JP2024015870A - Method for making car-t cell, kit for making car-t cell, and method for improving car-t cell content - Google Patents

Abstract

To provide a method for making CAR-T cells without using feeder cells, a cell making kit that is used in the method, and a method for improving a CAR-T cell content.SOLUTION: A method for making CAR-T cells according to one embodiment of the present invention includes causing chimera antibody receptor (CAR) gene-transduced cells to secrete CAR antigen proteins or their partial proteins extracellularly, thereby making a cell group with a high CAR-T cell content without using feeder cells.SELECTED DRAWING: Figure 1

Description

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 increasing CAR-T cell content.

CAR-T cells are used for cancer gene therapy. CAR-T cells are T cells that have a CAR chimeric antibody gene integrated into their genome. It recognizes and kills cancer cells by recognizing the antigen specifically expressed by the target cancer cells.

Usually, to obtain a cell population with a high content of CAR-T cells, cells are cultured on feeder cells lined in a culture system. CAR-T cells proliferate stably and efficiently by the action of secreted substances such as antigen proteins released from feeder cells (Non-Patent Document 1). However, such a method requires complicated operations and has low versatility. However, there is no method for producing CAR-T cells with high efficiency without using feeder cells.

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”

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 used in the method, and a method for increasing the content of CAR-T cells. It is to provide.

The method for producing CAR-T cells according to the embodiment is to secrete the CAR antigen protein or its partial protein from the cells during production of the CAR-T cells, thereby producing the desired CAR-T cells without using feeder cells. Prepare to form a group.

FIG. 2 is a schematic diagram showing the first embodiment. FIG. 3 is a schematic diagram showing a second embodiment. FIG. 7 is a schematic diagram showing a third embodiment. FIG. 7 is a schematic diagram showing a third embodiment. FIG. 4 is a schematic diagram showing a fourth embodiment. FIG. 4 is a schematic diagram showing a fourth embodiment. FIG. 7 is a schematic diagram showing a fifth embodiment. FIG. 7 is a schematic diagram showing a fifth embodiment. FIG. 7 is a schematic diagram showing a sixth embodiment. FIG. 7 is a schematic diagram showing a sixth embodiment. FIG. 7 is a schematic diagram showing a seventh embodiment. Schematic diagram showing the eighth embodiment Scatter diagram showing the detection results of Example 7. Graph showing the experimental results of Example 8.

Embodiments will be described below with reference to the accompanying drawings. In addition, in each embodiment, the same code|symbol may be attached|subjected to the substantially same component part, and some descriptions may be abbreviate|omitted. The drawings are schematic, and the relationship between the thickness of each part and the planar dimension, the ratio of the thickness of each part, etc. may differ from the actual one.

(First embodiment)
In the first embodiment, CAR antigen protein or its partial protein is secreted extracellularly from CAR gene-transfected cells to promote the proliferation of CAR-T cells, thereby eliminating the need to use feeder cells and containing CAR-T cells. This is a method for highly efficiently producing a cell group with a high cell density (S11 in Figure 1).

CAR gene-introduced cells are formed by integrating the CAR gene into material cells using known introduction means. The CAR gene may be introduced by, for example, liposome method, lipofection method, or electroporation method, but liposome method is preferable. For example, in the case of the liposome method, a nucleic acid encoding any CAR (chimeric antibody receptor) known per se is encapsulated in a liposome and delivered to the cytoplasm of the material cell, and from there to the nucleus of the material cell. It is structured so that it is integrated into the cell's genome after being programmed. By being integrated into the genome of the cell, the CAR is expressed on the cell membrane surface of the material cell.

The nucleic acid encoding the CAR is configured to be internalized into the nucleus and to express the CAR on the surface of the cells into which the material has been introduced. The nucleic acid encoding CAR is, for example, CAR-DNA, ie it is preferably double-stranded DNA encoding the CAR gene sequence. CAR-DNA may be in any form as long as it is incorporated into the nucleus and inserted into the genome of the cell to express CAR.

A nucleic acid encoding a CAR may be used in a form included in a gene set for producing CAR cells. For example, a gene set for producing CAR cells contains a nucleic acid encoding CAR, such as CAR-DNA, and may further contain a nucleic acid introduction promoter as an optional component. The nucleic acid introduction promoter includes a genome integration promoter such as a gene that promotes integration (insertion) of CAR-DNA into the genome. The genome integration promoter can be, for example, piggyBac gene such as PB-mRNA. Such enzyme genes are known as transposase (DNA transferase) genes. By introducing the CAR gene and transposase gene into T cells, which are material cells, CAR-T (cells with the CAR gene integrated into the T cell genome) can be stably produced. Typical transposases include piggyBac, which is derived from moths, and Sleeping Beauty, which is derived from fish. Any of these may be used, but is not limited thereto. Furthermore, it is also possible to use devices that exhibit other mechanisms for promoting genome integration. For example, a nucleic acid encoding a CAR may be incorporated into plasmid DNA and encapsulated in liposomes. Furthermore, the PB gene may be integrated into a plasmid and enclosed as a gene set for producing CAR cells. The PB gene may be DNA or mRNA, but mRNA is preferable.

Furthermore, the genome integration promoter may be a protein encoded by a gene that promotes integration (insertion) of CAR-DNA into the genome, such as piggyBac. Even when the genome integration promoter is a protein, it may be delivered to the cytoplasm of the material cell together with the nucleic acid encoding the CAR. In other words, the genome integration promoter may be a gene or protein that promotes genome integration (or insertion). Furthermore, for example, the genome integration promoter may be encapsulated in a liposome at the same time as the CAR gene and delivered to the material cells.

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 using T cells as material cells, a material cell group containing material cells in peripheral blood mononuclear cells (PBMCs) or lymphocyte fractions may be used. May be used as a minute. When CAR-T cells are used for treatment or diagnosis, they may be PBMCs collected from a subject for treatment, prevention, and/or diagnosis. Preparation of PBMC from peripheral blood may be performed by any method known per se, such as density gradient centrifugation such as ultracentrifugation using Ficoll.

The CAR antigen protein or its partial protein secreted from the CAR gene-introduced cells as described above is an antigen that is a target of the CAR. Secreting the CAR antigen protein or a partial protein thereof from the CAR gene-introduced cells may be performed before and/or after the introduction of the CAR-encoding nucleic acid into the material cells, and/or at the same time as the introduction. . Further, it may be performed at least simultaneously with and/or after the introduction of the CAR-encoding nucleic acid into the material cells.

In order to secrete the CAR antigen protein or a partial protein thereof from the CAR gene-introduced cells, secretion is performed on the material cells and/or the material cells and/or CAR-T cells into which the CAR-encoding nucleic acid has been introduced. This can be achieved by introducing a gene for a type CAR antigen protein or a partial protein thereof. Introduction of a gene for a CAR antigen protein or a partial protein thereof can be performed by contacting the cell with a liposome containing a nucleic acid encoding a secreted antigen protein. A nucleic acid encoding a secreted antigen protein may include a sequence encoding a CAR antigen protein or a partial protein thereof, and an extracellular secretion signal sequence. Alternatively, the nucleic acid encoding the secreted antigenic protein may be mRNA. A nucleic acid encoding a secreted antigen protein taken into the cytoplasm causes the antigen protein or a partial protein thereof to be secreted outside the cell. The division and proliferation of CAR-T cells is promoted by contact with the CAR antigen protein. The method for producing CAR-T cells according to the embodiment includes the production of CAR-T cells by introducing a nucleic acid into the genome, and the division and proliferation of CAR-T cells by contacting the produced CAR-T cells with a CAR antigen. By performing both of these steps, CAR-T cells are produced comprehensively as the desired "cell group with a high CAR-T cell content."

