows of each column for conditions 1-1, 1-2, 1-3, and 1-4) (Figure 14B, upper and middle rows of each column for conditions 2-1, 2-2, 2-3, and 2-4). Furthermore, most of the cells expressing NYESO1-TCR did not express WT1-TCR (Figure 14A, lower rows of each column for conditions 1-1, 1-2, 1-3, and 1-4) (Figure 14B, lower rows of each column for conditions 2-1, 2-2, and 2-4). These results suggest that the TCR expressed in Jurkat WT1-TCR cells was switched from WT1-TCR to NYESO1-TCR. These results demonstrate that genome editing can be used to switch the expression of an exogenously introduced T cell receptor (TCR) from the existing TCR to a different TCR.

Claims

1. Providing an effector cell or a precursor cell thereof, the effector cell or the precursor cell thereof having, in its genome, a cassette tape gene including a gene encoding a marker protein so that the marker protein can be expressed, and a cassette deck structure not including an exogenous drug resistance gene, one per cell; The method for simultaneously producing multiple types of effector cells expressing different proteins comprises the step of replacing the marker protein genes of the effector cells or their precursor cells with genes encoding multiple types of desired proteins.

2. The method according to claim 1, wherein a group of effector cells comprising a cassette tape gene-containing cassette deck structure containing a gene encoding a marker protein and a cassette tape gene containing recombinase target genes adjacent to the upstream and downstream thereof, and a plurality of cassette tape exchange vectors each containing genes encoding a plurality of proteins are simultaneously cultured in the presence of a recombinase to simultaneously produce a plurality of types of effector cells expressing distinct proteins.

3. The method according to claim 1 or 2, further comprising a step of selecting effector cells or their precursor cells expressing the desired protein by selecting marker protein-negative cells.

4. The method according to any one of claims 1 to 3, wherein the marker protein is a known ligand or receptor expressed on the surface of an effector cell or its precursor cell.

5. The method of claim 4, wherein the marker protein is a gene encoding a known rearranged T cell receptor (TCR) or a chimeric antigen receptor (CAR).

6. The method according to any one of claims 1 to 3, wherein the marker protein is a fluorescent protein.

7. The method according to any one of claims 1 to 6, wherein the step of replacing the marker protein genes of the effector cells or their precursor cells with genes encoding multiple types of desired proteins is carried out by a genome editing method.

8. The method according to any one of claims 1 to 7, wherein the effector cell is an immune cell.

9. The method according to claim 8, wherein the immune cells are NK cells or T cells.

10. The method according to any one of claims 1 to 9, wherein the protein to be expressed in an effector cell is TCR or CAR.

11. The method according to claim 10, wherein the TCR or CAR expressed in the effector cell is derived from a tumor-infiltrating lymphocyte.

12. The step of providing an effector cell or a precursor cell thereof, which contains, per cell, a cassette deck structure having a cassette tape gene containing a gene encoding a marker protein in a genome so that the marker protein can be expressed and does not contain an exogenous drug resistance gene, comprises: (1) providing a material cell having a cassette deck structure, the material cell having a differentiation potential into an effector cell, the material cell having a cassette tape gene including a gene encoding a marker protein in the genome of the material cell, such that the marker protein can be expressed in a cell obtained by inducing differentiation of the material cell into an effector cell or a precursor cell of the effector cell; (2) growing the material cells; (3) The method according to any one of claims 1 to 11, further comprising a step of inducing differentiation of the material cells into effector cells or their precursor cells.

13. The method of claim 12 , wherein the material cells are pluripotent stem cells.

14. The method according to claim 13 , wherein the material cells are ES cells or iPS cells.

15. A composition comprising multiple types of effector cells expressing distinct proteins obtained by the method according to any one of claims 1 to 14, for use in treating an immune-related disease in a subject.