Method for culturing t cells

Abstract

The present invention provides an exceptional T cell population. In the present invention, a method is used for improving the proportion of CD4+ T cells in a cell population, said method comprising a step for culturing a cell population containing human T cells in the presence of a human transforming growth factor-β (human TGFβ) component to obtain a cultured cell population. It is possible to optionally use a cell population containing CD3+ T cells in which the proportion of CD4+CD8- T cells among CD3+ T cells contained in the cell population is at least 50%.

Description

Method for culturing T cells

[0001] The technical field of the present invention relates to a method for culturing T cells and the like.

[0002] Chimeric antigen receptor T (CAR-T) cell therapy is a well-known treatment method using T cells. CAR-T cell therapy involves collecting a T cell-containing sample from a patient, modifying the T cells, expanding the T cells, and administering the CAR-T cells. Alpha-β T cells are broadly classified as helper T cells (CD4+) and killer T cells (CD8+). In recent years, the balance between CD4 and CD8 after culture has become increasingly important. For example, in the aforementioned CAR-T therapy, CD4-positive CAR-Ts exhibit similar antitumor activity to CD8-positive CAR-Ts, and it has been reported that the combination of CD4-positive CAR-Ts with CD8-positive CAR-Ts has a stronger antitumor effect than either CD4- or CD8-positive CAR-Ts alone (Non-Patent Document 1). Furthermore, CAR-T induction can sometimes result in a bias toward CD8 over CD4 after culture, which has been identified as a potential therapeutic challenge (Non-Patent Document 2).

[0003] Furthermore, in CAR-T therapy, it has been found that CD4-positive CAR-T has a higher killing effect on target cells than CD8-positive CAR-T, and the importance of CD4-positive cells has been pointed out (Non-Patent Document 3).

[0004] There are currently several CAR-T cell products on the market, but one that takes into account the CD4 / CD8 balance mentioned above is Breyangi Injection (lisocabtagene maraleucel). This product is designed to cultivate CD4-positive CAR-T and CD8-positive CAR-T separately, achieving a CD4:CD8 ratio of 1:1. While it has demonstrated a high therapeutic effect against the target B-cell lymphoma, it requires the operation of two culture systems, making it extremely expensive, costing tens of millions of yen.

[0005] Sommermeyer et al., Leukemia. 2016 Feb;30(2):492-500.Kiani et al., Arch Iran Med. 2019 Jan;22(1):7-10.Wang et al., JCI Insight. 2018 May 17;3(10):e99048.

[0006] When a cell population containing human T cells is cultured, CD8 + The proportion of T cells is high, and CD4 + The proportion of T cells tends to be low (see, for example, Figures 1 and 2 below). Therefore, there is room for improvement in the methods for producing T cell populations for use in regenerative medicine such as CAR-T cell therapy.

[0007] Meanwhile, in the course of their research, the inventors conducted an experiment in which they changed the culture conditions of human T cells. As a result, they surprisingly found that by culturing a cell population containing human T cells in the presence of human TGFβ components, CD4 + A cell population with an improved proportion of T cells was obtained. Based on these results, we succeeded in obtaining an excellent T cell population.

[0008] According to one aspect of the present invention, a method for detecting CD4 in a cell population, comprising culturing a cell population containing human T cells in the presence of a human transforming growth factor β (human TGFβ) component to obtain a cultured cell population, is provided. + A method for improving the proportion of T cells is provided, which results in a superior T cell population.

[0009] According to one aspect of the present invention, there is provided a cell population containing CD3-positive T cells, wherein the CD3-positive T cells contained in the cell population are CD4 + CD8 - A cell population containing 50% or more T cells is provided. This cell population can be used in regenerative medicine such as CAR-T cell therapy. According to one aspect of the present invention, a method for culturing a cell population containing human T cells in the presence of a human TGFβ component and CD4 + The present invention provides a method for producing a cell population, comprising the steps of: culturing a cell population containing human T cells in the presence of a human TGFβ component; and recovering a cultured cell population containing T cells at a rate of 40% or more. This method provides an excellent T cell population. According to one aspect of the present invention, the method comprises the steps of culturing a cell population containing human T cells in the presence of a human TGFβ component; and recovering CD4 + CD8 - The proportion of T cells is CD4 - CD8 + The present invention provides a method for producing a cell population, comprising the step of recovering a cultured cell population in which the proportion of T cells is 0.7 to 1.3 times that of T cells. This method allows for the production of an excellent T cell population. According to one aspect of the present invention, a method for producing a cell population in which the proportion of CD4 T cells in the cell population is 0.7 to 1.3 times that of T cells is provided, the method comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + T cells and CD8 + A method for approximating the proportion of T cells is provided. This method allows for the production of an excellent T cell population. According to one aspect of the present invention, a method for approximating the proportion of T cells is provided. The method includes culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + A method for producing a cell population with an improved proportion of T cells is provided. This method allows for the production of an excellent T cell population. According to one aspect of the present invention, a method for producing a CD4 T cell population is provided, which comprises the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + T cells and CD8 + A method for producing a cell population having a similar proportion of T cells is provided. This method allows for the production of an excellent T cell population. According to one aspect of the present invention, there is provided a method for improving the proportion of cells expressing stem cell memory T cell markers in a cell population, the method comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. This method allows for the production of an excellent T cell population. According to one aspect of the present invention, there is provided a method for producing a cell population having an improved proportion of stem cell memory T cells in the cell population, the method comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. This method allows for the production of an excellent T cell population. According to one aspect of the present invention, there is provided a method for improving the proportion of cells expressing stem cell memory T cell markers in a cell population, the method comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population.+ CCR7 + A method for improving the proportion of T cells is provided. By utilizing this method, a superior T cell population can be obtained. According to one aspect of the present invention, + A composition comprising a human TGFβ component is provided for use in producing a cell population with an improved proportion of T cells. Utilizing this composition, a superior T cell population can be obtained. In one aspect of the present invention, CD4 + T cells and CD8 + A composition containing a human TGFβ component is provided for use in producing a cell population with a similar proportion of T cells. Use of this method allows for the production of an excellent T cell population. According to one aspect of the present invention, a culture medium for T cells is provided, comprising 0.01 to 3 ng / ml of a TGFβ component. Use of this culture medium allows for the production of an excellent T cell population. According to one aspect of the present invention, a method for culturing T cells is provided, comprising the step of culturing T cells in the presence of 0.01 to 3 ng / ml of a TGFβ component. Use of this culture method allows for the production of an excellent T cell population.

