Method for producing car-t cells, kit for producing car-t cells, and method for increasing car-t cell content

Abstract

To provide a method for producing CAR-T cells without using feeder cells, to provide a cell-producing kit used for the method, and to provide a method for improving the content of CAR-T cells.SOLUTION: The method for producing CAR-T cells according to the embodiment improves the content of CAR-T cells by promoting the proliferation of CAR-T cells in a cell group without using feeder cells by co-culturing a cell group into which a secretory antigen protein gene in which an extracellular secretion signal sequence is added to the sequence of an antigen protein which is a target of CAR or a partial protein thereof is introduced and a cell group containing CAR-T cells at the time of producing CAR-T cells.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] Embodiments of the present invention relate to a method for producing CAR-T cells, a kit for producing CAR-T cells, and a method for improving the content of CAR-T cells. [Background technology]

[0002] CAR-T cells are used for cancer gene therapy. CAR-T cells are T cells with a CAR chimeric antibody gene integrated into their genome. They recognize and kill cancer cells by recognizing antigens specifically expressed in the target cancer cells.

[0003] Typically, to obtain a cell population with a high content of CAR-T cells, they are cultured on feeder cells laid down in a culture system. CAR-T cells proliferate stably and efficiently due to the action of secreted substances such as antigenic proteins released from the feeder cells (Non-Patent Document 1). However, this method requires complicated operations and is not very versatile. However, there is no method for producing CAR-T cells with high efficiency without using feeder cells. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Molecular Therapy: Method and Clinical Development, (US), March 2018, Vol.8, p.131-140, “Enhanced Expression of Anti-CD19 Chimeric Antigen Receptor in piggyback Transposon-Engineered T Cells” Summary of the Invention [Problem to be solved by the invention]

[0005] The problem to be solved by the present invention is to provide a method for producing CAR-T cells with high efficiency without using feeder cells, a cell production kit to be used in the method, and a method for increasing the CAR-T cell content. [Means for solving the problem]

[0006] In a method for producing CAR-T cells according to an embodiment, when producing CAR-T cells, a cell group containing CAR-T cells is co-cultured with a cell group into which a secretory antigen protein gene in which an extracellular secretion signal sequence has been added to the sequence of an antigen protein or a partial protein thereof that is the target of the CAR has been introduced, thereby promoting the proliferation of CAR-T cells in the cell group without using feeder cells, thereby improving the CAR-T cell content. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic diagram showing a first embodiment. [Figure 2] FIG. 10 is a schematic diagram showing a second embodiment. [Figure 3] FIG. 10 is a schematic diagram showing a third embodiment. [Figure 4] FIG. 10 is a schematic diagram showing a third embodiment. [Figure 5] FIG. 10 is a schematic diagram showing a fourth embodiment. [Figure 6] FIG. 10 is a schematic diagram showing a fourth embodiment. [Figure 7] FIG. 10 is a schematic diagram showing a fifth embodiment. [Figure 8] FIG. 10 is a schematic diagram showing a fifth embodiment. [Figure 9] FIG. 10 is a schematic diagram showing a sixth embodiment. [Figure 10] FIG. 10 is a schematic diagram showing a sixth embodiment. [Figure 11] FIG. 13 is a schematic diagram showing a seventh embodiment. [Figure 12] Schematic diagram showing an eighth embodiment. [Figure 13] Scatter plot showing the detection results of Example 7. [Figure 14] Graph showing experimental results of Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0008] Hereinafter, embodiments will be described with reference to the accompanying drawings. In each embodiment, substantially identical components are denoted by the same reference numerals, and some of their descriptions may be omitted. The drawings are schematic, and the relationship between the thickness of each component and the planar dimensions, the thickness ratio of each component, etc. may differ from the actual ones.

[0009] (First embodiment) The first embodiment is a method for efficiently producing a cell population with a high content of CAR-T cells without using feeder cells by secreting a CAR antigen protein or a partial protein thereof extracellularly from CAR gene-introduced cells and promoting the proliferation of CAR-T cells (S11 in Figure 1).

[0010] CAR gene-transfected cells are formed by incorporating a CAR gene into a source cell by a known transfection method. The CAR gene can be introduced by, for example, the liposome method, lipofection method, or electroporation method, but the liposome method is preferred. For example, in the case of the liposome method, a nucleic acid encoding any known CAR (chimeric antibody receptor) is encapsulated in a liposome and delivered to the cytoplasm of the source cell, from which it is transported to the nucleus of the source cell and then incorporated into the genome of the cell. By being incorporated into the genome of the cell, the CAR is expressed on the surface of the cell membrane of the source cell.

[0011] The nucleic acid encoding the CAR is configured to be incorporated into the nucleus and express the CAR on the surface of the introduced material cell. The nucleic acid encoding the CAR is, for example, CAR-DNA, i.e., it is preferably double-stranded DNA encoding the CAR gene sequence. The CAR-DNA may be in any form suitable for being incorporated into the nucleus and inserted into the genome of the cell to express the CAR.

[0012] The nucleic acid encoding the CAR may be used in the form contained in a gene set for producing CAR cells. For example, the gene set for producing CAR cells includes a nucleic acid encoding the CAR, such as CAR-DNA, and may further include a nucleic acid transfer promoter as an optional component. The nucleic acid transfer promoter includes a genome integration promoter, such as a gene that promotes the integration (insertion) of the CAR-DNA into the genome. The genome integration promoter may be, for example, a piggyBac gene such as PB-mRNA. Such an enzyme gene is known as a transposase (DNA transferase) gene. By introducing a CAR gene and a transposase gene into T cells, which are source cells, CAR-T cells (cells in which the CAR gene has been integrated into the T cell genome) can be stably produced. Representative transposases include piggyBac derived from moths and Sleeping Beauty derived from fish. Any of these may be used, but are not limited to these. Furthermore, devices that exhibit other genome integration promotion mechanisms may also be used. For example, a nucleic acid encoding the CAR may be incorporated into plasmid DNA and then encapsulated in liposomes. Furthermore, the PB gene may be integrated into a plasmid together with the PB gene and packaged as a gene set for producing CAR cells. The PB gene may be either DNA or mRNA, but mRNA is preferred.

[0013] The genome integration-promoting agent may also be a protein, such as piggyBac, encoded by a gene that promotes the integration (insertion) of CAR-DNA into the genome. Even when the genome integration-promoting agent is a protein, it may be delivered to the cytoplasm of the source cell together with the nucleic acid encoding the CAR. In other words, the genome integration-promoting agent may be a gene or protein that promotes genome integration (or insertion). Furthermore, for example, the genome integration-promoting agent may be encapsulated in a liposome together with the CAR gene and delivered to the source cell.

[0014] The material cells may be any cells from which CAR-T cells can be obtained by gene transfer. For example, the material cells are T cells. For example, when T cells are used as material cells, they may be used as a material fraction, which is a material cell group containing the material cells in a state contained in peripheral blood mononuclear cells (PBMCs) or a lymphocyte fraction. When CAR-T cells are used for therapy or diagnosis, they may be PBMCs collected from a subject for treatment, prevention, and / or diagnosis. PBMCs can be prepared from peripheral blood by any known method, such as density gradient centrifugation, such as ultracentrifugation using Ficoll.

[0015] The CAR antigen protein or a partial protein thereof secreted from the CAR gene-introduced cells as described above is an antigen that is the target of the CAR. The CAR antigen protein or a partial protein thereof may be secreted extracellularly from the CAR gene-introduced cells before and / or after, and / or simultaneously with, the introduction of a nucleic acid encoding a CAR into the source cells. This may also be done at least simultaneously with and / or after the introduction of a nucleic acid encoding a CAR into the source cells.

[0016] To secrete the CAR antigen protein or its partial protein extracellularly from CAR gene-introduced cells, a gene encoding a secreted CAR antigen protein or its partial protein can be introduced into source cells and / or source cells transfected with a nucleic acid encoding a CAR and / or CAR-T cells. The gene encoding the CAR antigen protein or its partial protein can be introduced by contacting the cells with a liposome containing a nucleic acid encoding the secreted antigen protein. The nucleic acid encoding the secreted antigen protein may include a sequence encoding the CAR antigen protein or its partial protein and an extracellular secretion signal sequence. Alternatively, the nucleic acid encoding the secreted antigen protein may be mRNA. The nucleic acid encoding the secreted antigen protein is incorporated into the cytoplasm, causing the antigen protein or its partial protein to be secreted extracellularly. Contact with the CAR antigen protein promotes the division and proliferation of CAR-T cells. The method for producing CAR-T cells according to the embodiment comprehensively produces CAR-T cells as the desired "cell group with a high CAR-T cell content" by both producing CAR-T cells by introducing nucleic acid into the genome and causing the produced CAR-T cells to divide and proliferate by contacting the CAR-T cells with a CAR antigen.