The target CAR-T cell group is a cell group containing CAR-T cells in a predetermined amount or number of cells, or a CAR group containing a predetermined ratio of other cells contained in the material cell group such as PBMC. -Can be a T cell population. For example, when a therapeutic CAR-T cell group is produced by the method according to the embodiment, the target CAR-T cell group may be the number of CAR-T cells necessary for the treatment. Here, the "target CAR-T cell group" may be, for example, a cell group containing CAR-T cells at 10% or more, more preferably at least 20%, for example from 20% to 80%. Further, such a CAR-T cell group can also be interpreted as a "cell group with a high CAR-T cell content."

As described above, the method of the first embodiment is characterized in that it does not use feeder cells. According to the method of the embodiment, CAR-T cells can be produced with high efficiency without using feeder cells. Feeder cells include cancer cells, typically supplied as specific established cell lines. When used, feeder cells are attached to the bottom of a culture container and co-cultured with target cells. By doing so, it is possible to create an environment more similar to that of a living body, and to stably culture and proliferate the target cells to be co-cultured. Therefore, feeders are widely used. In particular, feeder cells are considered essential for stably culturing primary human cells. Traditionally, it has also been required to generate cell populations with a high CAR-T cell content with high enough efficiency to be used for treatment, and for this purpose, established cell lines have been carefully passed down. ing. When CAR-T cells are produced using feeder cells, the feeder cells must be completely isolated prior to use of the CAR-T cells. Furthermore, before producing CAR-T cells, it is necessary to eliminate the proliferative ability of feeder cells by irradiating them with gamma rays. These complicated operations and the necessity of feeder cells make it difficult to generalize the production of CAR-T cells. According to the embodiment, CAR-T cells can be produced stably and with high efficiency without using such feeder cells. By secreting the CAR antigen protein or its partial protein from the CAR gene-introduced cells, the antigen protein or its partial protein binds to CAR, and the proliferation of CAR-T cells is promoted. Since it does not require the use of feeder cells, it is easy to operate and can be easily introduced by many medical institutions and research institutions. Therefore, it is expected to have remarkable effects in cancer treatment and prevention as well as basic research. be done.

(Second embodiment)
According to the second embodiment, a method for increasing CAR-T cell content during production of CAR-T cells is provided. This method involves introducing a secreted antigen protein gene in which an extracellular secretion signal sequence is added to the antigen protein, which is the target of CAR, or a partial sequence thereof (i.e., partial protein) to generate CAR-T cells. By co-cultivating a cell group containing CAR-T cells with a group of cells containing CAR-T cells, the proliferation of CAR-T cells is promoted without using feeder cells, thereby increasing the content of CAR cells in the cell group ( S21 in Figure 2).

The generation of CAR-T cells includes two situations. The first situation refers to a situation in which CAR-T cells are newly produced or formed from material cells by transfection into CAR-T cells by introducing a CAR gene into material cells. The second situation refers to a situation where activation causes division and proliferation, such as contact between multiple CAR-T cells or high density, and thereby increases the number of CAR-T cells. The second embodiment may be understood as a method for more efficiently increasing the number of CAR-T cells during the production of CAR-T cells. In other words, when producing CAR-T cells, cells are introduced with a secreted antigen protein gene in which an extracellular secretion signal sequence is added to the antigen protein that is the target of the CAR, or a partial sequence thereof (i.e., partial protein). This method promotes the proliferation of the CAR-T cells by co-culturing the cell group containing the CAR-T cells and the cell group containing the CAR-T cells, thereby increasing the CAR cell content of the cell group.

When the antigen protein or its partial protein released from the cell group into which the secreted antigen protein gene has been introduced binds to the CAR expressed on the surface of the CAR-T cell, the CAR-T cell is activated and the CAR -T cells divide and proliferate, effectively increasing their number.

The cell group into which the secreted antigen protein gene should be introduced may be the material cells of CAR-T cells, mononuclear cells other than the material cells included in the material cell group, or the material cell group (PBMC) itself. . Alternatively, it is preferable to select any other cell that is not a feeder cell and has no biotoxicity or toxicity, or a cell with relatively low biotoxicity or toxicity. "Cell group containing CR-T cells" to be co-cultured refers to CAR-T cells created by introducing the CAR gene and other cells contained in PBMCs when PBMCs are used as the material cell group. A group of cells containing The cell group into which the secreted antigen protein gene has been introduced includes CAR-T cells, T cells, and/or other mononuclear cells into which the secreted antigen protein gene has been introduced. Co-culture refers to a situation in which these cells and newly formed CAR-T cells into which the secreted antigen protein gene has not been introduced are cultured together in one culture system. The cell group to be co-cultured with the CAR-T cell group is non-toxic or non-toxic to CAR-T cells, or has relatively low toxicity or toxicity, and/or can support T-cell proliferation. cells, such as aK562 cells, a leukemia cell line, SJCRH30 cells, a rhabdomyosarcoma-derived cell line, and/or cells that express a high amount of protein from a secreted antigen protein gene, such as HEK293 cells, a human embryonic kidney cell. It will be done.

According to embodiments, the structural feature of not requiring feeder cells is also a significant advantage. Furthermore, according to the method for producing CAR-T cells and the method for increasing the content of CAR-T cells according to the embodiment, CAR-T cells can be produced with higher efficiency than when CAR-T cells are produced using feeder cells. It is possible to generate CAR-T cells.

(Third embodiment)
According to a third embodiment, a method for producing CAR-T cells without using feeder cells is provided. A third embodiment will be described using FIG. 3. This method comprises three stages, S31, S32 and S33. S31 includes two phases (a) and (b). In (a) of S31, in the first culture system, the CAR gene and the genome integration promoter are introduced into the material cells to obtain CAR-T cells. In (b), in a second culture system that is independent or non-independent of the first culture system, the antigen protein or a partial protein thereof that is the target of the CAR is secreted from the material-derived cells. (a) and (b) may be performed simultaneously or in stages. Furthermore, although the details will be described later, the relationship between the first culture system and the second culture system may be mutually independent or may be mutually non-independent. That is, the first culture system and the second culture system may be formed in separate containers, or may partially or completely overlap in one reaction container. That is, the "first culture system" may be equal to the "second culture system." In S32, the secreted antigen protein or a part thereof is brought into contact with CAR-T cells created by introducing the CAR gene of S31(a) and a genome integration promoter, and the CAR-T cells divide and proliferate. promote. In S33, a cell group with a high target CAR-T cell content is obtained by generating CAR-T cells through gene transfer and promoting the division and proliferation of CAR-T cells.