[0010] The analysis results of the CD4 / CD8 ratio when medium 1 was used are shown. The numbers in the upper left are CD4. - CD8 + T cells, the number on the bottom right is CD4 + CD8 - T cells, the number on the top right is CD4 + CD8 + The percentage of T cells (%) is shown (same for Figures 2 to 9, 19 to 23, 29 to 36 below). The analysis results of the CD4 / CD8 ratio when medium 2 was used are shown. The analysis results of the CD4 / CD8 ratio when medium 3 was used are shown. The analysis results of the CD4 / CD8 ratio when medium 4 was used are shown. The analysis results of the CD4 / CD8 ratio when medium 5 was used are shown. The analysis results of the CD4 / CD8 ratio when medium 6 was used are shown. The analysis results of the CD4 / CD8 ratio when medium 7 was used are shown. The analysis results of the CD4 / CD8 ratio when medium 8 was used are shown. The analysis results of the CD4 / CD8 ratio when medium 9 was used are shown. The analysis results of the CD45RA / CCR7 ratio when medium 1 was used are shown. The number in the upper left is CD45RA + CCR7 - T cells, numbers on the bottom right are CD45RA-CCR7+ T cells, the numbers in the upper right corner are CD45RA + CCR7 + The percentage (%) of T cells is shown (the same applies to Figures 11 to 18, 24 to 28, and 37 to 44 below). The results of analysis of the CD45RA / CCR7 ratio when medium 2 was used are shown. The results of analysis of the CD45RA / CCR7 ratio when medium 3 was used are shown. The results of analysis of the CD45RA / CCR7 ratio when medium 4 was used are shown. The results of analysis of the CD45RA / CCR7 ratio when medium 5 was used are shown. The results of analysis of the CD45RA / CCR7 ratio when medium 6 was used are shown. The results of analysis of the CD45RA / CCR7 ratio when medium 7 was used are shown. The results of analysis of the CD45RA / CCR7 ratio when medium 8 was used are shown. The results of analysis of the CD45RA / CCR7 ratio when medium 9 was used are shown. The results of analysis of the CD4 / CD8 ratio when medium 10 was used are shown. The results of analysis of the CD4 / CD8 ratio when medium 11 was used are shown. The results of analysis of the CD4 / CD8 ratio when medium 12 was used are shown. 1 shows the analysis results of the CD4 / CD8 ratio when medium 13 was used. 1 shows the analysis results of the CD4 / CD8 ratio when medium 14 was used. 1 shows the analysis results of the CD45RA / CCR7 ratio when medium 10 was used. 1 shows the analysis results of the CD45RA / CCR7 ratio when medium 11 was used. 1 shows the analysis results of the CD45RA / CCR7 ratio when medium 12 was used. 1 shows the analysis results of the CD45RA / CCR7 ratio when medium 13 was used. 1 shows the analysis results of the CD45RA / CCR7 ratio when medium 14 was used. 1 shows the analysis results of the CD4 / CD8 ratio when medium 15 was used. 1 shows the analysis results of the CD4 / CD8 ratio when medium 16 was used. 1 shows the analysis results of the CD4 / CD8 ratio when medium 17 was used. 1 shows the analysis results of the CD4 / CD8 ratio when medium 18 was used. 1 shows the analysis results of the CD4 / CD8 ratio when medium 19 was used. 1 shows the analysis results of the CD4 / CD8 ratio when medium 20 was used. 1 shows the analysis results of the CD4 / CD8 ratio when medium 21 was used. 2 shows the analysis results of the CD4 / CD8 ratio when medium 22 was used. 3 shows the analysis results of the CD45RA / CCR7 ratio when medium 15 was used. 4 shows the analysis results of the CD45RA / CCR7 ratio when medium 16 was used. 5 shows the analysis results of the CD45RA / CCR7 ratio when medium 17 was used. 6 shows the analysis results of the CD45RA / CCR7 ratio when medium 18 was used.Figure 4 shows the results of analysis of the CD45RA / CCR7 ratio when medium 19 was used. Figure 5 shows the results of analysis of the CD45RA / CCR7 ratio when medium 20 was used. Figure 6 shows the results of analysis of the CD45RA / CCR7 ratio when medium 21 was used. Figure 7 shows the results of analysis of the CD45RA / CCR7 ratio when medium 22 was used. Figure 8 shows the results of analysis of the proportion of CD3-positive T cells when medium 15 was used. The number in the upper right corner indicates the proportion (%) of CD3-positive T cells (the same applies to Figures 46 to 52 below). Figure 9 shows the results of analysis of the proportion of CD3-positive T cells when medium 16 was used. Figure 10 shows the results of analysis of the proportion of CD3-positive T cells when medium 17 was used. Figure 11 shows the results of analysis of the proportion of CD3-positive T cells when medium 18 was used. Figure 12 shows the results of analysis of the proportion of CD3-positive T cells when medium 19 was used. Figure 13 shows the results of analysis of the proportion of CD3-positive T cells when medium 20 was used. Figure 14 shows the results of analysis of the proportion of CD3-positive T cells when medium 21 was used. Figure 15 shows the results of analysis of the proportion of CD3-positive T cells when medium 22 was used. The analysis results of the CD4 / CD8 ratio when culture media 23 to 26 were used are shown.

[0011] Hereinafter, embodiments of the present invention will be described in detail, with the same contents omitted as appropriate to avoid repetition.

[0012] (1) Method According to one embodiment of the present invention, a method is provided which comprises a step of culturing cells in the presence of a human TGFβ component. This method comprises, for example, culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population, and + The present invention includes a method for improving the proportion of T cells. This method can be used in the process of culturing T cells in regenerative medicine such as CAR-T cell therapy. This method allows for the production of an excellent T cell population.

[0013] In one embodiment of the present invention, the superior T cell population comprises a superior T cell population for use in regenerative medicine, such as CAR-T cell therapy. In one embodiment of the present invention, the superior T cell population comprises CD4 + A cell population with an improved proportion of T cells, CD4 + T cells and CD8 + Cell population with a similar proportion of T cells, CD45RA+ CCR7 + Cell population with an improved proportion of T cells, cell population with an improved proportion of stem cell memory T cells, cell population that can survive for a long time when returned to the body, CD4 + T cells and CD8 + Controlled cell populations with a high proportion of T cells or CD4 + T cells and CD8 + It contains a cell population with an improved balance of T cell proportions.

[0014] According to one embodiment of the present invention, the method comprises culturing a cell population containing human T cells in the presence of a human TGFβ component, and culturing the population to generate CD4 + and recovering a cultured cell population in which the proportion of T cells is 40% or more. This method allows for the production of an excellent T cell population.

[0015] According to one embodiment of the present invention, the method comprises culturing a cell population containing human T cells in the presence of a human TGFβ component, and culturing CD8 + and recovering a cultured cell population in which the proportion of T cells is 60% or less. This method allows for the production of an excellent T cell population.

[0016] According to one embodiment of the present invention, the method comprises culturing a cell population containing human T cells in the presence of a human TGFβ component, and + CCR7 + and recovering a cultured cell population in which the proportion of T cells is 30% or more. This method allows for the production of an excellent T cell population.

[0017] According to one embodiment of the present invention, the method comprises culturing a cell population containing human T cells in the presence of a human TGFβ component, and culturing the population to generate CD4 + CD8 - The proportion of T cells is CD4 - CD8 + and recovering a cultured cell population with a ratio of 0.7 to 1.3 times the proportion of T cells. This method allows for the production of an excellent T cell population.

[0018] According to one embodiment of the present invention, a method for detecting CD4 in a cell population, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population, is provided. + T cells and CD8 + A method is provided for approximating the proportion of T cells, which results in a superior T cell population.

[0019] According to one embodiment of the present invention, a method for detecting CD4 in a cell population, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population, is provided. + T cells and CD8 + A method for improving the balance of T cell ratios is provided, which results in a superior T cell population.

[0020] According to one embodiment of the present invention, there is provided a method for producing a cell population, comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. This method allows the production of an excellent T cell population.

[0021] According to one embodiment of the present invention, a method for producing a CD4 T cell-containing cell population is provided, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + A method for producing a cell population with an improved proportion of T cells is provided, which allows for the production of an excellent T cell population.

[0022] According to one embodiment of the present invention, a method for producing a CD4 T cell-containing cell population is provided, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + T cells and CD8 + A method for producing a cell population with a similar proportion of T cells is provided, and this method allows for the production of an excellent T cell population.

[0023] According to one embodiment of the present invention, a method for producing a CD4 T cell-containing cell population is provided, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + A method for producing a cell population with a T cell ratio of 40% or more is provided, which allows for the production of an excellent T cell population.

[0024] According to one embodiment of the present invention, a method for producing a CD8 T cell-containing cell population is provided, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + A method for producing a cell population with a T cell ratio of 60% or less is provided, which allows for the production of an excellent T cell population.

[0025] According to one embodiment of the present invention, a method for producing a CD4 T cell-containing cell population is provided, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + CD8 - The proportion of T cells is CD4 - CD8 + A method for producing a cell population with a ratio of T cells of 0.7 times or more is provided, and this method yields an excellent T cell population.

[0026] One embodiment of the present invention provides a method for increasing the proportion of cells expressing stem cell memory T cell markers in a cell population, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. This method results in an excellent T cell population.

[0027] One embodiment of the present invention provides a method for producing a cell population with an improved proportion of stem cell memory T cells, comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. This method produces an excellent T cell population.