[0017] The target CAR-T cell population may be a cell population containing CAR-T cells in a predetermined amount or cell number, or a CAR-T cell population containing other cells that were contained in a source cell population such as PBMCs at a predetermined ratio. For example, when a therapeutic CAR-T cell population is produced by a method according to an embodiment, the target CAR-T cell population may be a cell population containing CAR-T cells with a cell number necessary for treatment. Here, the "target CAR-T cell population" may be, for example, a cell population containing CAR-T cells at 10% or more, more preferably 20% or more, e.g., 20% to 80%. Such a CAR-T cell population may also be interpreted as a "cell population with a high CAR-T cell content."

[0018] As described above, the method of the first embodiment is characterized by not using feeder cells. According to the method of the embodiment, highly efficient CAR-T cell production is possible without the use of feeder cells. Feeder cells are typically cancer cells or the like, supplied as a specific established cell line. When used, feeder cells are attached to the bottom of a culture vessel and co-cultured with target cells. This creates an environment closer to that of a living body, enabling stable culture and proliferation of the co-cultured target cells. For this reason, feeders are widely used. Feeder cells are considered essential, particularly for the stable culture of primary human cells. Traditionally, feeder cells have also been required for the efficient production of cell populations with a high CAR-T cell content sufficient for therapeutic use, and for this reason, established cell lines have been carefully passed down. When CAR-T cells are produced using feeder cells, they must be completely isolated prior to use. Furthermore, before producing CAR-T cells, the feeder cells must be irradiated with gamma rays to eliminate their proliferation ability. These complicated procedures and the need for feeder cells make it difficult to generalize the production of CAR-T cells. According to the embodiments, CAR-T cells can be produced stably and efficiently without using such feeder cells. By secreting the CAR antigen protein or a partial protein thereof extracellularly from CAR gene-transduced cells, the antigen protein or the partial protein thereof binds to the CAR, promoting the proliferation of CAR-T cells. Because no feeder cells are required, the procedure is simple and easy to implement in many medical and research institutions. Therefore, it is expected to have significant benefits in cancer treatment and prevention, as well as in basic research.

[0019] (Second embodiment) According to the second embodiment, there is provided a method for improving the CAR-T cell content during the production of CAR-T cells. This method involves co-culturing a cell population containing CAR-T cells with a cell population into which a secretory antigen protein gene, in which an extracellular secretion signal sequence has been added to an antigen protein that is the target of the CAR or a partial sequence thereof (i.e., a partial protein), has been introduced, thereby promoting the proliferation of CAR-T cells without using feeder cells, thereby improving the CAR cell content in the cell population (S21 in Figure 2).

[0020] The production of CAR-T cells encompasses two situations. The first situation refers to a situation in which a CAR gene is introduced into source cells, resulting in transfection of the CAR-T cells, thereby newly producing or forming CAR-T cells from the source cells. The second situation refers to a situation in which activation, such as contact between multiple CAR-T cells or high density, leads to division and proliferation, thereby increasing the number of CAR-T cells. The second embodiment may be interpreted as a method for more efficiently increasing the number of CAR-T cells during the production of CAR-T cells. In other words, this method involves co-culturing a cell population containing CAR-T cells with a cell population into which a secretory antigen protein gene, in which an extracellular secretion signal sequence has been added to the antigen protein targeted by the CAR or a partial sequence thereof (i.e., a partial protein), has been introduced during the production of CAR-T cells, thereby promoting the proliferation of the CAR-T cells and increasing the CAR cell content in the cell population.

[0021] When an antigen protein or a partial protein thereof released from a cell group into which a secretory antigen protein gene has been introduced binds to the CAR expressed on the surface of CAR-T cells, the CAR-T cells are activated, causing them to divide and proliferate, effectively increasing their numbers.

[0022] The cell population into which the secretory antigen protein gene is introduced may be source cells for CAR-T cells, mononuclear cells excluding the source cells contained in the source cell population, or the source cell population (PBMC) itself. Alternatively, it is preferable to select other cells that are not feeder cells and are non-biotoxic or toxic, or have relatively low biotoxicity or toxicity. When PBMCs are used as the source cell population, the "cell population containing CAR-T cells" to be co-cultured refers to a cell population containing CAR-T cells generated by CAR gene introduction and other cells contained in the PBMCs. The cell population into which the secretory antigen protein gene is introduced includes CAR-T cells, T cells, and / or other mononuclear cells, etc., into which the secretory antigen protein gene has been introduced. Co-culture refers to a situation in which these cells and newly formed CAR-T cells without the secretory antigen protein gene introduced are cultured together in a single culture system. Cells to be co-cultured with CAR-T cells include cells that are not toxic or harmful to CAR-T cells, or that have relatively low toxicity or toxicity, and / or that can support the proliferation of T cells, such as the leukemia cell line aK562 cells and the rhabdomyosarcoma-derived cell line SJCRH30 cells, and / or cells that express high amounts of proteins from secreted antigen protein genes, such as human embryonic kidney cells HEK293 cells.

[0023] According to the embodiments, the structural feature of not requiring feeder cells is also a significant advantage. Furthermore, according to the methods for producing CAR-T cells and the methods for increasing the content of CAR-T cells according to the embodiments, CAR-T cells can be produced more efficiently than when CAR-T cells are produced using feeder cells.

[0024] (Third embodiment) According to a third embodiment, a method for producing CAR-T cells without using feeder cells is provided. This third embodiment will be described with reference to FIG. 3. This method comprises three stages: S31, S32, and S33. S31 includes two phases (a) and (b). In S31 (a), a CAR gene and a genome integration promoter are introduced into source cells in a first culture system to obtain CAR-T cells. In S31 (b), an antigen protein or a partial protein thereof, which is the target of the CAR, is secreted from source cells in a second culture system, which may or may not be independent of the first culture system. Steps (a) and (b) may be performed simultaneously or in stages. Furthermore, as will be described in detail later, the first and second culture systems may be independent of each other or independent of each other. That is, the first and second culture systems may be formed in separate vessels, or may exist in a single reaction vessel, partially or completely overlapping each other. In other words, the "first culture system" may be equal to the "second culture system." In S32, the secreted antigen protein or a partial protein thereof is contacted with the CAR-T cells produced by the introduction of the CAR gene and a genome integration promoter in S31(a) to promote the division and proliferation of the CAR-T cells. In S33, CAR-T cells are produced by gene introduction and their division and proliferation is promoted to obtain a cell population with a high content of the desired CAR-T cells.

[0025] CAR-T cells are produced by gene transfer by contacting a first liposome containing a nucleic acid encoding a CAR and an enzyme gene or protein (genomic integration promoter) that integrates the nucleic acid into the cellular genome with source cells containing T cells, thereby introducing the nucleic acid into the source cells. Secretion of an antigen protein or a partial protein thereof is achieved by introducing a nucleic acid encoding the secreted antigen protein into the source cells and / or the source cells into which the nucleic acid encoding the CAR has been introduced. This introduction proceeds by contacting the cells with a second liposome. The second liposome contains a nucleic acid encoding a secreted antigen protein in which an extracellular secretion signal sequence has been added to the sequence encoding the antigen protein or a partial protein thereof. The production of CAR-T cells by the introduction of the CAR gene and the genome integration promoter, and the promotion of CAR-T cell division and proliferation, can be carried out simultaneously or in stages.

[0026] In other words, the third embodiment can be understood as a method having four stages as shown in FIG.

[0027] In the first stage, in a first culture system, a first liposome containing a nucleic acid encoding a CAR and a genome integration promoter is contacted with material cells containing T cells to introduce the nucleic acid encoding the CAR into the material cells, thereby producing CAR-T cells from the material cells (S41).

[0028] In the second stage, in a second culture system that is independent or not independent of the first culture system, the material cells and / or the material cells into which the nucleic acid encoding the CAR has been introduced are contacted with a second liposome containing a nucleic acid encoding a secretory antigen protein in which an extracellular secretion signal sequence has been added to a sequence encoding an antigen protein or a partial protein thereof that is the target of the CAR, thereby introducing the nucleic acid encoding the secretory antigen protein into the cells and causing the antigen protein or a partial protein thereof to be secreted from the cells (S42).

[0029] In the third stage, the secreted antigen protein or a partial protein thereof is contacted with the CAR-T cells formed by the introduction of the CAR gene and a genome integration promoter, thereby promoting the division and proliferation of the CAR-T cells (S43).