Creation of CAR-T cells by gene transfer involves the use of a first liposome containing a nucleic acid encoding CAR and an enzyme gene or protein (genome integration promoter) that integrates the nucleic acid into the genome of the cell, and material cells containing T cells. This is carried out by bringing the nucleic acid into contact with the cell material and introducing the nucleic acid into the material cell. Secreting an antigen protein or a partial protein thereof is performed by introducing a nucleic acid encoding the secreted antigen protein into a material cell and/or a material cell into which a nucleic acid encoding the CAR has been introduced. This introduction proceeds by contacting the cell with the second liposome. The second liposome includes a nucleic acid encoding a secreted antigen protein, which is obtained by adding an extracellular secretion signal sequence to a sequence encoding the antigen protein or a partial protein thereof. Generation of CAR-T cells and promotion of division and proliferation of CAR-T cells by introducing a CAR gene and a genome integration promoter are performed simultaneously or in stages.

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

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 brought into contact with a material cell containing T cells to obtain a nucleic acid encoding the CAR. is introduced into the material cells, and CAR-T cells are generated from the material cells (S41).

In the second stage, in a second culture system that is independent or non-independent of the first culture system, the CAR is introduced into the material cells and/or the material cells into which the nucleic acid encoding the CAR is introduced. A second liposome containing a nucleic acid encoding a secreted antigen protein in which an extracellular secretion signal sequence is added to a sequence encoding an antigen protein or a partial protein thereof that is a target of is introduced into the cells, and the antigen protein or a partial protein thereof is secreted from the cells (S42).

In the third stage, the secreted antigen protein or a partial protein thereof is brought into contact with the CAR-T cells formed by introducing the CAR gene and the genome integration promoter, and the CAR-T cells undergo division and proliferation. (S43).

In the fourth stage, CAR-T cells are generated by introducing the CAR gene and genome integration promoter, and the division and proliferation is promoted to create a cell group with a high content of the desired CAR-T cells. Create (S44).

According to the third embodiment, it is possible to stably and highly efficiently produce a cell group with a high CAR-T cell content without using feeder cells. Since there is no need to use feeder cells, it is easy to operate and can be easily introduced into many medical institutions and research institutions, and is therefore predicted to have a significant effect in cancer treatment and research.

(Fourth embodiment)
An example of a method for producing CAR-T cells without using feeder cells according to the fourth embodiment will be explained using FIGS. 5 and 6. This method is extremely efficient, as each liposome contains materials for all types of genes to be introduced, and the liposome addition and gene transfer operations are completed in one step. It is a one-step method that is easy to operate.

As shown in FIG. 5(a), the liposome 50 used includes a gene set for producing CAR cells and a secreted antigen protein gene. In the example of FIG. 5, specifically, the liposome 50 contains within the lipid particle 51 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. It is contained within. FIG. 5(b) is a schematic enlarged view of an example of nucleic acid 54 encoding a secreted antigen protein. Nucleic acid 54 encoding a secreted antigenic protein includes an antigenic protein partial sequence 54a, a secretion signal sequence 54b attached to one end thereof, and a modified sequence 54d connected to the other end via a linker 54c, for example, IgG Fc-tag sequence. Antigen proteins produced within cells are released outside the cells by secretion signals. The IgG Fc-tag is an arbitrary sequence and contributes to stabilization by imparting water affinity to proteins released outside the cell. One example of a CAR target is CD19. Thus, one example of an antigenic protein is CD19. The antigenic protein may be a portion of CD19, which may be long enough to be interpreted as a peptide. For example, one example of a partial protein of CD19 protein can be the extracellular domain sequence of CD19 protein. The extracellular secretion signal sequence can be, but is not limited to, the interleukin 6 signal sequence.

This liposome 50 is added to the CAR-T cell production medium 56 in the container 55. In the container 55, material cells 57 are prepared in advance in a medium 56 for producing CAR-T cells ((c) in FIG. 5). The material cells 57 may be T cells in PBMC as described above. PBMC can be prepared from peripheral blood previously collected from the patient being treated.

Isolation of peripheral blood mononuclear cells (PBMC) may be performed, for example, as follows. Mononuclear cells are separated from peripheral blood collected from humans by Ficoll density gradient centrifugation to obtain peripheral blood mononuclear cells (PBMCs). Specifically, PBMCs are centrifuged by density gradient centrifugation using, for example, Ficoll reagent for mononuclear cell separation (Ficoll-Paque Premium, Cytiva, etc.).

Cultivation of T cells contained in PBMC can be performed, for example, as follows. Cultivate T cells contained in the separated PBMC. T cells are cultured using a commercially available T cell culture medium (TexMACS, Miltenyi biotec, AlyS705, Cell Science Institute, etc.). The medium contains 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 (such as interleukin 7 and interleukin 15), as necessary. ) may be added.

The liposome 50 that has come into contact with the material cell 57 releases its contents in the cytoplasm after being taken into the material cell 57 by intracellular endocytosis. The CAR-encoding nucleic acid 52 from the liposome 50 taken up by the T cells, which are the material cells 57, enters the nucleus and is integrated into the genome, so that the T cells, which are the material cells 57, become CAR-T cells. It becomes 58. CAR59 is expressed on the cell membrane. On the other hand, the nucleic acid 54 encoding a secreted antigenic protein (if the nucleic acid is DNA, it is released from the liposome into the cytoplasm and then transferred to the cell nucleus, where it is transcribed into mRNA; After being released into the cytoplasm, it is translated into an antigen protein 60 in the cytoplasm and released into the medium 56 ((d) in FIG. 5). When antigen protein 60 binds to CAR 59, CAR-T cells 58 are activated and efficiently divide and proliferate. Figure 5(e) schematically shows the appearance of CAR-T cells 58 when expanded culture is performed for the purpose of proliferating the cells while preserving their properties. Cell division of the CAR-T cells stimulated and activated by the antigen protein is promoted, and additional CAR-T cells 61 are formed. In about 14 days after adding 50 liposomes to 56 PBMCs in a medium, CAR-T cells in the number necessary for treatment, for example, are produced.

A scheme of an example of a method for producing CAR-T cells without using feeder cells by such a one-step method is shown in FIG. First, one type of desired liposome is prepared (S51). In the case of the above example, a liposome 50 was prepared in which a lipid particle 51 contained a nucleic acid 52 encoding a CAR, PB-mRNA 53, and a nucleic acid 54 encoding a secreted antigen protein. Next, the liposomes are brought into the culture system containing the material cells (S52). Here, PBMC were prepared in CAR-T cell production medium 56, and liposomes were added to the medium. Next, by culturing under appropriate conditions, the desired CAR-T cell group is obtained without using feeder cells (S53). These steps accomplish one example of a method for producing CAR-T cells.

The differences in the CAR-T amplification effect obtained when using antigen-presenting feeder cells with antigens presented on the surface of the feeder cells and the method according to the embodiment are as follows. In CAR-T amplification, it is important to efficiently bring the antigen into contact with CAR-T (activate it). In the case of feeder cells, the antigen is presented on the cell 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. In the case of feeder cells, since the cells are attached to the bottom of the culture vessel, the contact surface with T cells is limited to one side of the T cells. On the other hand, since secreted antigenic peptides are dissolved in the culture medium in the incubator, the number of antigens that can be presented to T cells depends on the volume of the culture medium. Since the volume is (area x height), the secreted antigen peptide can present more antigen than the feeder cells by the (height) of the culture vessel. Furthermore, since the secreted antigenic peptide is dissolved in the culture medium, the contact surface with T cells is the entire surface of the cell, and the efficiency of contact with the cells is extremely high. Due to these characteristics, the method according to the embodiment has a higher efficiency of contacting CAR-T (activation efficiency) and a higher effect of amplifying CAR-T than antigen-presenting feeder cells.