[0028] According to one embodiment of the present invention, a method for detecting CD45RA in a cell population, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population, is provided. + CCR7 + A method for improving the proportion of T cells is provided, which results in a superior T cell population.

[0029] According to one embodiment of the present invention, a method for detecting CD45RA in a cell population, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population, is provided. + CCR7 + A method for producing a cell population with an improved proportion of T cells is provided, which allows for the production of an excellent T cell population.

[0030] According to one embodiment of the present invention, a method for producing a CD45RA T cell-containing cell population is provided, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + CCR7 + A method for producing a cell population with a T cell ratio of 30% or more is provided, which allows for the production of an excellent T cell population.

[0031] One embodiment of the present invention provides a culture method comprising culturing T cells in the presence of 0.01 to 3 ng / ml of TGFβ, which results in the production of a superior T cell population.

[0032] One embodiment of the present invention provides a culture method comprising culturing PBMCs or T cells in the presence of a human TGFβ component, which method results in an excellent T cell population.

[0033] According to one embodiment of the present invention, a method for detecting CD4 in a cell population, comprising culturing a cell population containing human T cells in a medium containing a human TGFβ component, is provided. + According to one embodiment of the present invention, a method for increasing the proportion of CD4 T cells in a cell population is provided, the method comprising culturing a cell population containing human T cells in a medium containing a human TGFβ component. + T cells and CD8 + According to one embodiment of the present invention, a method for approximating the proportion of CD4 T cells in a cell population is provided, the method comprising culturing a cell population containing human T cells in a medium containing a human TGFβ component. + T cells and CD8 + According to one embodiment of the present invention, a method for improving the balance of T cell ratios is provided, which comprises culturing a cell population containing human T cells in a medium containing a human TGFβ component. + According to one embodiment of the present invention, a method for producing a cell population with an improved proportion of T cells is provided, which comprises culturing a cell population containing human T cells in a medium containing a human TGFβ component. + T cells and CD8 + According to one embodiment of the present invention, a method for producing a cell population having a similar proportion of T cells is provided. + A method for producing a cell population containing 40% or more T cells is provided. According to one embodiment of the present invention, a method for producing a CD8 T cell population containing human T cells is provided, the method comprising the step of culturing a cell population containing human TGFβ in a medium containing a human TGFβ component. + A method for producing a cell population with a T cell percentage of 60% or less is provided. According to one embodiment of the present invention, a method for producing a cell population containing human T cells, comprising culturing the cell population in a medium containing a human TGFβ component, is provided. + CD8 - The proportion of T cells is CD4 - CD8 + A method for producing a cell population in which the proportion of stem cell memory T cell marker-expressing cells in the cell population is 0.7 times or more (e.g., 0.7 to 1.3 times) greater than the proportion of T cells is provided. According to one embodiment of the present invention, a method for increasing the proportion of cells expressing stem cell memory T cell markers in a cell population is provided, comprising the step of culturing a cell population containing human T cells in a medium containing a human TGFβ component. According to one embodiment of the present invention, a method for producing a cell population in which the proportion of stem cell memory T cells in the cell population is increased is provided, comprising the step of culturing a cell population containing human T cells in a medium containing a human TGFβ component. According to one embodiment of the present invention, a method for increasing the proportion of cells expressing stem cell memory T cell markers in the cell population is provided, comprising the step of culturing a cell population containing human T cells in a medium containing a human TGFβ component. + CCR7 + According to one embodiment of the present invention, a method for improving the proportion of T cells is provided, which comprises culturing a cell population containing human T cells in a medium containing a human TGFβ component. + CCR7 + According to one embodiment of the present invention, a method for producing a cell population having an improved proportion of T cells is provided, which comprises culturing a cell population containing human T cells in a medium containing a human TGFβ component. + CCR7 + Methods for producing cell populations with a proportion of T cells of 30% or more are provided.

[0034] According to one embodiment of the present invention, there is provided a method for treating a disease (e.g., cancer), comprising the step of administering to a patient a cell population obtained by any of the methods above, any of the cell populations described in (2) below, or T cells obtained from such a cell population.

[0035] According to one embodiment of the present invention, there is provided a CAR-T cell therapy comprising the steps of collecting a specimen from a patient (collection step), stimulating the specimen (e.g., CD3 stimulation) (stimulation step), modifying T cells (e.g., gene transfer) (modification step), culturing and expanding human T cells (expansion step), or administering CAR-T cells to a patient (administration step), wherein one or more of the stimulation step, modification step, and expansion step comprises culturing a cell population containing T cells in the presence of a TGFβ component.

[0036] (2) Cells According to one embodiment of the present invention, CD4 + CD8 - A cell population containing a certain percentage of T cells is provided. This cell population is, for example, a cell population containing CD3-positive T cells, and among the CD3-positive T cells contained in the cell population, CD4 + CD8 - This cell population contains more than 50% T cells. This cell population is suitable for regenerative medicine such as CAR-T cell therapy. Using this cell population can improve the therapeutic effect of regenerative medicine such as CAR-T cell therapy. Using this cell population, CD4 + T cells and CD8 + By using a cell population with a controlled proportion of T cells, superior treatment with CAR-T cell therapy can be achieved.

[0037] According to one embodiment of the present invention, CD4 - CD8 + A cell population containing a certain percentage of T cells is provided. This cell population is, for example, a cell population containing CD3-positive T cells, and the CD3-positive T cells contained in the cell population are CD4 - CD8 + This cell population contains a cell population with a T cell ratio of 50% or less. This cell population is suitable for regenerative medicine such as CAR-T cell therapy. Using this cell population can improve the therapeutic effect of regenerative medicine such as CAR-T cell therapy. Using this cell population, CD4 + T cells and CD8 + By using a cell population with a controlled proportion of T cells, superior treatment with CAR-T cell therapy can be achieved.

[0038] According to one embodiment of the present invention, CD4 + CD8 - The proportion of T cells is CD4 - CD8 + A cell population containing at least a certain multiple of the proportion of CD3-positive T cells is provided. This cell population is, for example, a cell population containing CD3-positive T cells, and the CD4 + CD8 - The proportion of T cells is CD4 - CD8 + This cell population contains more than 0.9 times the proportion of T cells. This cell population is suitable for regenerative medicine such as CAR-T cell therapy. Using this cell population can improve the therapeutic effects of regenerative medicine such as CAR-T cell therapy. Using this cell population, CD4 + T cells and CD8 + By using a cell population with a controlled proportion of T cells, superior treatment with CAR-T cell therapy can be achieved.

[0039] According to one embodiment of the present invention, CD45RA + CCR7 + A cell population containing a certain percentage of cells is provided. This cell population may contain, for example, CD45RA + CCR7 + This cell population contains a cell population with a ratio of CD45RA cells of 35% or more. This cell population is suitable for regenerative medicine such as CAR-T cell therapy. Using this cell population can improve the therapeutic effect of regenerative medicine such as CAR-T cell therapy. Using this cell population, + CCR7 + By using a cell population with a controlled cell ratio, superior treatment with CAR-T cell therapy can be achieved.

[0040] According to one embodiment of the present invention, CD4 + CD8 - T cell ratio >50%, CD4 - CD8 + T cell percentage less than 50% and CD45RA + CCR7 + This cell population contains a cell population with a CD4+ cell ratio of 35% or more. This cell population is suitable for regenerative medicine such as CAR-T cell therapy. Using this cell population can improve the therapeutic effects of regenerative medicine such as CAR-T cell therapy. Using this cell population, + T cells, CD8 + T cells and CD45RA + CCR7 + By using a cell population with a controlled cell ratio, superior treatment with CAR-T cell therapy can be achieved.

[0041] (3) Composition According to one embodiment of the present invention, a composition containing a human TGFβ component is provided. This composition is, for example, a CD4 + This includes a composition containing a human TGFβ component for use in producing a cell population with an improved proportion of T cells. By culturing a cell population containing human T cells using this composition, a superior T cell population can be produced.

[0042] According to one embodiment of the present invention, CD4 + T cells and CD8 + A composition containing a human TGFβ component is provided for use in producing a cell population with a similar proportion of T cells. By culturing a cell population containing human T cells using this composition, a superior T cell population can be produced.