[0030] In the fourth stage, CAR-T cells are generated by introducing the CAR gene and a genome integration promoter, and the division and proliferation of the cells is promoted, thereby creating a cell population with a high content of the desired CAR-T cells (S44).

[0031] According to the third embodiment, it is possible to stably and efficiently produce a cell population with a high content of CAR-T cells without using feeder cells. Because there is no need to use feeder cells, the operation is simple and easy to implement in many medical and research institutions. Therefore, it is expected to have a significant effect on cancer treatment and research.

[0032] (Fourth embodiment) An example of the method for producing CAR-T cells without using feeder cells according to the fourth embodiment will be described with reference to Figures 5 and 6. This method is a very simple one-step method in which a single type of liposome is used, each liposome encapsulating substances for all types of genes to be introduced, thereby completing the addition of liposomes and gene introduction in a single step.

[0033] As shown in Figure 5(a), the liposome 50 used contains a gene set for producing CAR cells and a secreted antigen protein gene. In the example shown in Figure 5, the liposome 50 specifically encapsulates a nucleic acid 52 encoding a CAR, a genome integration promoter 53, such as PB-mRNA, and a nucleic acid 54 encoding a secreted antigen protein within a lipid particle 51. Figure 5(b) is a schematic enlarged view of an example of the nucleic acid 54 encoding a secreted antigen protein. The nucleic acid 54 encoding the secreted antigen protein comprises an antigen protein subsequence 54a, a secretory signal sequence 54b attached to one end, and a modification sequence 54d, such as an IgG Fc-tag sequence, linked to the other end via a linker 54c. The antigen protein produced within the cell is released extracellularly by the secretory signal. The IgG Fc-tag is an arbitrary sequence that confers hydrophilicity to the released protein, contributing to its stabilization. One example of a CAR target is CD19. Accordingly, CD19 is an example of an antigen protein. The antigen protein may be a portion of CD19, which may have a length that can be considered a peptide. For example, a partial protein of the CD19 protein may be the extracellular domain sequence of the CD19 protein. The extracellular secretory signal sequence may be, but is not limited to, the signal sequence of interleukin-6.

[0034] The liposomes 50 are added to a medium 56 for producing CAR-T cells in a container 55. Material cells 57 are prepared in advance in the medium 56 for producing CAR-T cells in the container 55 (FIG. 5(c)). The material cells 57 may be T cells in PBMCs, as described above. The PBMCs can be prepared from peripheral blood collected in advance from a patient to be treated.

[0035] Peripheral blood mononuclear cells (PBMCs) can be isolated, for example, as follows: Mononuclear cells are isolated from human peripheral blood by Ficoll density gradient centrifugation to obtain peripheral blood mononuclear cells (PBMCs). Specifically, PBMCs are centrifuged by density gradient centrifugation using, for example, a Ficoll reagent for mononuclear cell isolation (e.g., Ficoll-Paque Premium, Cytiva).

[0036] T cells contained in PBMCs can be cultured, for example, as follows: T cells contained in isolated PBMCs are cultured. T cells are cultured using a commercially available T cell culture medium (e.g., TexMACS, Miltenyi Biotec, or AlyS705, Cell Science Institute). If necessary, the medium may contain components that support the survival and proliferation of T cells, such as artificial serum (artificially prepared serum that does not contain animal-derived components) and cytokines (e.g., interleukin-7 and interleukin-15).

[0037] Liposome 50 that comes into contact with material cell 57 is taken up by material cell 57 by intracellular endocytosis and then releases its contents in the cytoplasm. The CAR-encoding nucleic acid 52 from liposome 50 taken up by the T cell, which is material cell 57, enters the nucleus and is integrated into the genome, causing the T cell, which is material cell 57, to become a CAR-T cell 58. CAR 59 is expressed on the cell membrane. Meanwhile, nucleic acid 54 encoding a secreted antigen protein (if the nucleic acid is DNA, it is released from the liposome into the cytoplasm, then translocated to the cell nucleus, where it is transcribed into mRNA; if the nucleic acid is mRNA, it is released from the liposome into the cytoplasm) is translated into antigen protein 60 in the cytoplasm and released into the medium 56 (Figure 5(d)). When antigen protein 60 binds to CAR 59, CAR-T cell 58 is activated and begins to divide and proliferate efficiently. Figure 5(e) shows a schematic of CAR-T cells 58 during expansion culture, which aims to proliferate the cells while maintaining their properties. CAR-T cells are stimulated and activated by antigen proteins, which promotes cell division, resulting in the formation of additional CAR-T cells 61. Approximately 14 days after adding liposomes 50 to PBMCs in culture medium 56, CAR-T cells in the number required for treatment, for example, are produced.

[0038] Figure 6 shows a schematic diagram of an example of a one-step method for generating CAR-T cells without using feeder cells. First, a desired type of liposome is prepared (S51). In the example described above, liposomes 50 are prepared, each containing a lipid particle 51 encapsulating a nucleic acid 52 encoding a CAR, PB-mRNA 53, and a nucleic acid 54 encoding a secreted antigen protein. Next, the liposomes are introduced into a culture system containing source cells (S52). In this example, PBMCs are prepared in a CAR-T cell production medium 56, and liposomes are added to the medium. Next, the desired CAR-T cell population is obtained without using feeder cells by culturing under appropriate conditions (S53). These steps complete an example of a method for generating CAR-T cells.

[0039] The difference in the CAR-T expansion effect obtained by the method according to the embodiment compared to the case using antigen-presenting feeder cells that present antigens on their surface is as follows. In CAR-T expansion, the key factor is how efficiently antigens can be brought into contact with (activated by) CAR-T. Feeder cells present antigens on their surface while attached to the bottom of the culture vessel. Therefore, the maximum amount of antigen that feeder cells can present to T cells is limited by the area of ​​the bottom of the culture vessel. Furthermore, since feeder cells are attached to the bottom of the culture vessel, their contact surface with T cells is limited to one side of the T cell. In contrast, since secretory antigen peptides are dissolved in the culture medium in the culture vessel, the number of antigens that can be presented to T cells depends on the volume of the culture medium. Since volume is (area × height), secretory antigen peptides can present more antigens than feeder cells due to the height of the culture vessel. Furthermore, since secretory antigen peptides are dissolved in the culture medium, their contact surface with T cells is the entire cell surface, resulting in extremely high cell contact efficiency. Due to these characteristics, the method according to the embodiment has a higher contact efficiency (activation efficiency) with CAR-T and a higher CAR-T amplification effect than antigen-presenting feeder cells.

[0040] According to the fourth embodiment, CAR-T cells can be produced stably and efficiently without using feeder cells. Furthermore, because it is a one-step method, it is easy to produce CAR-T cells and minimizes the possibility of contamination.

[0041] (Fifth embodiment) The one-step method for producing CAR-T cells without using feeder cells shown in the fourth embodiment may be carried out using two types of liposomes. The method according to the fifth embodiment can be carried out in the same manner as the fourth embodiment except that two types of liposomes are used. The two types of liposomes will be described using Figures 7 and 8.

[0042] The first liposome 70A used in this method contains a lipid particle 51 containing a nucleic acid 52 encoding a CAR and a genome integration promoter 53. The second liposome 70B contains a nucleic acid 54 encoding a secreted antigen protein (Figure 7(a)). These two types of desired liposomes are prepared. Material cells 57 are added to a container 55 containing a medium 56, and the two types of liposomes are then added simultaneously (Figure 7(b)). This is then cultured under appropriate conditions to obtain the desired CAR-T cell population (Figure 7(c)). Although not shown, continued expansion promotes CAR-T cell division, as shown in Figure 5(e). Approximately 14 days after adding the liposome 50 to the PBMCs in the medium 56, CAR-T cells, for example, in the number required for treatment, are produced.

[0043] Figure 8 shows a schematic diagram of an example of a one-step method for generating CAR-T cells without using feeder cells. First, two types of desired liposomes are prepared (S61). In the example described above, liposome 70A, which contains a CAR-encoding nucleic acid 52 and PB-mRNA 53 within lipid particle 51, and liposome 70B, which encapsulates a nucleic acid 54 encoding a secreted antigen protein, are prepared. Next, these liposomes are introduced into a culture system containing source cells (S62). In this example, PBMCs are prepared in CAR-T cell production medium 56, and liposomes are added to the medium. Next, by culturing under appropriate conditions, the desired CAR-T cell population is obtained without using feeder cells (S63). These steps complete an example of a method for generating CAR-T cells.