According to the fourth embodiment, CAR-T cells can be produced stably and with high efficiency without using feeder cells. Furthermore, since it is a one-step method, it is simple and the possibility of contamination can be reduced as much as possible.

(Fifth embodiment)
The one-step method for producing CAR-T cells without using feeder cells shown in the fourth embodiment may be performed 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 for using two types of liposomes. Two types of liposomes will be explained using FIGS. 7 and 8.

The first liposome 70A used in this method includes a nucleic acid 52 encoding a CAR and a genome integration promoter 53 in a lipid particle 51. The second liposome 70B includes a nucleic acid 54 encoding a secreted antigenic protein (FIG. 7(a)). These two types of desired liposomes are prepared. Two types of liposomes are added simultaneously to a container 55 containing a medium 56 and material cells 57 added thereto (FIG. 7(b)). By culturing this under appropriate conditions, the desired CAR-T cell group is obtained (Figure 7(c)). Although not shown in the figure, continued expansion culture in this case also promotes the division of CAR-T cells as shown in Figure 5(e). In about 14 days after adding 50 liposomes to 56 PBMCs in a medium, CAR-T cells in the number necessary for treatment, for example, are produced.

FIG. 8 shows a scheme of an example of a method for producing CAR-T cells without using feeder cells by such a one-step method. First, two types of desired liposomes are prepared (S61). In the case of the above example, a liposome 70A containing a nucleic acid 52 encoding CAR and PB-mRNA 53 in a lipid particle 51, and a liposome 70B containing a nucleic acid 54 encoding a secreted antigen protein were prepared. Next, these liposomes are brought into a culture system containing material cells (S62). Here, PBMC were prepared in CAR-T cell production medium 56, and liposomes were added to the medium. Next, by culturing under appropriate conditions, the desired CAR-T cell group is obtained without using feeder cells (S63). These steps accomplish one example of a method for producing CAR-T cells.

According to the fifth embodiment, it is possible to stably and highly efficiently produce a cell group with a high CAR-T cell content without using feeder cells. Furthermore, since it is a one-step method, it is simple and the possibility of contamination can be reduced as much as possible. Furthermore, since the nucleic acid 52 encoding CAR and the nucleic acid 54 encoding a secreted antigen protein are separately encapsulated in the first liposome 70A and the second liposome 70B, diversification is possible by combining them. It has excellent versatility and is advantageous when used as a commercial product or when conducting various research.

(Sixth embodiment)
By using the two types of liposomes shown in the fifth embodiment, a two-step method for producing CAR-T cells can be achieved. This will be explained using FIG. 9. In Figure 9, each process and culture of the A series (a-1) to (a-2) as the first step and the B series (b-1) to (b-4) as the second step are shown. FIG. 10 schematically shows the state of the system and shows the overall flow as a scheme.

Two types of liposomes as described in the fifth embodiment are prepared (FIG. 9(a-1), (b-1), FIG. 10 (S71)). In series A, the first liposome 70A is added to the medium 56 of the container 55A, which is the first culture system. The first liposome is taken into contact with the material cells 57 in the medium 56A, and as a result, CAR-T cells are produced (FIG. 9(a-2), (S72) and (S73) in FIG. 10). . On the other hand, in the B series, the second liposome 70B is added to the medium 66 contained in the container 55B, which is a second culture system independent of the first culture system. The medium 66 contains arbitrary cells 77 (FIG. 9(b-2), (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 or after adding the first liposome 70A to the container 55A, which is the first culture system. They may be present or may be present at the same time. It 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), FIG. 10(S73)) . Next, the medium 66 is collected and added to the container 55A which is the first culture system (FIG. 9(a-2), (S74) in FIG. 10). FIG. 9(a-2) shows the state of the first culture system when the addition of the first liposome and the addition of the medium 66 containing the antigen protein 60 obtained from the B series were performed simultaneously.

Here, an example is shown in which the antigen protein 60 is brought in by collecting the medium 66 and adding it to the first culture system. By constructing the antigen protein to be released so that it is water-soluble, the efficiency of contact between the antigen protein and CAR-T cells increases, making it possible to proceed with amplification with higher efficiency. The arbitrary cell 77 may be a material cell or material cell group used in the first culture system, or any known cell that can be used to more advantageously carry out other antigen proteins. Good too. Furthermore, cells known to be harmless to living organisms are preferred. The medium 66 may be selected depending on the cell type of the arbitrary cells 77 used.

According to the sixth embodiment, it is possible to produce a desired amount of antigen protein at a desired timing. This may be done in conjunction with the production of CAR-T cells by CAR-T gene introduction, or may be carried out prior to the addition of the first liposome. Furthermore, since the cell type can be arbitrarily selected, the B lineage may be performed in combination with the one-step method in cases where the antigen protein production ability of the material cells and material cell groups is low, or in preparation for such a case.

(Seventh embodiment)
According to the seventh embodiment, a cell production kit for use in a method for producing CAR-T cells without using feeder cells is provided. The cell production kit may include the following liposome group A and/or B together with a substance that improves the complementation stability of liposomes;
・Liposome group A:
A first liposome containing a nucleic acid encoding a chimeric antibody receptor (CAR), and encoding a secreted antigen protein in which an extracellular secretion signal sequence is added to a sequence encoding an antigen protein or a partial protein thereof that is a target of the CAR. a second liposome comprising a nucleic acid;
・Liposome group B:
A liposome containing both a nucleic acid encoding the CAR and a nucleic acid encoding a secreted antigenic protein obtained by adding an extracellular secretion signal sequence to a sequence encoding an antigenic protein or a partial protein thereof that is a target of the CAR.

An example of the first liposome included in the liposome group A is the first liposome 70A shown in FIG. 7(a), and an example of the second liposome is the second liposome 70B shown in FIG. 7(a). be. The liposome group A is a group containing a plurality of first liposomes and a plurality of second liposomes.

An example of a liposome included in the liposome group B is the liposome shown in FIG. 5(a). This is a group of liposomes that contain elements that enable expression of both antigen protein genes in one liposome. Furthermore, the liposome may contain further elements that make it possible to introduce and express the CAR gene.

The liposomes 70A and 70B will be further explained with reference to FIG. The lipid particles 51 constituting the liposomes 70A and 70B may be composed of, for example, a lipid membrane formed by non-covalently arranging a plurality of lipid molecules that are the material thereof. The lipid particle 51 is hollow 82, and 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 enclosed in the hollow portion 82. The lipid particles 51 may be of any type as long as they have the property of being taken into cells and decomposed, and examples of their constituent components include those containing at least the first lipid 81a and the second lipid 81b. 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 shown in formula (I) is FFT10, and the second lipid compound shown in formula (II) is FFT20.