[0043] According to one embodiment of the present invention, there is provided a composition containing a human TGFβ component for use in any of the methods described in (1) above. When a cell population containing human T cells is cultured using this composition, an excellent T cell population can be produced.

[0044] According to one embodiment of the present invention, there is provided a composition or pharmaceutical composition comprising a cell population obtained by any of the methods in (1) above, any of the cell populations described in (2) above, or T cells obtained from these cell populations. This composition can be used in regenerative medicine such as CAR-T cell therapy (e.g., regenerative medicine for cancer). Use of this composition can enhance the therapeutic effect of regenerative medicine such as CAR-T cell therapy. Use of this composition can enhance the therapeutic effect of CD4 + T cells, CD8 + T cells, or CD45RA + CCR7 + By using a cell population with a controlled cell ratio, superior treatment with CAR-T cell therapy can be achieved.

[0045] According to one embodiment of the present invention, CD4 + Cell population with a T cell ratio of 40% or more, CD8 + Cell population with a T cell ratio of 60% or less, CD4 + CD8 - The proportion of T cells is CD4 - CD8 + Cell population 0.7 times or more than the proportion of T cells, or CD45RA + CCR7 + Compositions are provided that include a cell population with a T cell percentage of 30% or more, and may include, for example, a human TGFβ component, ascorbic acid, a derivative or a salt thereof, PLT, or IL-2 component.

[0046] According to one embodiment of the present invention, the composition contains a CD4 + CD8 - T cell ratio >50%, CD4 - CD8 + T cell percentage <50% or CD45RA + CCR7 + Compositions are provided that have a cell percentage of 35% or greater. The compositions may include, for example, a human TGFβ component, ascorbic acid, a derivative or salt thereof, PLT, or IL-2 component.

[0047] According to one embodiment of the present invention, there is provided a composition comprising a human TGFβ component, ascorbic acid, a derivative or a salt thereof, PLT, or IL-2 component.

[0048] One embodiment of the present invention provides a composition for T cells, comprising 0.01 to 3 ng / ml of a TGFβ component. Culturing a cell population containing human T cells using this composition enables the production of an excellent T cell population.

[0049] (4) Container According to one embodiment of the present invention, there is provided a container containing a cell population obtained by any of the methods described in (1) above, any of the cell populations described in (2) above, or any of the compositions described in (3) above.

[0050] (5) Use According to one embodiment of the present invention, there is provided use of any of the cell populations described in (2), any of the compositions described in (3), any of the containers described in (4), or a human TGFβ component for any of the methods described in (1).

[0051] An embodiment of each of the methods, cells, compositions, containers, and uses described above in (1) to (5) is described in more detail below. The embodiments and explanations of each term described below can be applied to each of the methods, cells, compositions, containers, and uses described above in (1) to (5).

[0052] In one embodiment of the present invention, the method comprises, for example, + a step of recovering a cell population having a T cell ratio of 40% or more, CD8 + a step of recovering a cell population in which the proportion of T cells is 60% or less; + CD8 - The proportion of T cells is CD4 - CD8 + a step of recovering a cell population with a ratio of 0.7 to 1.3 times that of T cells; + CCR7 + a step of recovering a cell population in which the proportion of T cells is 30% or more, or a step of recovering CD4 + T cell ratio ≥ 40%, CD8 + T cell percentage <60%, CD45RA + CCR7 + The method may include the steps of recovering a cell population containing 30% or more T cells, producing a cell population enriched in CD3-positive T cells from human PBMCs, producing a cell population enriched in αβ T cells from human PBMCs, stimulating CD3 in human PBMCs, stimulating CD3 in T cells in human PBMCs, inducing CD3-mediated signaling in human PBMCs, inducing CD3-mediated signaling in T cells in human PBMCs, stimulating human PBMCs with an anti-CD3 antibody, and stimulating T cells in human PBMCs with an anti-CD3 antibody. The step of stimulating T cells with an anti-CD3 antibody may be carried out, for example, by contacting the T cells with the anti-CD3 antibody. In one embodiment of the present invention, the method may include, for example, a step of collecting a sample (e.g., a cell population containing T cells (e.g., PBMCs)) from a patient, a step of concentrating (e.g., by centrifugation) a composition containing PBMCs or a cell population containing T cells, a step of mixing PBMCs or T cells with a medium containing a human TGFβ component, a step of modifying PBMCs or T cells (e.g., by gene transfer with a vector), a step of culturing PBMCs or T cells until a certain number is reached, a step of producing CAR-T cells, a step of purifying, sorting, or purifying T cells from the cell population, a step of recovering T cells from the cell population, a step of testing the transduction rate of T cells (e.g., by flow cytometry), or a step of administering T cells to a patient.

[0053] In one embodiment of the present invention, the method includes, for example, a step of cell sorting (e.g., cell sorting to identify CD4 +The method may not include a step of sorting T cells. Even in such a case, an excellent T cell population can be obtained by using the above method. In addition, for example, when the method does not include cell sorting of a desired cell population according to biomarkers expressed by the cells, it has advantages such as reduced costs, a reduced number of cells to be prepared, and no exogenous antibodies compared to when cell sorting is included. Sorting includes, for example, isolating and obtaining a subject, or isolating a subject to obtain a composition that is more concentrated than before separation. In this embodiment, for example, it includes a step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population, and cell sorting can be performed to obtain CD4 + A method for producing a cell population can be provided that does not include a step of sorting T cells.

[0054] In one embodiment of the present invention, the method may be performed in the absence of exogenous antibodies, e.g., by cell sorting to identify CD4 + When sorting T cells, exogenous antibodies may be contained in the product. However, exogenous antibodies used for cell sorting may transmit unknown signals to cells or may alter the properties of the cells themselves (side effects). Therefore, it is advantageous to perform the sorting in the absence of exogenous antibodies. Furthermore, cell sorting requires individual instrument settings, and failure to properly configure the settings may result in a decrease in the viability of the sorted cells. Even in the case of column cell sorting, it is unclear whether cells expressing CD4 or CD8 antigens at various intensities compared to before culture can be properly separated. In another embodiment, the above method may not include a step of contacting or binding cells with an exogenous antibody or a label. In another embodiment, the CD4 in the above method may be separated. + T cells react with CD4 + The foreign antibody contained in the cell population or composition may be a CD4 T cell. + The percentage of T cells can be 50, 60, 70, 80, 90, 95, 98, 99, or 100%, or can be within a range of any two of those values. Foreign means, for example, obtained from a patient different from the patient from which the cells were derived.

[0055] In one embodiment of the present invention, the cells may be, for example, T cells. The T cells may be, for example, CD3-positive T cells or αβ T cells. αβ T cells include cells expressing αβ TCR. CD3-positive T cells or αβ T cells can be detected, counted, or selected, for example, by flow cytometry. In the Examples described below, a cell population enriched for CD3-positive T cells or αβ T cells was obtained by stimulating human PMBCs with an anti-CD3 antibody. In this case, CD3-positive T cells actively proliferated due to CD3 stimulation, resulting in an increase in the proportion of CD3-positive T cells or αβ T cells.