[0044] According to the fifth embodiment, it is possible to stably and efficiently produce a cell population with a high content of CAR-T cells without using feeder cells. Furthermore, because it is a one-step method, it is easy to do and the possibility of contamination can be minimized. Furthermore, because the nucleic acid 52 encoding the CAR and the nucleic acid 54 encoding the secretory antigen protein are separately encapsulated in the first liposome 70A and the second liposome 70B, diversification by combination is possible, making it highly versatile and advantageous for commercialization and various research applications.

[0045] (Sixth embodiment) By using the two types of liposomes described in the fifth embodiment, a two-step method for producing CAR-T cells can be achieved. This will be explained using Figure 9. Figure 9 shows a schematic diagram of each process and culture system, including the first step, A series (a-1) to (a-2), and the second step, B series (b-1) to (b-4). The overall flow is shown as a scheme in Figure 10.

[0046] Two types of liposomes are prepared as described in the fifth embodiment (FIGS. 9(a-1), (b-1), and FIG. 10(S71)). In series A, a first liposome 70A is added to a culture medium 56 in a container 55A, which is a first culture system. The first liposome comes into contact with and is taken up by material cells 57 in the culture medium 56A, resulting in the production of CAR-T cells (FIG. 9(a-2), and (S72) and (S73) in FIG. 10). On the other hand, in series B, a second liposome 70B is added to a culture medium 66 contained in a container 55B, which is a second culture system independent of the first culture system. The culture medium 66 contains arbitrary cells 77 (FIGS. 9(b-2), and (S72) in FIG. 10). The timing of adding the second liposome 70B to the container 55B, which is the second culture system, may be before, after, or simultaneously with the addition of the first liposome 70A to the container 55A, which is the first culture system. The second liposome 70B may also be added at multiple times. Upon contact with the liposome 70B, the nucleic acid 54 encoding the secreted antigen protein is introduced into any cell 77, and the antigen protein 60 is released into the medium 66 (FIG. 9(b-3) and FIG. 10(S73)). The medium 66 is then collected and added to the container 55A, which is the first culture system (FIG. 9(a-2) and FIG. 10(S74)). FIG. 9(a-2) shows the state of the first culture system when the addition of the first liposome and the medium 66 containing the antigen protein 60 obtained from the B lineage are simultaneously performed.

[0047] Here, an example is shown in which antigen protein 60 is introduced by recovering medium 66 and adding it to the first culture system. By constructing the released antigen protein so that it is water-soluble, the contact efficiency between the antigen protein and CAR-T cells increases, enabling more efficient amplification. The arbitrary cells 77 may be the material cells or material cell groups used in the first culture system, or any other known cells capable of more effectively expressing antigen proteins. Cells known to be harmless to living organisms are also preferred. The medium 66 may be selected depending on the cell type of the arbitrary cells 77 used.

[0048] According to the sixth embodiment, it is possible to produce a desired amount of antigen protein at a desired timing. This may be performed in conjunction with the production of CAR-T cells by CAR-T gene transfer, or may be performed prior to the addition of the first liposome. Furthermore, since cell types can be selected arbitrarily, if the antigen protein production ability of the source cells or source cell group is low, or in preparation for such a case, the B lineage may be performed in combination with the one-step method.

[0049] (Seventh embodiment) According to a seventh embodiment, there is provided a cell production kit for use in a method for producing CAR-T cells without using feeder cells. The cell production kit may be a kit comprising the following liposome group A and / or B together with a substance that improves the complementary stability of the liposome: liposome group A: a first liposome comprising a nucleic acid encoding a chimeric antibody receptor (CAR); and A second liposome containing a nucleic acid encoding a secretory antigen protein in which an extracellular secretion signal sequence has been added to a sequence encoding an antigen protein or a partial protein thereof that is the target of the CAR, and a liposome group B: a nucleic acid encoding the CAR; A liposome that encapsulates a nucleic acid encoding a secretory antigen protein in which an extracellular secretion signal sequence has been added to a sequence encoding an antigen protein or a partial protein thereof that is the target of the CAR.

[0050] An example of the first liposome contained in liposome group A is first liposome 70A shown in Fig. 7(a), and an example of the second liposome is second liposome 70B shown in Fig. 7(a). Liposome group A is a group containing a plurality of first liposomes and a plurality of second liposomes.

[0051] An example of liposomes included in liposome group B is the liposome shown in Figure 5(a). Liposome group B is, for example, a group that contains elements of the one-step, one-liposome method, i.e., elements that enable expression of both the CAR gene and the antigen protein gene in one liposome. Furthermore, this liposome may contain additional elements that enable introduction and expression of the CAR gene.

[0052] Liposomes 70A and 70B will be further described with reference to FIG. 11. Lipid particles 51 constituting liposomes 70A and 70B may be composed of, for example, a lipid membrane formed by non-covalently arranging a plurality of lipid molecules, which are the material of the liposomes 70A and 70B. Lipid particles 51 have a hollow space 82, in which a nucleic acid 52 encoding a CAR, an optional genome integration promoter 53, and / or a nucleic acid 54 encoding a secreted antigen protein are encapsulated. The lipid particle 51 may be any lipid particle as long as it has the property of being taken up by cells and decomposed. Examples of the lipid particle 51 include those containing at least a first lipid 81a and a second lipid 81b as components. The first lipid 81a is a lipid compound having the following formula (I), and the second lipid 81b is a lipid compound having the following formula (II). The first lipid compound represented by formula (I) is FFT10, and the second lipid compound represented by formula (II) is FFT20.

[0053] [ka]

[0054] In addition to liposomes, the cell production kit may further contain a substance that improves the storage stability of liposomes. Examples of substances that improve storage stability include, but are not limited to, glycoproteins such as albumin, lipoproteins, apolipoproteins, and globulins; pH adjusters, buffering agents, tonicity adjusters; pharmaceutically acceptable agents that bring the composition closer to physiological conditions, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride; lipophilic free radical quenchers such as α-tocopherol that suppress damage caused by free radicals; and lipid-protecting agents, such as water-soluble chelators such as ferrioxamine, that suppress lipid peroxidation damage and improve storage stability.

[0055] The cell production kit may include liposomes in a liquid composition contained in a suitable carrier. The carrier may be, for example, water, a saline solution such as physiological saline, an aqueous glycine solution, or a buffer solution. Alternatively, the liposomes may be provided in the cell production kit as a dry powder composition. The powder composition can be used by the user by adding a suitable liquid such as the carrier. Furthermore, any of these liposomes may be contained in a suitable container or small bag. It is also preferable that such compositions and liposomes are sterilized by a known method.

[0056] The cell production kit may further include other elements as desired, such as sterilized containers such as culture dishes, petri dishes, and tubes, culture media, pH-adjusting reagents, antibiotics, growth factors, vitamins, salts, cleaning compositions, sterilized ultrapure water, reagents for adjusting PBMCs, lipid compositions for preparing liposomes, and various instructions.

[0057] The lipid particle 51 may contain additional lipids in addition to the first lipid 81a and the second lipid 81b. Among the lipid molecular materials constituting the lipid particle 51, a fraction consisting of the first lipid 81a and the second lipid 81b will be referred to as the "first fraction" below. Furthermore, a fraction consisting of lipid molecular materials other than the first lipid 81a and the second lipid 81b will be referred to as the "second fraction" below. The lipids contained in the second fraction will also be collectively referred to as the "third lipid 81c" below.

[0058] The terms "first fraction" and "second fraction" refer to the composition of the constituent components of lipid particle 51, and do not refer to the physical location of the lipids contained therein. For example, the constituent components of the first fraction and the second fraction do not need to be each a single entity within lipid particle 51, and the lipids contained in the first fraction and the lipids contained in the second fraction may exist as a mixture. The blending ratio of the first fraction to the total lipid material constituting lipid particle 51 is preferably 30% or more and less than 50% (molar ratio).

[0059] The proportion of the second lipid 81b in the first fraction is preferably 40% or more. In this case, the T cell tumor cell specificity of nucleic acid transfer and the transfer efficiency of the substance encapsulated in the liposome can be improved. Furthermore, if the proportion of the second lipid 81b in the first fraction is 50% or more, the amount of the substance to be transferred encapsulated in the liposome, particularly in vivo, can be improved, which is more preferable. It is even more preferable if the proportion of the second lipid 81b is 60% or more. The upper limit of the proportion of the second lipid 81b can be 80%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0060] The particle size and cellular permeability of the lipid particles 51 may vary depending on the blending ratio of the first lipid 81a and the second lipid 81b in the first fraction. For example, the larger the amount of the second lipid 81b, the larger the particle size of the lipid particles 51. The average particle size of the lipid particles 51 can be changed depending on the application, and is preferably adjusted to, for example, about 50 nm to about 300 nm. For example, when used in vivo, it may be preferable to set the average particle size to about 70 nm to about 100 nm.