In addition to the liposome, the cell production kit may further contain a substance that improves the storage stability of the liposome. 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, etc.; sodium acetate; , pharmaceutically acceptable agents such as sodium lactate, sodium chloride, potassium chloride, calcium chloride, which bring the composition closer to physiological conditions; lipophilic agents such as α-tocopherol, which inhibit free radical damage; free radical quenchers; lipid protectants such as water-soluble chelators such as ferrioxamine to inhibit peroxidative damage to lipids and improve storage stability;

The cell production kit may be provided as a liquid composition containing liposomes in a suitable carrier. Examples of the carrier include water, a saline solution such as physiological saline, an aqueous glycine solution, or a buffer solution. Alternatively, the liposome may be included in a cell production kit as a dry powder composition. Powdered compositions are ready for use by the user by adding a suitable liquid, such as the carrier described above. Further, any such liposomes may be contained in a suitable container, sachet, or the like. It is also preferred that such compositions and liposomes be sterilized by known methods.

The cell production kit further includes sterile containers such as culture dishes, petri dishes, and tubes, culture media, pH adjustment reagents, antibiotics, growth factors, vitamins, salts, cleaning compositions, sterile ultrapure water, and PBMC adjustment reagents. , a lipid composition for liposome preparation, various instructions, and other desired elements may be further provided.

The lipid particles 51 may contain additional lipids in addition to the first lipid 81a and the second lipid 81b. In the composition of the lipid molecule material constituting the lipid particle 51, the fraction consisting of the first lipid 81a and the second lipid 81b is hereinafter referred to as a "first fraction." Further, a fraction consisting of lipid molecule materials other than the first lipid 81a and the second lipid 81b will be hereinafter referred to as a "second fraction". The lipids contained in the second fraction are also collectively referred to as "third lipid 81c" hereinafter.

The terms "first fraction" and "second fraction" refer to the composition of the constituent components of the lipid particles 51, and do not indicate 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 in one unit in the lipid particles 51, and the lipid contained in the first fraction and the lipid contained in the second fraction are They can exist mixed together. The blending ratio of the first fraction to the entire lipid material constituting the lipid particles 51 is preferably 30% or more and less than 50% (molar ratio).

The blending ratio of the second lipid 81b in the first fraction is preferably 40% or more. In that case, the T-cell tumor cell specificity due to nucleic acid introduction and the efficiency of introduction of substances contained in liposomes can be improved. Further, it is more preferable that the blending ratio of the second lipid 81b in the first fraction is 50% or more, since the amount of the substance to be introduced to be included in the liposome in the living body, that is, in vivo, can be improved. . It is further preferable that the blending ratio of the second lipid 81b is 60% or more. The upper limit of the blending ratio of the second lipid 81b may be 80%, 90%, 95%, 96%, 97%, 98% or 99%.

Depending on the blending ratio of the first lipid 81a and the second lipid 81b in the first fraction, the particle diameter and cell permeability of the lipid particles 51 may change. For example, the particle size of the lipid particles 51 may become larger as the second lipid 81b increases. The average particle diameter 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, for in vivo use, about 70 nm to about 100 nm may be preferred.

Although the type of the third lipid 81c contained in the second fraction of the lipid particles 51 is not limited, for example, the second fraction contains a base lipid. As the base lipid, for example, a lipid that is a main component of biological membranes can be used. The base lipid is a phospholipid or sphingolipid, such as diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin or cerebroside, or a combination thereof.

For example, as a base lipid,
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-dimethylammonium propane (DODAP),
1,2-dimyristoyl-3-dimethylammoniumpropane (14:0 DAP),
1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP),
1,2-distearoyl-3-dimethylammonium propane (18:0 DAP),
N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane (DOBAQ),
1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP),
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), or cholesterol,
Alternatively, it is preferable to use a combination of any of these.

It is particularly preferable to use a cationic lipid or a neutral lipid as the base lipid, and the acid dissociation constant of the lipid particles 51 can be adjusted depending on the content thereof. It is preferable to use DOTAP as the cationic lipid, and it is preferable to use DOPE as the neutral lipid.

It is also preferred that the second fraction contains a lipid that prevents aggregation of the lipid particles 51. For example, lipids that prevent aggregation may include PEG-modified lipids such as polyethylene glycol (PEG), dimyristoylglycerol (DMG-PEG), polyamide oligomers derived from omega-amino (oligoethylene glycol) alkanoic acid monomers (U.S. Pat. No. 6,320,017) or monosialoganglioside.

The second fraction further includes a relatively low toxicity lipid for adjusting toxicity; a lipid having a functional group that binds a ligand to the lipid particle 51; and suppressing leakage of inclusions such as sterols, such as cholesterol. It may also contain lipids such as lipids for In particular, it is preferable to include cholesterol.

The type of lipid used in the second fraction and its composition are determined by the acid dissociation constant (pKa) of the target lipid particles 51, the particle size of the lipid particles 51, the type of substance to be encapsulated, the stability in cells, etc. be selected appropriately taking into account the

For example, when the second fraction contains DOPE, DOTAP, cholesterol, and DMG-PEG, the gene nucleic acid to be introduced, for example, the nucleic acid encoding CAR 52, the genome integration promoter 53, the secretion This method is preferable because the delivery efficiency of the nucleic acid 54 encoding the type antigen protein is particularly excellent. Hereinafter, for convenience, the nucleic acid 52 encoding CAR, any nuclear introduction promoter 53, and the nucleic acid 54 encoding a secreted antigen protein will also be referred to as "gene nucleic acids 52 to 54 to be introduced".

In addition to the gene nucleic acids 52 to 54 to be introduced, additional components may be included in the lipid particle 51 as necessary. Further components are, for example, pH regulators, osmotic pressure regulators or gene activators. Examples of the pH adjuster include organic acids such as citric acid and salts thereof. Osmotic pressure regulators include sugars or amino acids. The gene activator will be described later.

The lipid particles 51 encapsulating the gene nucleic acids 52 to 54 to be introduced and other substances as necessary can be prepared by, for example, known methods used for encapsulating small molecules in lipid particles, such as the Bangham method, organic solvents, etc. It can be produced using an extraction method, a surfactant removal method, a freeze-thaw method, or the like. For example, a lipid mixture obtained by impregnating materials for the lipid particles 51 in a desired ratio in an organic solvent such as alcohol, and an aqueous buffer solution containing components to be encapsulated, such as gene nucleic acids to be introduced, are prepared. Add aqueous buffer to the mixture. The resulting mixture is stirred and suspended to encapsulate the gene nucleic acid to be introduced, for example, the nucleic acid 52 encoding CAR, the genome integration promoter 53, and the nucleic acid 54 encoding the secreted antigen protein, as desired. Lipid particles 51 are formed.

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 mixing ratio of the components of the lipid particles 51 may be approximately the same as the mixing ratio of each material in the lipid mixture. Furthermore, the ratio of the amounts of the substances contained in the lipid particles 51 can be easily adjusted by changing the ratio of the two substances in the aqueous buffer.

By bringing the lipid particles 51 into contact with T cells, they can be taken up into the cells, for example, by endocytosis. Then, the gene nucleic acids to be introduced, such as nucleic acid 52 encoding CAR, genome integration promoter 53, and nucleic acid 54 encoding secreted antigen protein, can be released into the cells. Since the lipid particles 51 include the first lipid 81a and the second lipid 81b, they have the property of being easily taken up by T cells and difficult to be taken up by other cells. Therefore, by simply enclosing the gene nucleic acid to be introduced into the lipid particle 51 and bringing it into contact with T cells, the gene nucleic acid to be introduced, for example, encoding the CAR, can be encoded without using antibodies or receptors as in the conventional method. The nucleic acid 52 encoding the nuclear introduction promoter 53, and the nucleic acid 54 encoding the secreted antigen protein can be combined as desired and efficiently introduced into T cells. Thereby, the lipid particles 51 can deliver the gene nucleic acid to be selectively or specifically introduced into T cells without using feeder cells.