[0056] In one embodiment of the present invention, the cell population includes, for example, a cell population containing T cells. In one embodiment of the present invention, the cell population containing T cells may be, for example, a cell population containing CD3-positive T cells. The cell population containing T cells may have a percentage of CD3-positive T cells of 90% or more. This percentage may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be any of these values ​​or higher, or may be within a range of any two of these values. This percentage is, for example, preferably 95% or higher, more preferably 97% or higher, even more preferably 98% or higher, and particularly preferably 99% or higher. This percentage may be, for example, 90-100, 95-100, 97-100, 98-100, or 99-100%. The cell population containing the T cells may have a percentage of CD3-positive T cells of 90% or more of the T cells contained in the cell population (i.e., when the number of T cells is taken as the population). This percentage may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be any of these values ​​or higher, or may be within a range of any two of these values. This percentage is, for example, preferably 95% or higher, more preferably 97% or higher, even more preferably 98% or higher, and particularly preferably 99% or higher. This percentage may be, for example, 90-100, 95-100, 97-100, 98-100, or 99-100%. The cell population containing the T cells may also be, for example, a cell population containing αβ T cells. The cell population containing the T cells may have a percentage of αβ T cells of 90% or higher. This percentage may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be equal to or greater than any of these values, or may be within a range of any two of these values. This percentage is, for example, preferably 95% or greater, more preferably 97% or greater, even more preferably 98% or greater, and particularly preferably 99% or greater. This percentage may be, for example, 90 to 100, 95 to 100, 97 to 100, 98 to 100, or 99 to 100%.The cell population containing the T cells may have, for example, an αβ T cell ratio of 90% or more of the T cells contained in the cell population. This ratio may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be any of these values ​​or greater, or may be within a range of any two of these values. This ratio is, for example, preferably 95% or greater, more preferably 97% or greater, even more preferably 98% or greater, and particularly preferably 99% or greater. This ratio may be, for example, 90-100, 95-100, 97-100, 98-100, or 99-100%. The cell population containing the T cells may be, for example, PBMCs. The cell population containing the T cells may be composed of a cell population containing, for example, T cells, B cells, NK cells, monocytes, dendritic cells, or the like. The cell population containing T cells may be, for example, 70, 80, 90, 95% or more, or 100% of the total cells are T cells. The cell population containing T cells may be, for example, an apheresis specimen.

[0057] In one embodiment of the present invention, PBMCs include, for example, human PBMCs. PBMCs may be composed of a cell population including, for example, T cells, B cells, NK cells, monocytes, or dendritic cells. PBMCs may be obtained by removing plasma components, red blood cells, platelets, and granulocytes from human or animal blood by density gradient centrifugation, followed by separation and recovery. PBMCs may be primarily composed of lymphocytes, monocytes, and dendritic cells (e.g., accounting for 80% or more of the total cells). PBMCs may contain 70% or more of T cells. In one embodiment of the present invention, the above cells may exist as a cell population. In one embodiment of the present invention, a population obtained by culturing cells or the total cells in the medium after cell culture is referred to as a cultured cell population.

[0058] In one embodiment of the present invention, CD4 in the cultured cell population or in the total cells in the composition + T cells or CD4 + CD8 - The percentage of T cells may be, for example, 35% or higher. This percentage may be, for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 52, 55, 57, 60, 62, 65, 77, 80, 82, or 85%, or may be equal to or higher than any of these values, or may be within a range of any two of these values. This percentage may be, for example, 35-70, 35-65, 40-60, 45-55, or 47-55%. From the viewpoint of improving suitability for regenerative medicine, 35% or higher is preferred, 40% or higher is more preferred, 45% or higher is more preferred, and 47% or higher is even more preferred. In one embodiment of the present invention, the CD4 + The proportion of T cells may be, for example, 1.5 times or more compared to before culture. This fold increase may be, for example, 1.5, 1.7, 2, 2.2, 2.5, 2.7, 3, or 4 times, or may be any of these values ​​or more, or may be within a range of any two of these values. The cultured cell population or total cells may contain, for example, CD3-positive T cells or αβ-type T cells (for example, 90% or more). The CD4 + The proportion of T cells can be determined, for example, by calculating the percentage of CD4 T cells among the CD3-positive T cells or αβ T cells contained in the cultured cell population or total cells (i.e., when the number of CD3-positive T cells or αβ T cells is taken as the population). + It may also be the proportion of T cells.

[0059] In one embodiment of the present invention, CD8 in the cultured cell population or in the total cells in the composition + T cells or CD4 - CD8 + The percentage of T cells may be, for example, 60% or less. This percentage may be, for example, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 37, 35, 32, 30, 27, 25, 22, or 20%, or may be equal to or less than any of these values, or may be within a range of any two of these values. This percentage may be, for example, 60-30, 60-40, 55-45, 55-40, 50-30, or 44-30%. From the viewpoint of improving suitability for regenerative medicine, 60% or less is preferable, 55% or less is more preferable, 50% or less is more preferable, and 44% or less is even more preferable. In one embodiment of the present invention, the percentage of CD8 T cells in the cultured cell population is 60% or less, 55% or less is more preferable, 50% or less is even more preferable, and 44% or less is even more preferable. + The proportion of T cells may be, for example, 0.8 times or less compared to before culture. This proportion may be, for example, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 times, or may be any of these values ​​or more, or may be within a range of any two of these values. The cultured cell population or total cells may contain, for example, CD3-positive T cells or αβ-type T cells (for example, 90% or more). The CD8 + The proportion of T cells can be determined by, for example, measuring the percentage of CD8+ T cells among CD3+ T cells or αβ-type T cells contained in the cultured cell population or total cells. + It may also be the proportion of T cells.

[0060] In one embodiment of the present invention, CD4 in the cultured cell population or in the total cells in the composition + CD8 - The proportion of T cells, e.g., CD4 - CD8 + The ratio may be 0.7 to 1.3 times the ratio of T cells. This ratio may be, for example, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.05, 1.1, 1.15, 1.2, 1.25, or 1.3 times, or may be equal to or greater than any one of these values, or may be within a range of any two of these values. This ratio may be, for example, 0.8 to 1.3, 0.9 to 1.2, 0.9 to 1.1, or 0.95 to 1.05 times. From the viewpoint of improving suitability for regenerative medicine, a ratio of 0.8 times or more is preferable, 0.9 times or more is more preferable, and 1 time or more is even more preferable.

[0061] In one embodiment of the present invention, CD45RA in the cultured cell population or in the total cells in the composition + CCR7 + The percentage of T cells may be, for example, 30% or more. This percentage may be, for example, 30, 32, 35, 37, 40, 42, 45, 47, 50, 52, 55, 57, 60, 62, 65, 77, 80, 82, 85, 87, or 90%, or may be any of these values ​​or more, or may be within a range of any two of these values. This percentage may be, for example, 30-90, 40-90, 50-90, or 60-90%. From the viewpoint of improving suitability for regenerative medicine, 40% or more is preferable, 50% or more is more preferable, and 60% or more is even more preferable. In one embodiment of the present invention, the CD45RA expression level in the cultured cell population is 40% or more, more preferably 50% or more, and even more preferably 60% or more. + CCR7 + The proportion of T cells may be, for example, 1.2 times or more compared to before culture. This fold increase may be, for example, 1.2, 1.5, 1.7, 2, 2.2, 2.5, 2.7, or 3 times, or may be any of these values ​​or more, or may be within a range of any two of these values. The cultured cell population or total cells may contain, for example, CD3-positive T cells or αβ-type T cells (for example, 90% or more). The CD45RA + CCR7 + The proportion of T cells can be determined by, for example, determining the percentage of CD45RA T cells among CD3-positive T cells or αβ-type T cells contained in the cultured cell population or total cells. + CCR7 + It may also be the proportion of T cells.

[0062] In one embodiment of the present invention, the proportion of cells expressing stem cell memory T cell markers among the total cells in the cultured cell population or composition may be, for example, 30% or more. This proportion may be, for example, 30, 32, 35, 37, 40, 42, 45, 47, 50, 52, 55, 57, 60, 62, 65, 77, 80, 82, 85, 87, or 90%, or may be any of these values ​​or greater, or may be within a range of any two of these values. This proportion may be, for example, 30-90, 40-90, 50-90, or 60-90%. From the perspective of improving suitability for regenerative medicine, 40% or greater is preferred, 50% or greater is more preferred, and 60% or greater is even more preferred. In one embodiment of the present invention, the proportion of cells expressing stem cell memory T cell markers in the cultured cell population may be, for example, 1.2-fold or greater than that before culture. This multiple may be, for example, 1.2, 1.5, 1.7, 2, 2.2, 2.5, 2.7, or 3, or may be equal to or greater than any of these values, or may be within a range between any two of these values. The cultured cell population or total cells may contain, for example, CD3-positive T cells or αβ T cells (e.g., 90% or more). The proportion of stem cell memory T cell marker-expressing cells may be, for example, the proportion of stem cell memory T cell marker-expressing cells among CD3-positive T cells or αβ T cells contained in the cultured cell population or total cells.