[0061] The type of third lipid 81c contained in the second fraction of the lipid particle 51 is not limited, but for example, the second fraction includes a base lipid. For example, a lipid that is a main component of a biological membrane can be used as the base lipid. The base lipid is a phospholipid or a sphingolipid, such as diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, or cerebroside, or a combination thereof.

[0062] For example, as base lipids, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-stearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-di-O-octadecyl-3-trimethylammoniumpropane (DOTMA), 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP), 1,2-dimyristoyl-3-dimethylammoniumpropane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammoniumpropane (16:0 DAP), 1,2-distearoyl-3-dimethylammoniumpropane (18:0 DAP), 1,2-dioleoyl-3-dimethylammoniumpropane (18:0 DAP), 1,2-dioleoyl-3-dimethylammoniumpropane (14:0 DAP), 1,2-dipalmitoyl-3-dimethylammoniumpropane (16:0 DAP), 1,2-distearoyl-3-dimethylammoniumpropane (18:0 DAP), 1,2-dioleoyl-3-dimethylammoniumpropane (14:0 DAP), 1,2-dioleoyl-3-dimethylammoniumpropane (16 ... It is preferable to use 1,2-dioleoyl-sn-glycero-3-phosphochlorin (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphochlorin (DLPC), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), cholesterol, or any combination thereof.

[0063] As the base lipid, it is particularly preferable to use a cationic lipid or a neutral lipid, and the content thereof can adjust the acid dissociation constant of the lipid particle 51. It is preferable to use DOTAP as the cationic lipid, and it is preferable to use DOPE as the neutral lipid.

[0064] It is also preferred that the second fraction contains a lipid that prevents aggregation of the lipid particles 51. For example, the lipid that prevents aggregation preferably further contains a PEG-modified lipid, such as polyethylene glycol (PEG) dimyristoylglycerol (DMG-PEG), a polyamide oligomer derived from an omega-amino(oligoethylene glycol)alkanoic acid monomer (U.S. Pat. No. 6,320,017), or a monosialoganglioside.

[0065] The second fraction may further contain lipids such as a lipid with relatively low toxicity for adjusting toxicity, a lipid having a functional group for binding a ligand to the lipid particle 51, and a lipid for suppressing leakage of encapsulated substances such as sterols, e.g., cholesterol. In particular, it is preferable to include cholesterol.

[0066] The type and composition of lipids used in the second fraction are appropriately selected taking into consideration the acid dissociation constant (pKa) or particle size of the target lipid particles 51, the type of substance to be encapsulated, or stability in cells.

[0067] For example, when the second fraction contains DOPE, DOTAP, cholesterol, and DMG-PEG, this is preferred because it provides particularly excellent delivery efficiency for the genetic nucleic acid to be introduced, such as the nucleic acid 52 encoding CAR, the genome integration promoter 53, and the nucleic acid 54 encoding a secreted antigen protein. Hereinafter, for convenience, the nucleic acid 52 encoding CAR, the optional nuclear transfer promoter 53, and the nucleic acid 54 encoding a secreted antigen protein will also be referred to as the "genetic nucleic acids 52 to 54 to be introduced."

[0068] In addition to the gene nucleic acids 52 to 54 to be introduced, further components may be encapsulated in the lipid particle 51 as needed. The further components are, for example, a pH adjuster, an osmotic pressure adjuster, or a gene activator. Examples of pH adjusters include organic acids such as citric acid and their salts. Examples of osmotic pressure adjusters include sugars or amino acids. Gene activators will be described later.

[0069] Lipid particles 51 encapsulating the genetic nucleic acids 52-54 to be introduced and, if necessary, other substances can be produced using known methods used for encapsulating small molecules in lipid particles, such as the Bangham method, organic solvent extraction, surfactant removal, or freeze-thawing. For example, a lipid mixture obtained by incorporating the materials for lipid particles 51 in an organic solvent such as alcohol at a desired ratio and an aqueous buffer containing the components to be encapsulated, such as the genetic nucleic acid to be introduced, are prepared, and the aqueous buffer is added to the lipid mixture. The resulting mixture is stirred and suspended to form lipid particles 51 encapsulating the genetic nucleic acid to be introduced, such as a nucleic acid encoding a CAR 52, a genome integration promoter 53, or a nucleic acid encoding a secreted antigen protein 54, as desired.

[0070] The blending ratio of the components of the lipid particles 51 can be easily adjusted by changing the blending ratio of each material in the lipid mixture. For example, the blending ratio of the components of the lipid particles 51 can be approximately the same as the blending ratio of each material in the lipid mixture. Furthermore, the quantitative ratio of the substances encapsulated in the lipid particles 51 can be easily adjusted by changing the quantitative ratio of both in the aqueous buffer solution.

[0071] By contacting the lipid particles 51 with T cells, the lipid particles 51 can be taken up into the cells by, for example, endocytosis. Then, the genetic nucleic acid to be introduced, e.g., the nucleic acid 52 encoding a CAR, the genome integration promoter 53, and the nucleic acid 54 encoding a secreted antigen protein, can be released into the cells. By containing the first lipid 81a and the second lipid 81b, the lipid particles 51 have the property of being easily taken up by T cells but not easily taken up by other cells. Therefore, by the simple procedure of encapsulating the genetic nucleic acid to be introduced into the lipid particles 51 and contacting them with T cells, the genetic nucleic acid to be introduced, e.g., the nucleic acid 52 encoding a CAR, the nuclear transfer promoter 53, and the nucleic acid 54 encoding a secreted antigen protein, can be efficiently introduced into T cells in any desired combination without using antibodies or receptors as in the conventional method. This allows the lipid particles 51 to selectively or specifically deliver the genetic nucleic acid to be introduced into T cells without using feeder cells.

[0072] According to the cell production kit of the seventh embodiment, it is possible to simply and easily carry out a method for producing a cell population with a high content of CAR-T cells without using feeder cells.

[0073] (Eighth embodiment) The method for producing CAR-T cells without using feeder cells according to any of the above-described embodiments can be used to treat cancer with CAR-T cells. Here, an example of autoimmune cell therapy using the one-step method for producing CAR-T cells according to the fourth embodiment and shown in FIG. 5 will be described with reference to FIG. 12. First, liposomes are prepared (S91). For example, as shown in FIG. 5, liposomes 50 are prepared, each of which contains a lipid particle 51 containing a nucleic acid 52 encoding a CAR, a genome integration promoter 53, such as PB-mRNA, and a nucleic acid 54 encoding a secreted antigen protein. Next, a material cell population is collected from a subject to be treated (S92). The material cell population is seeded in an appropriate medium, and liposomes are added thereto (S93). The material cell population is cultured under appropriate conditions to produce a CAR-T cell population (S94). The obtained CAR-T cell population is subjected to any desired test (S95). The CAR-T cell population is then administered to a subject (S96). Administration to a subject can be performed intravenously, with the dosage determined based on the CAR-T content of the resulting CAR-T cell population.

[0074] The resulting CAR-T cell population may also be cryopreserved at -180°C until use. CAR-T cells can be cryopreserved using any known cryopreservation method for live cells. For example, they can be suspended in a cryopreservation solution and cryopreserved in a cryopreservation bag, for example, in a -150°C freezer. The number of cells to be cryopreserved can be determined based on the CAR-T cell content obtained by measuring the total viable cell count for all types of cells contained in the final CAR-T cell population or the viable cell count for CAR-T cells. Furthermore, CAR-T cells can also be separated from other cells. For example, they can be separated based on cell surface antigens specific to CAR-T cells / T cells. Separation of T cells / CAR-T cells can be performed using an antibody against CD3 (anti-CD3 antibody), a specific antigen on the surface of T cells, using methods such as MACS (magnetic cell sorting). Separation of CAR-T cells can be performed using a CAR-specific idiotypic antibody, using methods such as MACS.

[0075] The liposome may be any of the liposomes described above. The subject may be any animal, including humans, pets, and livestock, in need of treatment. The material cell population may be any cell population containing T cells, such as PBMCs or lymphocyte fractions. For example, the material cell population may be obtained by collecting peripheral blood using a syringe or the like, and then isolating the mononuclear cell fraction by gradient centrifugation or the like, or collecting the lymphocyte fraction using a blood component separator.

[0076] The appropriate medium may be any medium suitable for the material cell group used.