According to the cell production kit of the seventh embodiment, a method for producing a cell group with a high CAR-T cell content can be carried out simply and easily without using feeder cells. is possible.

(Eighth embodiment)
The method for producing CAR-T cells without using feeder cells according to any of the embodiments described above can be used to treat cancer using CAR-T cells. Here, for example, an example of autoimmune cell therapy using the fourth embodiment and the one-step method for producing CAR-T cells shown in FIG. 5 will be described with reference to FIG. 12. First, liposomes are prepared (S91). For example, as shown in FIG. 5, a liposome encapsulating within a lipid particle 51 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. Prepare 50. Next, a material cell group is collected from the subject to be treated (S92). Material cell groups are 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). Any test is performed on the obtained CAR-T cell group as desired (S95). Administer the CAR-T cell group to the subject (S96). Administration to a subject may be performed by intravenous administration, with the dosage determined based on the CAR-T content of the obtained CAR-T cell group.

Furthermore, the obtained CAR-T cell population may be stored frozen at -180°C until it is used. As a cryopreservation method for CAR-T cells, any known cryopreservation method for living cells can be used. For example, it is suspended in a cryopreservation solution and stored frozen in a cryopreservation bag, for example, in a -150°C freezer. The number of cells to be subjected to cryopreservation is determined by measuring the total number of living cells of all types of cells included in the finally obtained CAR-T cell group and the number of living CAR-T cells. It can be determined based on the obtained CAR-T cell content. Furthermore, it is also possible to separate CAR-T cells from other cells. For example, CAR-T cells/T cells can be separated based on cell surface antigens specific to them. To isolate T cells/CAR-T cells, use an antibody against CD3 (anti-CD3 antibody), which is a specific antigen on the surface of T cells, and use a method such as MACS (magnetic cell separation). . When separating CAR-T cells, they can be separated using a method such as the above-mentioned MACS using an idiotype antibody specific to CAR.

The liposome may be any of the liposomes mentioned above. The subject can be any animal, including humans, companion animals, livestock, etc., in need of treatment. The material cell group may be any cell group containing T cells, and may be, for example, PBMC or lymphocyte fraction. For example, it can be obtained by collecting peripheral blood with a syringe or the like, and then isolating a mononuclear cell fraction using a gradient centrifugation method or the like, or collecting a lymphocyte fraction using a blood component separation device.

As an appropriate medium, any medium suitable for the material cell group used may be used.

Any desired test may be a safety test performed prior to administering the CAR-T cell population to a subject. Tests can be conducted taking into account patient safety from biological, morphological, clinical, non-clinical, and pharmaceutical perspectives. In addition, various tests may be conducted as required by the standards of the country, government, academic society, or organization or organization of experts. Further, although not shown in FIG. 12, various tests including safety tests on materials may be performed before implementing the method or before using each material.

This treatment method improves QOL because it treats cancer, prevents recurrence, delays cancer progression, prevents cancer, improves self-healing ability, prevents viral infection, induces immunity, and is used in combination with chemotherapy. May be used for such purposes. Further, the treatment method may be used prophylactically for subjects who are in a healthy state or a pre-symptomatic state.

Although the above example uses autoimmune cells, this embodiment is not limited to autologous material cells, and also includes the use of allogeneic material cells. In that case, the method can be carried out in the same manner, except that the step S92 in FIG. 12 is "collect material cell group from donor" or "prepare material cell group from donor".

According to the eighth embodiment, it is possible to provide simpler and safer CAR-T cell therapy. Here, one example of a treatment method using the method shown in the fourth embodiment has been shown, but instead of the fourth embodiment, any other embodiment described herein may also be used. It is possible to carry out the same method and obtain the same effect.

(Example 1)
Example 1 Synthesis of the DNA sequence of the secreted antigen protein (sCD19-Fc) gene The antigen protein is CD19, which is the target of CD19.CAR-T, and the DNA sequence of the secreted CD19 antigen protein (sCD19-Fc) (SEQ ID NO: 1 ) was designed and the DNA sequence was synthesized. The structure of the designed DNA sequence (SEQ ID NO: 1) is as shown in FIG. 5(b). The amino acid sequence of the extracellular secretion signal of human interleukin 6 (IL-6) ( A DNA sequence (SEQ ID NO: 6) encoding SEQ ID NO: 5) was ligated. The amino acid sequence of the Fc region of immunoglobulin (SEQ ID NO: 9) is attached to the 3' end of the DNA sequence (SEQ ID NO: 4) via a DNA sequence (SEQ ID NO: 8) encoding a linker sequence (SEQ ID NO: 7). The encoding DNA sequence (SEQ ID NO: 10) was ligated. The "secretion signal sequence,""antibody protein partial sequence," and "IgG Fc-tag sequence" are connected in the order from the 5' end to the 3' end.

Example 2 Preparation of Plasmid A plasmid was prepared by incorporating the sCD19-Fc DNA sequence obtained in Example 1 into pGEM-GL-pA as a template DNA for preparing RNA of the secreted CD19 antigen gene. pGEM-GL-pA is a commercially available RNA synthesis template plasmid DNA: pGEM-4Z (Promega) into which a T7 promoter and a poly(A) sequence have been integrated, and a human β-globin leader sequence (Globin leader) and pSP64 pA. It was created by incorporating the poly(A) sequence of vector (Promega). The sCD19-Fc DNA sequence obtained in Example 1 was integrated between the β-globin leader sequence and poly(A) sequence of pGEM-GL-pA, and this was used as template DNA for RNA preparation.

Example 3 Synthesis of mRNA Using the template DNA obtained in Example 2, mRNA was synthesized as follows using an mRNA synthesis kit: T7 mScript Standard Production System (Cellscript). The RNA synthesis procedure followed the kit's protocol. After the template DNA of Example 2 was purified by cleaving it with the restriction enzyme Eco RI, an in vitro transcription reaction solution containing 1.0 μg of template DNA was prepared and reacted in a constant temperature bath at 37° C. for 2 hours. After the reaction was completed, the synthesized RNA was purified by ammonium acetate precipitation, and then a Cap structure and a poly(A) structure were added to the 5' and 3' ends of the RNA to convert it into mRNA. After incubating the synthesized RNA at 65°C for 10 minutes, it was rapidly cooled on ice to destroy the secondary structure of the RNA, and 100 μL of the kit's capping reaction solution was prepared. A Cap structure was added to the 5' end of the RNA by reacting for 1 hour in a constant temperature bath at 37°C. Subsequently, the kit's poly(A) structure-adding reagent was added to the same solution, and the mixture was reacted for 2 hours in a constant temperature bath at 37°C 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 concentration of RNA was calculated by measuring absorbance (A260) using a spectrophotometer (BioSpec-mini, Shimadzu) as OD1.0 = 40.0 μg/mL RNA.