[0063] In one embodiment of the present invention, the culture may be performed in the presence of, for example, 0.01 ng / mL or more of a human TGFβ component, and the composition may contain, for example, 0.01 ng / mL or more of a human TGFβ component, which may be, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12, 0.15, 0.17, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.5, 1.7, 2, 2.2, 2.5, 3, 4, 5, 6, 7, or 8 ng / mL, or any value greater than or equal to, or within a range between any two of these values. This concentration may be, for example, 0.01 to 8, 0.01 to 4, 0.01 to 3, 0.01 to 2, 0.05 to 8, 0.05 to 4, 0.05 to 3, 0.05 to 2, 0.1 to 2.5, 0.1 to 2, 0.1 to 1, 0.2 to 2, 0.4 to 2.4, 0.4 to 2, 0.4 to 1.5, or 0.4 to 1 ng / ml. From the viewpoint of improving suitability for regenerative medicine, 0.2 ng / ml or more is preferable, 0.3 ng / ml or more is more preferable, 0.4 ng / ml or more is even more preferable, and 0.5 ng / ml or more is particularly preferable. From the viewpoint of improving suitability for regenerative medicine, 3 ng / ml or less is preferable, 2.5 ng / ml or less is more preferable, 2 ng / ml or less is more preferable, 1.7 ng / ml or less is more preferable, 1.5 ng / ml or less is even more preferable, and 1.3 ng / ml or less is particularly preferable. The concentration of the human TGF-β component may be, for example, 0.21 pM or higher. This concentration may be, for example, 0.2, 0.5, 0.8, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 65, 68, 70, 80, 90, 100, or 150 pM, or may be any of these values ​​or higher, or may be within a range of any two of these values. This concentration may be, for example, 0.2-150, 0.2-70, 0.2-50, 0.2-30, 2-70, 2-50, 2-30, 2-70, 4-70, 4-50, 4-30, or 4-20 ng / ml. From the viewpoint of improving suitability for regenerative medicine, a concentration of 4 ng / ml or higher is preferred, 6 ng / ml or higher is more preferred, 8 ng / ml or higher is even more preferred, and 10 ng / ml or higher is particularly preferred.From the viewpoint of improving suitability for regenerative medicine, the concentration is preferably 70 pM or less, more preferably 60 ng / ml or less, more preferably 50 ng / ml or less, more preferably 40 ng / ml or less, even more preferably 30 ng / ml or less, and particularly preferably 20 ng / ml or less. By adjusting the concentration of human TGF-β component during culture, CD4 in the cell population can be reduced. + T cells or CD8 + The proportion of T cells can be adjusted, which allows for the differentiation of CD4 + T cells and CD8 + The balance of T cell ratios can be improved.

[0064] In one embodiment of the present invention, the human TGFβ component includes, for example, a molecule having TGFβ activity and derived from human TGFβ. The human TGFβ component may be, for example, (a) a protein having an amino acid sequence set forth in any of SEQ ID NOS: 1 to 3 or a biologically active fragment thereof, or (b) a protein having an amino acid sequence with 90% or greater identity to the amino acid sequence set forth in any of SEQ ID NOS: 1 to 3 or a biologically active fragment thereof. The human TGFβ component may be, for example, a protein having an amino acid sequence with one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence set forth in any of SEQ ID NOS: 1 to 3 or a biologically active fragment thereof. TGFβ activity includes, for example, the activity of binding to a human TGFβ receptor (e.g., TGFβR2) and promoting TGFβ signaling. TGFβ signaling may involve, for example, phosphorylation of SMAD2 / SMAD3. The biologically active fragment includes, for example, a polypeptide having TGFβ activity but lacking a portion of human TGFβ not involved in TGFβ activity.

[0065] In one embodiment of the present invention, the human TGFβ includes a protein also referred to as human transforming growth factor β or human TGF-β. Human TGFβ includes the isoforms human TGFβ-1, human TGFβ-2, and human TGFβ-3. Details of the amino acid sequence of human TGFβ can be found on websites such as NCBI or UniProt. The primary accession numbers for human TGFβ listed in UniProt are, for example, P01137, P61812, and P10600 for each isoform. The amino acid sequences of human TGFβ-1, TGFβ-2, and TGFβ-3 include, for example, the amino acid sequences set forth in SEQ ID NOs: 1 to 3, respectively.

[0066] In one embodiment of the present invention, the culture may be carried out in the presence of, for example, ascorbic acid, a derivative thereof, or a salt thereof, and the composition may contain, for example, ascorbic acid, a derivative thereof, or a salt thereof. The concentration of ascorbic acid, a derivative thereof, or a salt thereof may be, for example, 1 μM or higher. This concentration may be, for example, 1, 2, 5, 8, 10, 20, 50, 80, 100, 120, 150, 200, 500, 700, 1000, 1200, 1500, or 2000 μM, or may be any value or higher, or may be within a range of any two values. This ratio may be, for example, 1 to 2000, 5 to 1500, 10 to 1000, 50 to 500, or 80 to 200 μM. From the perspective of improving suitability for regenerative medicine, a concentration of 5 μM or higher is preferred, 10 μM or higher is more preferred, and 50 μM or higher is even more preferred.

[0067] In one embodiment of the present invention, the ascorbic acid includes, for example, a compound designated by the IUPAC nomenclature (2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one. Ascorbic acid includes a compound with CAS Registry Number 50-81-7. Ascorbic acid includes the L-isomer and the D-isomer. In one embodiment of the present invention, the ascorbic acid derivative includes, for example, AA2G or AA2P. AA2G includes, for example, a molecule also designated as AA-2G, L-ascorbic acid 2-glucoside, or (2R)-2-[(1S)-1,2-dihydroxyethyl]-3-hydroxy-4-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2H-furan-5-one. AA2P includes, for example, a molecule also known as L-ascorbic acid 2-phosphate or [(2R)-2-[(1S)-1,2-dihydroxyethyl]-3-hydroxy-5-oxo-2H-furan-4-yl] dihydrogen phosphate. In one embodiment of the present invention, the salt of ascorbic acid or a derivative thereof includes, for example, a sodium salt or a calcium salt.

[0068] In one embodiment of the present invention, the culture may be carried out in the presence of PLT, and the composition may contain PLT. The concentration of PLT may be, for example, 0.05% or more. This concentration may be, for example, 0.05, 0.07, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.5, 1.7, 2, 2.2, 2.5, 3, 4, 5, 6, 7, or 8%, or may be any value or more thereof, or may be within a range of any two values ​​thereof. This concentration may be, for example, 0.1 to 4, 0.2 to 3, 0.3 to 2, 0.5 to 1.5, or 0.7 to 1.5%. From the viewpoint of improving suitability for regenerative medicine, 0.2% or more is preferable, 0.3% or more is more preferable, and 0.7% or more is even more preferable.

[0069] In one embodiment of the present invention, the PLTs include a component also referred to as platelet lysate. The PLTs may be human PLTs. The PLTs may include a growth factor such as PDGF, IGF-1, or EGF.

[0070] In one embodiment of the present invention, the culture may be performed in the presence of, for example, an IL-2 component, and the composition may contain, for example, an IL-2 component. The concentration of the IL-2 component may be, for example, 1 ng / mL or higher. This concentration may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / mL, or any value higher than these values, or within a range of any two values ​​thereof. This ratio may be, for example, 1 to 20, 5 to 20, 5 to 15, or 8 to 12 ng / mL. From the viewpoint of improving suitability for regenerative medicine, a concentration of 2 ng / mL or higher is preferable, 5 ng / mL or higher is more preferable, and 8 ng / mL or higher is even more preferable. The concentration of the IL-2 component may be, for example, 65 pM or higher. This concentration may be, for example, 65, 100, 200, 300, 400, 500, 600, 700, 800, 900, 100, 1100, 1200, or 1300 ng / mL, or any value greater than or equal to these values, or within a range of any two values ​​between these values. This ratio may be, for example, 65 to 1300, 100 to 1000, 300 to 800, 500 to 800, or 600 to 700 pM. From the viewpoint of improving suitability for regenerative medicine, a concentration of 100 ng / mL or greater is preferable, 300 ng / mL or greater is more preferable, and 500 ng / mL or greater is even more preferable.