[0077] The optional tests performed as desired may be tests to confirm safety before administering the CAR-T cell population to a subject. Tests may be performed taking into consideration patient safety from biological, morphological, clinical, non-clinical, and pharmaceutical perspectives. Various tests may also be performed as required by the standards of the country, government, academic society, or expert organization or group where the test is performed. Furthermore, although not shown in Figure 12, various tests, including safety tests on materials, may be performed before implementing the method or using each material.

[0078] The treatment method may be used for cancer treatment, prevention of recurrence, delay of cancer progression, cancer prevention, improvement of self-healing ability, prevention of viral infection, induction of immunity, combination therapy with chemotherapy, improvement of QOL, etc. The treatment method may also be used prophylactically for subjects in a healthy state or a pre-disease state.

[0079] While the above example shows the use of autologous immune cells, the present embodiment is not limited to autologous material cells, and also includes the use of allogeneic material cells. In this case, the method can be carried out in the same manner, except that step S92 in Figure 12 becomes "collecting a material cell population from a donor" or "preparing a material cell population from a donor."

[0080] According to the eighth embodiment, it is possible to provide a simple and safe CAR-T cell therapy. Here, one example of a treatment method using the method shown in the fourth embodiment is shown, but it is also possible to carry out the same treatment using any of the other embodiments described herein instead of the fourth embodiment, and to obtain the same effects.

[0081] Example 1 Example 1: Synthesis of the DNA sequence of the secreted antigen protein (sCD19-Fc) gene The antigen protein was CD19, the target of CD19 CAR-T. The DNA sequence (SEQ ID NO: 1) of the secreted CD19 antigen protein (sCD19-Fc) was designed and synthesized. The structure of the designed DNA sequence (SEQ ID NO: 1) is shown in Figure 5(b). The DNA sequence (SEQ ID NO: 4) encoding the amino acid sequence of the extracellular domain of human CD19 (SEQ ID NO: 3) was linked to the 5' end of the DNA sequence (SEQ ID NO: 6) encoding the amino acid sequence of the extracellular secretion signal of human interleukin-6 (IL-6) (SEQ ID NO: 5). The DNA sequence (SEQ ID NO: 10) encoding the amino acid sequence of the Fc region of immunoglobulin (SEQ ID NO: 9) was linked to the 3' end of the DNA sequence (SEQ ID NO: 4) via the DNA sequence (SEQ ID NO: 8) encoding the linker sequence (SEQ ID NO: 7). The "secretory signal sequence," "antibody protein partial sequence," and "IgG Fc-tag sequence" were linked in this order from the 5' end to the 3' end.

[0082] Example 2: Plasmid construction A plasmid was constructed by incorporating the sCD19-Fc DNA sequence obtained in Example 1 into pGEM-GL-pA as a template DNA for preparing RNA from the secreted CD19 antigen gene. pGEM-GL-pA was constructed by incorporating the human β-globin leader sequence and the poly(A) sequence of the pSP64 pA vector (Promega) into pGEM-4Z (Promega), a commercially available RNA synthesis template plasmid DNA containing a T7 promoter and poly(A) sequence. The sCD19-Fc DNA sequence obtained in Example 1 was inserted between the β-globin leader sequence and poly(A) sequence of pGEM-GL-pA, and this was used as the template DNA for RNA preparation.

[0083] Example 3: Synthesis of mRNA Using the template DNA obtained in Example 2, mRNA was synthesized using the mRNA synthesis kit T7 mScript Standard Production System (Cellscript) as follows. The RNA synthesis procedure followed the kit's protocol. The template DNA from Example 2 was digested with the restriction enzyme EcoRI and purified. An in vitro transcription reaction mixture containing 1.0 μg of template DNA was then prepared and incubated in a 37°C incubator for 2 hours. After the reaction was completed, the synthesized RNA was purified by ammonium acetate precipitation, and a cap structure and poly(A) structure were added to the 5' and 3' ends of the RNA, respectively, to produce mRNA. The synthesized RNA was incubated at 65°C for 10 minutes, then rapidly cooled on ice to disrupt the RNA secondary structure. 100 μL of the capping reaction mixture from the kit was then prepared. The mixture was then incubated in a 37°C incubator for 1 hour to add a cap structure to the 5' end of the RNA. Next, the poly(A) structure-adding reagent from the kit was added to the same solution, and the mixture was incubated in a 37°C incubator for 2 hours to add a poly(A) structure to the 3' end. After the reaction was completed, the mRNA in the reaction solution was purified by ammonium acetate precipitation. The RNA concentration was determined by measuring the absorbance (A260) using a spectrophotometer (BioSpec-mini, Shimadzu) and calculating OD1.0 = 40.0 μg / mL RNA.

[0084] Example 4: Preparation of sCD19-Fc mRNA-encapsulating liposomes and piggyBac-mRNA / CAR-pDNA co-encapsulating liposomes (liposomes for producing CAR-T using the two-step, two-liposome method) As liposomes to be used for CAR-T production using the 2-step-2 liposome method, sCD19-Fc mRNA-encapsulating liposomes (sCD19 liposomes) and liposomes for CAR-T production (PB / CAR liposomes) were prepared.

[0085] The sCD19-Fc mRNA solution was added to an ethanolic lipid solution (FFT20 / FFT10 / DOTAP / DOPE / cholesterol / PEG-DMG = 0.35 / 0.702 / 0.09 / 0.21 / 0.875 / 0.105 nmol / μL ratio), and then a 6.6-fold volume of 10 mM HEPES (pH 7.3) was gently added to prepare a liposome solution.

[0086] The liposomes for CAR-T production were designed to be suitable for CAR-T production using the moth transposase piggyBac. The PiggyBac encapsulated in the liposomes was mRNA of TS-HyPB (SEQ ID NO: 11), a highly activated version of wild-type piggyBac. TS-HyPB is a piggyBac constructed by linking, in this order from the 5' end, the nuclear localization signal of the human immunodeficiency virus TAT ​​protein, the nuclear localization signal of the simian virus 40 large T antigen protein, and a highly activated HyPB mutated into wild-type piggyBac, with codon usage optimized for humans. The liposomes for CAR-T production co-encapsulated piggyBac mRNA and the piggyBac genome integration donor DNA (CAR-pDNA), a CD19.CAR transposon-integrated plasmid DNA (pIRII-CAR.CD19(CD28)). Liposomes for CAR-T production were prepared by adding piggyBac mRNA solution and CAR-pDNA to an ethanol lipid solution (FFT20 / FFT10 / DOTAP / DOPE / cholesterol / PEG-DMG = 0.35 / 0.702 / 0.09 / 0.21 / 0.875 / 0.105 nmol / μL ratio), followed by gently adding 6.6 volumes of 10 mM HEPES (pH 7.3) to prepare the liposome solution.

[0087] After preparation, the liposome solution was concentrated by centrifugation at 14,000 × g using a centrifugal ultrafiltration tube (Amicon Ultra 0.5 mL, Ultracel-50K, Millipore). 10 mM HEPES (pH 7.3) was added, and the solution was simultaneously concentrated and buffer exchanged using the same centrifugal ultrafiltration tube. The amounts of RNA and DNA encapsulated in the liposomes were measured using the QuantiFluor™ RNA System (Promega) and Quant-iT™ PicoGreen™ dsDNA Assay Kits.

[0088] Example 5: Preparation of sCD19-Fc mRNA / piggyBac-mRNA / CAR-pDNA co-encapsulating liposomes (liposomes for producing CAR-T using the one-step-one liposome method) Liposomes containing sCD19-Fc mRNA, piggyBac-mRNA, and CAR-pDNA (sCD19 / PB / CAR liposomes) were prepared for use in CAR-T production using the one-step-one liposome method (see Example 4 for piggyBac-mRNA and CAR-pDNA).

[0089] The sCD19-Fc mRNA solution, piggyBac mRNA solution, and CAR-pDNA were added to an ethanol lipid solution (FFT20 / FFT10 / DOTAP / DOPE / cholesterol / PEG-DMG = 0.35 / 0.702 / 0.09 / 0.21 / 0.875 / 0.105 nmol / μL ratio), and then a 6.6-fold volume of 10 mM HEPES (pH 7.3) was gently added to prepare a liposome solution.

[0090] After preparation, the liposome solution was concentrated by centrifugation at 14,000 × g in a centrifugal ultrafiltration tube (Amicon Ultra 0.5 mL, Ultracel-50K, Millipore). 10 mM HEPES (pH 7.3) was then added, and the solution was subjected to buffer exchange and concentration simultaneously in the same centrifugal ultrafiltration tube. The amounts of RNA and DNA encapsulated in the liposomes were measured using the QuantiFluor™ RNA System (Promega) and Quant-iT™ PicoGreen™ dsDNA Assay Kits, as in Example 4.