Example 4 Preparation of sCD19-Fc mRNA-containing liposome and piggyBac-mRNA/CAR-pDNA co-containing liposome (liposome for producing CAR-T by 2-step-2 liposome method)
As liposomes used for CAR-T production using the 2-step-2 liposome method, sCD19-Fc mRNA-containing liposomes (sCD19 liposomes) and liposomes for CAR-T production (PB/CAR liposomes) were prepared.

After adding the sCD19-Fc mRNA solution to the 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), 6.6 times the amount of The liposome solution was prepared by gently adding 10 mM HEPES (pH 7.3).

The liposome for CAR-T production was a liposome that can be applied to CAR-T production using piggyBac, a moth transposase. The PiggyBac encapsulated in the liposome was the mRNA of TS-HyPB (SEQ ID NO: 11), which is highly activated wild-type piggyBac. TS-HyPB contains the nuclear import signal of the TAT protein of the human immunodeficiency virus HIV, the nuclear import signal of the large T antigen protein of simian virus 40, and highly activated HyPB, which is a mutated version of wild-type piggyBac, on the 5' end. This is piggyBac, which is linked in this order from 1 to 2 and has the codon usage frequency optimized for humans. The liposome for CAR-T production co-encapsulated piggyBac mRNA and CD19.CAR transposon integration plasmid DNA (pIRII-CAR.CD19 (CD28)), which is piggyBac genome integration donor DNA (CAR-pDNA). Liposomes for CAR-T production are made by adding piggyBac mRNA solution and CAR- to 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). After adding pDNA, the liposome solution was prepared by gently adding 6.6 times the amount of 10 mM HEPES (pH 7.3).

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), and then 10 mM HEPES (pH 7.3) was added. Buffer exchange and concentration were performed simultaneously using a similar centrifugal ultrafiltration tube.
The amounts of RNA and DNA encapsulated in the liposomes were measured using QuantiFluorTM RNA System (Promega) and Quant-iTTM PicoGreenTM dsDNA Assay Kits.

Example 5 Preparation of sCD19-Fc mRNA/piggyBac-mRNA/CAR-pDNA co-encapsulating liposome (liposome for producing CAR-T by 1 step-1 liposome method)
A liposome co-encapsulating sCD19-Fc mRNA/ piggyBac-mRNA/CAR-pDNA (sCD19/PB/CAR liposome) was prepared as a liposome used for CAR-T production using the 1-step-1 liposome method (piggyBac-mRNA). and CAR-pDNA, see Example 4).

Add sCD19-Fc mRNA solution, piggyBac mRNA solution, and CAR-pDNA to 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). was added, then 6.6 times the amount of 10 mM HEPES (pH 7.3) was gently added to prepare the liposome solution.

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), and then 10 mM HEPES (pH 7.3) was added. Buffer exchange and concentration were performed simultaneously using a similar centrifugal ultrafiltration tube.
The amounts of RNA and DNA encapsulated in the liposomes were measured in the same manner as in Example 4 using QuantiFluorTM RNA System (Promega) and Quant-iTTM PicoGreenTM dsDNA Assay Kits.

Example 6 Production of CAR-T using sCD19-Fc by two-liposome method and one-liposome method
CAR-T was produced using PBMC (Peripheral Blood Mononuclear Cells, purchased from Lonza), which are human peripheral blood mononuclear cells. After the PBMCs were woken up, a cell suspension of 1.0 x 10 ⁶ cells/mL was prepared using TexMACS medium supplemented with cytokines (IL-7, 10 ng/mL, IL-15, 5 ng/mL). 200 μL of this suspension was seeded on a 96-well round bottom plate coated with anti-CD3/CD28 antibody, and cultured at 37° C. in a 5% CO ₂ atmosphere to activate T cells. For the culture plate, add 150 μL of anti-CD3/CD28 antibody solution diluted 100 times with phosphate buffered saline (PBS) per well to a 96-well round bottom plate (Non-tissue culture treated, Nunc) and incubate at 37°C. , and coated by standing in a 5% CO ₂ atmosphere for more than 2 hours.

In the 2-step-2 liposome method, PB/CAR liposomes (Example 4) were administered at 2.0 μg/well 24 hours after T cell activation, and culture was continued at 37° C. in a 5% CO ₂ atmosphere. In parallel, sCD19 liposomes were administered at 2.0 μg/well to wells separate from the PB/CAR liposomes, and cultured at 37° C. in a 5% CO ₂ atmosphere.

24 hours after administration of sCD19 liposomes, the culture medium was collected from the wells, cells were precipitated by centrifugation, and the supernatant was collected as conditioned medium. The cells in the wells to which the liposomes for CAR production were administered were collected by pipetting, the collected culture medium was transferred to a 48-well plate, the above-mentioned conditioned medium was added thereto, and the cells were incubated at 37°C in a 5% CO ₂ atmosphere. Culture was continued.

In the 1-step-1 liposome method, 24 hours after T cell activation, sCD19/PB/CAR liposomes (Example 5) are administered at 2.0 μg/well and culture is continued at 37°C in a 5% CO ₂ atmosphere. did. Thereafter, in both the two-step method and the one-step method, CAR-T was produced by changing the medium and expanding the culture depending on the growth state of the cells, and continuing the cell culture for two weeks.

Example 7 Detection of CAR-T Two weeks after liposome administration, PBMCs were removed from the incubator and the production rate of CAR-T was examined using a fluorescence-activated cell sorter (FACS). For FACS, FACSVerse (BD Biosciences) was used. Collect PBMC from the culture plate, wash once with PBS, suspend the cells in 50 μL of PBS, and add biotin-labeled anti-human IgG (H+L) antibody [Biotin F(ab')2 Fragment Goat Anti- 2 μL of Human IgG (H+L) antibody, The Jackson Laboratory] was added and reacted at 4° C. for 15 minutes. After the reaction, wash once with PBS, suspend the cells in 50 μL of PBS, and add anti-human CD3 antibody (V450 Mouse Anti-Human CD3, Clone UCHT1, BD Biosciences) and APC-labeled streptavidin (APC Streptavidin, BD Biosciences). ) was added to each cell, reacted for 15 minutes at 4°C, washed the cells with PBS, suspended in PBS, and analyzed by FACS. FACS detected red fluorescence (APC) of CAR-expressing cells and blue fluorescence (V450) of CD3-expressing T cells. CAR-T is a cell that is co-positive for red fluorescence and blue fluorescence (CAR-positive and CD3-positive cell). The CAR-T production rate was defined as the ratio of CAR-T to all detected cells.

A scatter diagram of the results is shown in FIG. 13. The vertical axis is the fluorescence intensity of APC (CAR), and the horizontal axis is the fluorescence intensity of V450 (T cells). CAR-T is the cell in the UR region (CAR-positive and CD3-positive region) of the scatter diagram. The CAR-T production rate is shown in the graph of FIG. 13. In the cell group in which CAR-T was produced using the 2-step-2 liposome method (cell group cultured in conditioned medium), the production rate was 36.3%. In the cell group in which CAR-T was produced using the 1-step-1 liposome method, the production rate was 39%. The cell group cultured without sCD19 had a production rate of 19.7%. This result showed that the use of sCD19 improved the production rate of CAR-T.