[0071] In one embodiment of the present invention, the IL-2 component includes, for example, a molecule having IL-2 activity and derived from IL-2. The IL-2 component may be, for example, (a) a protein having the amino acid sequence set forth in SEQ ID NO: 4 or a biologically active fragment thereof, or (b) a protein having an amino acid sequence with 90% or more identity to the amino acid sequence set forth in SEQ ID NO: 4 or a biologically active fragment thereof. The IL-2 component may be, for example, a protein having an amino acid sequence with one to several amino acid deletions, substitutions, insertions, or additions relative to the amino acid sequence set forth in SEQ ID NO: 4 or a biologically active fragment thereof. IL-2 activity includes, for example, the activity of promoting IL-2 signal transduction. The biologically active fragment includes, for example, a polypeptide having IL-2 activity, in which a portion of IL-2 not involved in IL-2 activity has been deleted.

[0072] In one embodiment of the present invention, IL-2 includes, for example, a component also referred to as interleukin-12. Details of IL-2, such as its amino acid sequence, can be found on websites such as NCBI or UniProt. The primary accession number for IL-2 listed in UniProt is, for example, P60568. The amino acid sequence of IL-2 includes, for example, the amino acid sequence shown in SEQ ID NO: 4. IL-2 includes, for example, human-derived IL-2.

[0073] In one embodiment of the present invention, the culturing may be performed in the absence of exogenous antibodies, including, for example, exogenous anti-CD4, anti-CD8, anti-CD45RA, or anti-CCR7 antibodies.

[0074] In one embodiment of the present invention, the culture may be performed in a serum-free environment. The Examples described below demonstrate that the percentage of CD4(+)CD8(-) T cells is improved when cultured in the presence of TGFβ and bovine serum-free conditions compared to when cultured in the presence of TGFβ and bovine serum. Examples of bovine serum include, for example, fetal bovine serum. "Free" includes a state in which the target component is not added to the medium, a state in which the medium is completely free of the target component, a state in which the medium is substantially free of the target component, or a state in which the medium does not contain the component at a concentration above the detection limit. "Serum-free" includes being substantially serum-free. The serum may include bovine or mammalian (e.g., human or mouse) serum. "Serum-free" does not necessarily include, for example, human serum-free. Bovine serum-free is preferred from the viewpoint of producing a superior T cell population. According to another embodiment, a serum-free medium containing TGFβ is provided.

[0075] In one embodiment of the present invention, the cell population is, for example, CD4 + CD8 - The cell population may have a T cell percentage of 50% or more. This percentage may be, for example, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 65, 77, 80, 82, 85, 87, or 90%, or may be any of these values ​​or more, or may be within a range of any two of these values. This percentage may be, for example, 50-90, 50-80, 50-70, 50-65%, or 55-70%. From the viewpoint of suppressing the decline in T cell function, 50% or more is preferable, 53% or more is more preferable, and 55% or more is even more preferable. The cell population may be, for example, a cell population containing CD3-positive T cells or αβ-type T cells (e.g., 90% or more). The CD4 + CD8 - The proportion of T cells can be determined, for example, by calculating the CD4 T cell count among the CD3-positive T cells or αβ T cells contained in the cell population (i.e., when the number of CD3-positive T cells or αβ T cells is taken as the population). + CD8 - It may also be the proportion of T cells.

[0076] In one embodiment of the present invention, the cell population is, for example, CD4 - CD8 + The cell population may be a cell population containing 50% or less T cells. This percentage may be, for example, 50, 49, 48, 47, 46, 45, 42, 40, 37, 35, 32, 30, 27, 25, 22, or 20%, or may be equal to or less than any of these values, or may be within a range of any two of these values. This percentage may be, for example, 20-50, 25-45, 25-40, or 30-40%. From the viewpoint of suppressing the decline in T cell function, 50% or less is preferable, 45% or less is more preferable, and 40% or less is even more preferable. The cell population may be, for example, a cell population containing CD3-positive T cells or αβ-type T cells (e.g., 90% or more). The CD4 - CD8 + The proportion of T cells can be determined by, for example, measuring the CD4+ T cells among CD3+ T cells or αβ-type T cells contained in the cell population. - CD8 + It may also be the proportion of T cells.

[0077] In one embodiment of the present invention, the cell population is, for example, CD4 + CD8 - The proportion of T cells is CD4 - CD8 + The cell population may be a cell population with a ratio of 0.9 or more times the ratio of T cells. This ratio may be, for example, 0.9, 0.95, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, or 2.5 times, or may be any of these values ​​or more, or may be within a range of any two of these values. This ratio may be, for example, 0.9 to 2.5, 1 to 2, 1.5 to 2, or 1.5 to 2.5 times. From the viewpoint of suppressing the decline in T cell function, a ratio of 0.9 or more is preferable, 1 or more is more preferable, and 1.5 or more is even more preferable. The cell population may be, for example, a cell population containing CD3-positive T cells or αβ T cells (e.g., 90% or more).

[0078] In one embodiment of the present invention, the cell population is, for example, CD45RA + CCR7 + The cell population may contain 35% or more CD45RA T cells. This percentage may be, for example, 35, 40, 45, 50, 55, 60, 65, 70, 75, 77, 80, 81, 82, 83, 84, 85, 87, 90, 92, or 95%, or may be any of these values ​​or greater, or may be within a range of any two of these values. This percentage may be, for example, 35-95, 40-95, 50-95, 70-90, 75-85, or 80-85%. From the viewpoint of suppressing the decline in T cell function, 60% or greater is preferable, 70% or greater is more preferable, and 80% or greater is even more preferable. The cell population may be, for example, a cell population containing CD3-positive T cells or αβ-type T cells (e.g., 90% or greater). The CD45RA T cells may be, for example, 90% or greater. + CCR7 + The percentage of cells can be determined by, for example, determining whether the CD45RA T cells are CD3-positive or αβ T cells contained in the cell population. + CCR7 + It may also be a percentage of cells.

[0079] In one embodiment of the present invention, the cell population may be a cell population in which the proportion of cells expressing stem cell memory T cell markers is 35% or more. This proportion may be, for example, 35, 40, 45, 50, 55, 60, 65, 70, 75, 77, 80, 81, 82, 83, 84, 85, 87, 90, 92, or 95%, or may be equal to or greater than any of these values, or may be within a range of any two of these values. This proportion may be, for example, 35-95, 40-95, 50-95, 70-90, 75-85, or 80-85%. From the viewpoint of suppressing the decline in T cell function, a proportion of 60% or more is preferable, 70% or more is more preferable, and 80% or more is even more preferable. The cell population may be, for example, a cell population containing CD3-positive T cells or αβ T cells (e.g., 90% or more). The proportion of cells expressing a stem cell memory T cell marker may be, for example, the proportion of cells expressing a stem cell memory T cell marker among CD3-positive T cells or αβ T cells contained in the cell population.

[0080] In one embodiment of the present invention, the percentage of CD3-positive T cells or αβ T cells among the total cells in the cell population, cultured cell population, or composition may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be equal to or greater than any of these values, or may be within a range of any two of these values.

[0081] In one embodiment of the present invention, the cells may be, for example, cells to which no exogenous antibodies or labels are bound. In one embodiment of the present invention, the cell population may be, for example, free of exogenous antibodies or labels. Exogenous antibodies include, for example, exogenous anti-CD4 antibodies, anti-CD8 antibodies, anti-CD45RA antibodies, or anti-CCR7 antibodies. Labels include, for example, GFP.

[0082] In one embodiment of the present invention, the CD4 + T cells, for example, CD4 + CD8 - In one embodiment of the present invention, the above-mentioned CD8 + T cells, for example, CD4 - CD8 + Contains T cells.