[0091] Example 6: Preparation of CAR-T using sCD19-Fc by the two-liposome method and the one-liposome method CAR-T was produced using human peripheral blood mononuclear cells (PBMCs, purchased from Lonza). After activating PBMCs, they were cultured at a density of 1.0 x 10 in TexMACS medium supplemented with cytokines (IL-7, 10 ng / mL, IL-15, 5 ng / mL). 6 A cell suspension was prepared at 100 cells / mL. 200 μL of this suspension was seeded onto a 96-well round-bottom plate coated with anti-CD3 / CD28 antibodies and cultured at 37°C in a 5% CO2 atmosphere to activate the T cells. The anti-CD3 / CD28 antibody solution was diluted 100-fold with phosphate-buffered saline (PBS) and added at 150 μL per well to a 96-well round-bottom plate (Nunc, non-tissue culture treated). The plate was then coated by incubating at 37°C in a 5% CO2 atmosphere for at least 2 hours.

[0092] In the two-step-two-liposome method, 24 hours after T cell activation, PB / CAR liposomes (Example 4) were administered at 2.0 μg / well, and the cells were cultured at 37°C in a 5% CO atmosphere. In parallel with this, sCD19 liposomes were administered at 2.0 μg / well to wells separate from the PB / CAR liposomes, and the cells were cultured at 37°C in a 5% CO atmosphere.

[0093] Twenty-four hours after administration of sCD19 liposomes, the culture medium was collected from the wells, the cells were centrifuged, and the supernatant was collected as conditioned medium. From the wells administered with liposomes for CAR production, the cells were collected by pipetting, and the collected culture medium was transferred to a 48-well plate. The above-mentioned conditioned medium was added to the wells, and the culture was continued at 37°C in a 5% CO2 atmosphere.

[0094] In the one-step-one liposome method, 24 hours after T cell activation, sCD19 / PB / CAR liposome (Example 5) was administered at 2.0 μg / well, and the cells were cultured at 37°C in a 5% CO2 atmosphere. After that, in both the two-step and one-step methods, the medium was changed and the cells were expanded depending on the cell growth status, and the cells were cultured for two weeks to produce CAR-T.

[0095] Example 7: CAR-T detection Two weeks after liposome administration, PBMCs were removed from the incubator and the CAR-T production rate was examined using a fluorescence-activated cell sorter (FACS) on a BD Biosciences FACS system. PBMCs were collected from the culture plate, washed once with PBS, and then suspended in 50 μL of PBS. 2 μL of biotin-labeled anti-human IgG (H+L) antibody [Biotin F(ab')2 Fragment Goat Anti-Human IgG (H+L) antibody, The Jackson Laboratory] was added and incubated at 4°C for 15 minutes. After the incubation, the cells were washed once with PBS and suspended in 50 μL of PBS. 2 μL each of anti-human CD3 antibody (V450 Mouse Anti-Human CD3, Clone UCHT1, BD Biosciences) and APC-conjugated streptavidin (APC Streptavidin, BD Biosciences) were added and incubated at 4°C for 15 minutes. The cells were then washed with PBS, suspended in PBS, and analyzed by FACS. FACS detected red fluorescence (APC) from CAR-expressing cells and blue fluorescence (V450) from CD3-expressing T cells. CAR-T cells were defined as cells that were both red and blue fluorescent (CAR-positive and CD3-positive cells). The CAR-T production rate was calculated as the ratio of CAR-T cells to the total number of detected cells.

[0096] The scatter plot of the results is shown in Figure 13. The vertical axis represents the fluorescence intensity of APC (CAR) and the horizontal axis represents the fluorescence intensity of V450 (T cells). CAR-T cells are in the UR region of the scatter plot (CAR-positive and CD3-positive region). The CAR-T production rate is shown in the graph in Figure 13. The cell group in which CAR-T was produced using the 2-step 2 liposome method (cell group cultured in conditioned medium) had a production rate of 36.3%. The cell group in which CAR-T was produced using the 1-step 1 liposome method had a production rate of 39%. The cell group cultured without sCD19 had a production rate of 19.7%. These results demonstrate that the use of sCD19 improves the production rate of CAR-T.

[0097] Example 8: Evaluation of tumor cell killing ability by CAR-T In Example 6, CAR-T prepared by the 2-step-2 liposome method and the human precursor B-cell leukemia cell line NALM6 (purchased from ATCC) were mixed at a mixing ratio of 1:1, with a total cell count of 1.0 × 10 5 CAR-T and NALM6 cells were seeded into a 96-well plate at 1000 cells / well. After co-culture for 5 days at 37°C and 5% CO2, the cells were harvested and washed with PBS. Then, 2 μL each of anti-human CD3 antibody (V450 Mouse Anti-Human CD3, Clone UCHT1, BD Biosciences) and anti-human CD19 antibody (FITC Mouse Anti-Human CD19, BD Biosciences), suspended in 50 μL of PBS, was added and incubated for 30 minutes at 4°C. After incubation, the cells were washed with PBS, suspended in PBS, and analyzed by FACS. The blue fluorescence of CD3-expressing T cells (V450) and the green fluorescence of CD19-expressing NALM6 cells (FITC) were detected by FACS.

[0098] The scatter plots of the results are shown in Figures 14(a-1) and (a-2), and the tumor cell killing rate is shown in Figure 14(b). The vertical axis of the graphs (a-1) and (a-1) represents the FITC fluorescence intensity, which is an indicator of the presence of NALM6. The horizontal axis represents the V450 fluorescence intensity, which is an indicator of the presence of T cells. As shown in (a-2), in the coculture of PBMCs not expressing CAR with NALM6, approximately 93% of the cells were FITC-positive NALM6(UL; upper left fraction in the figure). In contrast, in the CAR-expressing group shown in (a-1), approximately 95% of the T cells were CAR-T-containing T cell groups (LR) (Figures 14(a-1) and (a-2)). NALM6(UL) was present in only approximately 3%, and the tumor cell killing rate was approximately 93% (Figure 14(b)). The CAR-T prepared in Example 6 was shown to kill CD19-expressing B cell tumor cells.

[0099] [Table 1-1] [Table 1-2] [Table 1-3]

[0100] Although several embodiments of the present invention have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These novel embodiments can be embodied in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, and are also included in the scope of the invention and its equivalents as defined in the claims. The inventions described in the original claims of this application are set forth below. [1] A method for producing chimeric antibody receptor (CAR) T cells, which involves producing CAR T cell populations without using feeder cells by secreting CAR antigen protein or its partial protein extracellularly from cells transfected with the CAR gene. [2] In a first culture system, contacting a first liposome encapsulating a nucleic acid encoding a CAR with a material cell comprising a T cell to introduce the nucleic acid encoding the CAR into the material cell; In a second culture system that is independent or not independent of the first culture system, the material cells and / or the material cells into which the nucleic acid encoding the CAR has been introduced are contacted with a second liposome containing a nucleic acid encoding a secretory antigen protein in which an extracellular secretion signal sequence has been added to a sequence encoding an antigen protein or a partial protein thereof that is the target of the CAR, thereby introducing the nucleic acid encoding the secretory antigen protein into the cytoplasm of the cells and secreting the antigen protein or a partial protein thereof from the cells; contacting the secreted antigen protein or a partial protein thereof with the CAR-T cells produced by the introduction of the nucleic acid encoding the CAR-T, thereby promoting the division and proliferation of the CAR-T cells; and A method for producing CAR-T cells, comprising producing the CAR-T cells by introducing a nucleic acid encoding the CAR-T and promoting the division and proliferation of the CAR-T cells to produce a desired population of the CAR-T cells. [3] A method for producing CAR-T cells according to [2], in which the production of CAR-T cells by introducing a nucleic acid encoding the CAR-T and the promotion of the division and proliferation of the CAR-T cells are carried out simultaneously or in stages. [4] The method for producing CAR-T cells according to [2], wherein the second culture system is independent of the first culture system, and the antigen protein or a partial protein thereof is brought into contact with the produced CAR-T cells by adding a medium from the second culture system to the first culture system. [5] The method for producing CAR-T cells according to [2], wherein the first culture system and the second culture system are independent culture systems. [6] The method for producing CAR-T cells described in [2], wherein the first culture system and the second culture system are independent culture systems, the first liposome further contains a nucleic acid encoding the secreted antigen protein, and the second liposome further contains a nucleic acid encoding the CAR, and therefore the liposome used in the method is of a single type overall. [7] A method for producing CAR-T cells according to [6], in which the introduction of a nucleic acid encoding the CAR into the cell and the introduction of a nucleic acid encoding the secreted antigen protein into the cell are carried out simultaneously for one cell using the same type of liposome. [8] The method for producing CAR-T cells described in [6], wherein the first culture system and the second culture system are independent culture systems, the first liposome produces the CAR-T cells by introducing a nucleic acid encoding the CAR-T into the material cells via the nucleic acid encoding the CAR contained therein, and the second liposome secretes the antigen protein or a partial protein thereof from cells present in the single culture system and that have taken up the second liposome via the nucleic acid encoding the secreted antigen protein contained therein. [9] A method for producing CAR-T cells according to any one of [2] to [8], wherein the target of the CAR is CD19, the antigen protein is a CD19 protein, the partial protein is an extracellular domain sequence of the CD19 protein, and the extracellular secretory signal sequence is an interleukin-6 signal sequence.