Example 8 Evaluation of tumor cell killing performance by CAR-T In Example 6, CAR-T produced 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 The cells were seeded in a 96-well plate at a ratio of 1:1 for a total number of cells of 1.0×10 ⁵ cells/well. After CAR-T and NALM6 were co-cultured for 5 days at 37°C in a 5% CO ₂ atmosphere, the cells were collected 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 were added. The mixture was reacted at 4°C for 30 minutes. After the reaction, the cells were washed with PBS, suspended in PBS, and analyzed by FACS. FACS detected blue fluorescence (V450) of CD3-expressing T cells and green fluorescence (FITC) of CD19-expressing NALM6 cells.

The scatter diagram of the results is shown in FIGS. 14(a-1) and (a-2), and the tumor cell killing rate is shown in FIG. 14(b). The vertical axis of the graphs (a-1) and (a-1) is the fluorescence intensity of FITC, which is an indicator of the presence of NALM6. The horizontal axis is the fluorescence intensity of V450, which is an indicator of the presence of T cells. As shown in (a-2), in the co-culture of PBMCs that do not express CAR and 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 cells were a T cell group (LR) containing CAR-T (Figure 14 (a-1), ( a-2)). Only about 3% of NALM6 (UL) was contained, and the tumor cell killing rate was about 93% (Figure 14(b)). The CAR-T produced in Example 6 was shown to kill CD19-expressing B-cell tumor cells.

Although several embodiments of the invention have been described, these embodiments are presented by way of example and are not intended to limit the scope of the invention. These novel embodiments can be implemented in various other forms, and various omissions, substitutions, and changes can be made without departing from the gist of the invention. These embodiments and their modifications are included within the scope and gist of the invention, as well as within the scope of the invention described in the claims and its equivalents.

50... Liposome, 51... Lipid particle, 52... Nucleic acid encoding CAR, 53... Nuclear import promoter, 54... Nucleic acid encoding secreted antigen protein, 54a... Antigen protein partial sequence, 54b Secretion signal sequence, 54c... Linker , 54d... IgG Fc-tag sequence, 55, 55A, 55B... Container, 56... CAR-T cell production medium, 57... Material cells, 58... CAR-T cells, 59... CAR, 60... Antigen protein, 61... CAR-T cell, 66...medium, 70A...first liposome, 70B...second liposome, 77...any cell, 81a...first lipid, 81b...second lipid, 81c...third lipid

Claims (20)

Creating CAR-T cells, which involves creating a CAR-T cell group without using feeder cells, by secreting the CAR antigen protein or its partial protein extracellularly from chimeric antibody receptor (CAR) gene-transfected cells. Method. In a first culture system, bringing a first liposome containing a nucleic acid encoding a CAR into contact with material cells containing T cells to introduce the nucleic acid encoding the CAR into the material cells;
In a second culture system that is independent or non-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 treated with an antigen protein or the target of the CAR. A second liposome containing a nucleic acid encoding a secreted antigen protein in which an extracellular secretion signal sequence is added to the sequence encoding the partial protein is brought into contact with the nucleic acid encoding the secreted antigen protein into the cytoplasm of the cell. introducing the antigen protein or a partial protein thereof from the cell;
contacting the secreted antigen protein or a partial protein thereof to the CAR-T cells produced by introducing the nucleic acid encoding the CAR-T, and promoting the division and proliferation of the CAR-T cells; A method for producing CAR-T cells comprising producing the CAR-T cells by introducing a nucleic acid encoding CAR-T and producing the desired CAR-T cell group by promoting the division and proliferation. . CAR-T according to claim 2, wherein the generation of the CAR-T cells by introducing the nucleic acid encoding the CAR-T and the promotion of division and proliferation of the CAR-T cells proceed simultaneously or in stages. How to make cells. The second culture system is independent from the first culture system, and contacting the antigen protein or its partial protein with the produced CAR-T cells is a step of the second culture system. 3. The method for producing CAR-T cells according to claim 2, which is carried out by adding a medium to the first culture system. 3. The method for producing CAR-T cells according to claim 2, wherein the first culture system and the second culture system are one culture system that is independent of each other. The first culture system and the second culture system are one culture system independent of each other, the first liposome further contains a nucleic acid encoding the secreted antigen protein, and the second liposome further contains a nucleic acid encoding the secreted antigen protein. 3. The method for producing CAR-T cells according to claim 2, further comprising a nucleic acid encoding the CAR, and thus using only one type of liposome in total. According to claim 6, the introduction of the nucleic acid encoding the CAR into the cell and the introduction of the nucleic acid encoding the secreted antigen protein into the cell proceed simultaneously with respect to one cell using the one type of liposome. How to generate CAR-T cells. The first culture system and the second culture system are one independent culture system, and the first liposome converts the material cells into CAR-T using the nucleic acid encoding the CAR contained therein. The CAR-T cells are produced by introducing a nucleic acid encoding the secreted antigen protein, and the second liposome is present in the first culture system due to the nucleic acid encoding the secreted antigen protein contained therein, and the second liposome 7. The method for producing CAR-T cells according to claim 6, wherein the antigen protein or a partial protein thereof is secreted from cells that have taken up the antigen protein. 2. 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 secretion signal sequence is an interleukin-6 signal sequence. A method for producing CAR-T cells according to any one of items 8 to 8. The method for producing CAR-T cells according to claim 9, wherein the sequence encoding the antigen protein or a partial protein thereof is mRNA. 10. The method for producing CAR-T cells according to claim 9, wherein the nucleic acid encoding the CAR is double-stranded DNA encoding the CAR gene sequence. The method for producing CAR-T cells according to any one of claims 2 to 8, wherein the first liposome contains a genome integration promoter together with the nucleic acid encoding the CAR, and does not use feeder cells. The method for producing CAR-T cells according to any one of claims 2 to 8, wherein the liposome further contains mRNA encoding the piggyBac gene together with the nucleic acid encoding the CAR. 14. The method for producing CAR-T cells according to claim 13, wherein the mRNA encoding the piggyBac gene is mRNA encoding piggyBac transposase. 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).

The method for producing CAR-T cells according to any one of claims 2 to 8, comprising: 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).

The method for producing CAR-T cells according to claim 9, comprising: 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).

13. The method for producing CAR-T cells according to claim 12. The following liposome groups A and/or B, together with a substance that improves the storage stability of liposomes;
・Liposome group A:
A first liposome containing a nucleic acid encoding a CAR, and a second liposome containing a nucleic acid encoding a secreted antigenic protein obtained by adding an extracellular secretion signal sequence to a sequence encoding an antigenic protein or a partial protein thereof that is a target of the CAR. 2 liposomes,
・Liposome group B:
A liposome containing both a nucleic acid encoding the CAR and a nucleic acid encoding a secreted antigen protein obtained by adding an extracellular secretion signal sequence to a sequence encoding an antigen protein or a partial protein thereof that is a target of the CAR;
A kit for producing CAR-T cells for use in the method for producing CAR-T cells according to any one of claims 1 to 8. The lipid particles constituting any of the liposomes include the first lipid compound (FFT10) represented by formula (I) and/or the second lipid compound (FFT20) represented by formula (II).

The cell production kit according to claim 18, comprising: A cell group into which a secreted antigen protein gene, in which an extracellular secretion signal sequence is added to the sequence of the antigen protein or its partial protein that is the target of the CAR, was introduced during the production of CAR-T cells, and cells containing the CAR-T cells. A method of increasing the CAR-T cell content by co-culturing the CAR-T cells in the cell group without using feeder cells.

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