[10] The method for producing CAR-T cells according to [9], wherein the sequence encoding the antigen protein or a partial protein thereof is mRNA.

[11] A method for producing CAR-T cells according to [9], wherein the nucleic acid encoding the CAR is double-stranded DNA encoding the CAR gene sequence.

[12] The method for producing CAR-T cells according to any one of [2] to [8], wherein the first liposome contains a genome integration promoter together with the nucleic acid encoding the CAR, and no feeder cells are used.

[13] A method for producing CAR-T cells according to any one of [2] to [8], wherein the liposome further contains mRNA encoding the piggyBac gene in addition to the nucleic acid encoding the CAR.

[14] The method for producing CAR-T cells according to

[13] , wherein the mRNA encoding the piggyBac gene is mRNA encoding the piggyBac transposase.

[15] The lipid particles constituting the first and second liposomes are a first lipid compound (FFT10) represented by formula (I) and / or a second lipid compound (FFT20) represented by formula (II): [ka] A method for producing CAR-T cells according to any one of [2] to [8], comprising:

[16] The lipid particles constituting the first and second liposomes are a first lipid compound (FFT10) represented by formula (I) and / or a second lipid compound (FFT20) represented by formula (II): [ka] [9] A method for producing CAR-T cells, comprising:

[17] The lipid particles constituting the first and second liposomes are a first lipid compound (FFT10) represented by formula (I) and / or a second lipid compound (FFT20) represented by formula (II): [ka]

[12] A method for producing CAR-T cells, comprising:

[18] Liposomes of the following groups A and / or B, together with a substance that improves the storage stability of the liposomes: Liposome group A: a first liposome comprising a nucleic acid encoding a CAR; and a second liposome comprising a nucleic acid encoding a secretory antigen protein in which an extracellular secretion signal sequence has been added to a sequence encoding an antigen protein or a partial protein thereof that is the target of the CAR; Liposome group B: a nucleic acid encoding the CAR; A kit for producing CAR-T cells to be used in the method for producing CAR-T cells described in any one of [1] to [8], comprising: a liposome encapsulating a nucleic acid encoding a secretory antigen protein in which an extracellular secretion signal sequence has been added to a sequence encoding an antigen protein or a partial protein thereof that is the target of the CAR.

[19] The lipid particles constituting any of the liposomes contain a first lipid compound (FFT10) represented by formula (I) and / or a second lipid compound (FFT20) represented by formula (II). [ka] The cell production kit according to

[18] , comprising:

[20] A method for increasing the CAR-T cell content by co-culturing a cell group containing CAR-T cells with a cell group into which a secretory antigen protein gene has been introduced, in which an extracellular secretion signal sequence has been added to the sequence of the antigen protein or partial protein that is the target of the CAR, which is the target of the CAR, is added. This method promotes the proliferation of the CAR-T cells in the cell group without using feeder cells. [Explanation of symbols]

[0101] 50...liposome, 51...lipid particle, 52...nucleic acid encoding CAR, 53...nuclear transfer promoter, 54...nucleic acid encoding secretory antigen protein, 54a...antigen protein partial sequence, 54b secretory signal sequence, 54c...linker, 54d...IgG Fc-tag sequence, 55, 55A, 55B...container, 56...medium for producing CAR-T cells, 57...material cells, 58...CAR-T cells, 59...CAR, 60...antigen protein, 61...CAR-T cells, 66...medium, 70A...first liposome, 70B...second liposome, 77...any cell, 81a...first lipid, 81b...second lipid, 81c...third lipid

Claims

1. In a first culture system, contacting a first liposome containing a nucleic acid encoding a CAR with a source cell containing a T cell to introduce the nucleic acid encoding the CAR into the source cell; In a second culture system independent of the first culture system, contacting cells with a second liposome containing a nucleic acid encoding a secretory antigen protein in which an extracellular secretion signal sequence has been added to a sequence encoding an antigen protein or a partial protein thereof that is the target of the CAR, thereby introducing the nucleic acid encoding the secretory antigen protein into the cytoplasm of the cells and secreting the antigen protein or the partial protein from the cells; contacting the secreted antigen protein or the partial protein with CAR-T cells generated by introducing a nucleic acid encoding the CAR, thereby promoting the division and proliferation of the CAR-T cells; and A method for producing a population of CAR-T cells, comprising producing the CAR-T cells by introducing a nucleic acid encoding the CAR and promoting the division and proliferation of the CAR-T cells to produce a desired population of CAR-T cells.

2. The method according to claim 1, wherein the production of CAR-T cells by introducing a nucleic acid encoding the CAR and the promotion of the division and proliferation of the CAR-T cells are carried out simultaneously or in a stepwise manner.

3. The method of claim 1, wherein contacting the antigen protein or the partial protein with the produced CAR-T cells is carried out by adding the medium of the second culture system to the first culture system.

4. The method according to any one of claims 1 to 3, wherein the target of the CAR is CD19, the antigen protein is a CD19 protein, the partial protein is an extracellular domain sequence of the CD19 protein, and the extracellular secretory signal sequence is an interleukin-6 signal sequence.

5. The method according to claim 4, wherein the sequence encoding the antigen protein or the partial protein is mRNA.

6. The method of claim 4, wherein the nucleic acid encoding the CAR is a double-stranded DNA encoding a CAR gene sequence.

7. The method of any one of claims 1 to 3, wherein the first liposome contains a genome integration promoter together with the nucleic acid encoding the CAR, and no feeder cells are used.

8. The method of any one of claims 1 to 3, wherein the first liposome further comprises mRNA encoding the piggyBac gene together with the nucleic acid encoding the CAR.

9. The method according to claim 8, wherein the mRNA encoding the piggyBac gene is an mRNA encoding a piggyBac transposase.

10. The lipid particles constituting the first and second liposomes are a first lipid compound (FFT10) represented by formula (I) and / or a second lipid compound (FFT20) represented by formula (II): 【Chemistry 1】 The method according to any one of claims 1 to 3, comprising:

11. The lipid particles constituting the first and second liposomes are a first lipid compound (FFT10) represented by formula (I) and / or a second lipid compound (FFT20) represented by formula (II): 【Chemistry 2】 5. The method of claim 4, comprising:

12. The lipid particles constituting the first and second liposomes are a first lipid compound (FFT10) represented by formula (I) and / or a second lipid compound (FFT20) represented by formula (II): 【Transformation 3】 8. The method of claim 7, comprising:

13. together with substances that improve the storage stability of liposomes. a first liposome comprising a nucleic acid encoding the CAR; and A second liposome comprising a nucleic acid encoding the secretory antigen protein in which an extracellular secretion signal sequence has been added to a sequence encoding the antigen protein or partial protein that is the target of the CAR. A kit for producing CAR-T cells for use in the method according to any one of claims 1 to 3, comprising:

14. The lipid particles constituting the first liposome and the second liposome also contain a first lipid compound (FFT10) represented by formula (I) and / or a second lipid compound (FFT20) represented by formula (II). 【Chemistry 4】 The cell production kit according to claim 13, comprising:

15. A method for increasing the CAR-T cell content by co-culturing a cell group containing CAR-T cells with a cell group into which a secretory antigen protein gene has been introduced, in which an extracellular secretion signal sequence has been added to the sequence of the antigen protein or partial protein that is the target of the CAR, which is the target of the CAR, is added. This method promotes the proliferation of the CAR-T cells in the cell group without using feeder cells.

16. A method for producing a pharmaceutical composition containing the CAR-T cells produced using the method of claim 1 or 15.

17. 17. A pharmaceutical composition prepared using the method of claim 16.

Images