[0083] In one embodiment of the present invention, after the protein name + and (+) indicates positive, - and (-) means negative. For example, CD4 + , CD8 + , CD45RA + , or CCR7 + The above-mentioned CD4 - , CD8 - includes negative gene expression for each gene. Positivity / negative may be determined, for example, by a cell surface antigen test (e.g., flow cytometry).

[0084] In one embodiment of the present invention, the culturing includes incubating the cells under conditions suitable for proliferation or maintenance. The culturing may include contacting a medium with the cells to produce a cell-containing composition, and incubating the cell-containing composition. The incubating step may be performed while the cell-containing composition is left standing or stirred. The incubation may be performed at about 37°C and in an atmosphere of about 5% CO2. The contacting step may include, for example, seeding the cells in the medium or mixing the cells with the medium. The culturing may be performed in a bovine serum-free medium. The proliferation may include, for example, expanding the cells 10-fold or more from the start of the culture. This proliferation may be 10, 50, 100, 200, 300, 400, or 500-fold, or may be any of these values ​​or more, or may be within a range of any two of these values.

[0085] In one embodiment of the present invention, the cell population includes a plurality of cells generated by, for example, cell division. The cell population may be the total cells contained in a cell fraction.

[0086] In one embodiment of the present invention, the culture time may be, for example, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 17, 18, 20, 30, or 35 days, or may be any value greater than or equal to any two of these values. This culture time may be, for example, 1 to 20 days, 3 to 17 days, 4 to 16 days, or 5 to 14 days. Culturing includes the time for incubating a medium containing cells.

[0087] In one embodiment of the present invention, the composition or medium may be a medium used for culturing cells. Any common basal medium may be used as a base component in the medium, and the composition of the basal medium is not limited. The basal medium may contain, for example, amino acids (e.g., glutamine), peptides, proteins, inorganic salts, vitamins, minerals, or a carbon source (e.g., glucose). Examples of basal media include RPMI 1640 (e.g., Nacalai Tesque), OpTmizer (Gibco TM CTS TM OpTmizer TMT Cell Expansion SFM, bottle format (Brand: Gibco TM A1048501), DMEM (Dulbecco's Modified Eagle Medium) (e.g., Thermo Fisher Scientific Inc.), DMEM F12 (e.g., Thermo Fisher Scientific Inc.), DMEM, high glucose (e.g., Thermo Fisher Scientific Inc.), E-MEM (e.g., Fujifilm Wako Pure Chemical Corporation), G-MEM (e.g., Fujifilm Wako Pure Chemical Corporation), MEMα (e.g., Fujifilm Wako Pure Chemical Corporation), MEM (e.g., Thermo Fisher Scientific Inc.), Ham's F-12 (e.g., Thermo Fisher Scientific Inc.), Ham's F-12K (e.g., Fujifilm Wako Pure Chemical Corporation), D-MEM / Ham's F-12 (e.g., Fujifilm Wako Pure Chemical Corporation), IMDM (e.g., Fujifilm Wako Pure Chemical Corporation), Leibovitz's L-15 (e.g., Fujifilm Wako Pure Chemical Corporation), LymphoONE™ T-Cell Expansion Xeno-Free Medium (e.g., Takara Bio), iMediam for T (e.g., GC LYMPHOTEC Inc.), PRIME-XV T Cell CDM (e.g., FUJIFILM Irvine Scientific), or media supplemented with amino acids, peptides, proteins, inorganic salts, vitamins, minerals, or carbon sources may also be used. + From the viewpoint of increasing the proportion of T cells, the medium is preferably a bovine serum-free medium. The medium may be a medium for culturing T cells or PBMCs. The medium may be a liquid medium or a solid medium.

[0088] In one embodiment of the present invention, the container includes a culture container or a medical container. The culture container may be, for example, a plate-type, a petri dish-type, a flask-type, or a bottle-type. The medical container may be, for example, a flask-type, a bottle-type, a tube-type, a bag-type, or a syringe-type.

[0089] In one embodiment of the present invention, the recovering step may include, for example, transferring the desired cells, cell population, or cell fraction from a container (e.g., a culture container) containing a composition containing the cells or cell population to another container (e.g., a medical container). The recovering step may include a separation, sorting, or purification step. The transfer may be performed using a syringe or pipette, or by tilting the container. The recovering step may be performed in a sterile environment.

[0090] In one embodiment of the present invention, the composition may be, for example, an additive. The additive may be an additive for supplying additional nutritional components to a basal medium. The additive may be, for example, an additive containing a human TGFβ component.

[0091] In one embodiment of the present invention, the pharmaceutical composition may be prepared by mixing cells (e.g., T cells) with one or more pharmaceutically acceptable carriers using any method known in the art of pharmaceuticals. The pharmaceutical composition may also contain a stabilizer, buffer, or pH adjuster. The dosage, administration interval, administration method, and administration route are not particularly limited and can be appropriately selected depending on the patient's age, weight, symptoms, target organ, etc. The pharmaceutical composition preferably contains a therapeutically effective amount, or an effective amount of T cells that exerts the desired effect. In one embodiment of the present invention, a therapeutically effective amount includes the amount necessary to clinically observe improvement or suppression of symptoms in a patient. In one embodiment of the present invention, pharmaceutically acceptable means a state suitable for use within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. The pharmaceutical composition may not contain an exogenous antibody. The exogenous antibody may include, for example, an exogenous anti-CD4 antibody, anti-CD8 antibody, anti-CD45RA antibody, or anti-CCR7 antibody. The pharmaceutical composition may be in a frozen state. The pharmaceutical composition may also contain a cryoprotectant (e.g., DMSO).

[0092] In one embodiment of the invention, the subject (including the patient) includes a human, and may be a patient diagnosed with or in need of treatment for a disease (e.g., cancer).

[0093] In one embodiment of the present invention, culturing in the presence of a particular component includes culturing in a state in which the particular component is included in the medium.

[0094] In one embodiment of the present invention, the proportion of a particular cell may be the proportion of the total cells in a composition containing the cells, or the proportion of the total cells in a container containing the cells.

[0095] In one embodiment of the present invention, exogenous components include components derived from an organism (individual) other than the organism (individual) from which the cells (e.g., PBMCs or T cells) used for culture were derived, or components not produced by the cells used for culture.

[0096] In one embodiment of the present invention, enriched includes, for example, a state of high concentration or a state in which the proportion of a particular cell in a cell population is high.

[0097] In one embodiment of the present invention, the homology (%) may be calculated by, for example, calculating the percentage of homologous amino acids between multiple amino acid sequences according to methods known in the art. Before calculating the percentage, the sequences to be compared are aligned, and gaps are introduced into the sequences, if necessary, to maximize the percentage of homologous / identical amino acids. Alignment methods, percentage calculation methods, and related computer programs are well known in the art. Homology may be calculated using, for example, global or local alignment. The former may be calculated using the Needleman-Wunsch algorithm (Needleman et al., J. Mol. Biol. 1970 Mar;48(3):443-53) or EMBOSS Needle (Rice et al., EMBOSS User's Guide: Practical Bioinformatics, 25 July 2011). The default settings for EMBOSS Needle may include, for example, MATRIX: BLOSUM62, Gap Open Penalty: 10, Gap Extend Penalty: 0.5, Output formats: pair, End Gap Penalty: false, End Gap Open Penalty: 10, End Gap Extend Penalty: 0.5. The latter may be determined using the BLAST algorithm (Altschul et al., J Mol Biol. 1990 Oct 5;215(3):403-10.) or calculated using blastp (The BLAST Sequence Analysis Tool. The NCBI Handbook, 2nd edition, March 15, 2013.). The default settings for blastp may include, for example, MATRIX: BLOSUM62, Gap Open Penalty: 11, Extension: 1, Compositional adjustments: Conditional compositional score matrix adjustment.Homology may be expressed as (number of homologous amino acids / number of amino acids in the amino acid sequence to be compared) × 100. Homology is preferably calculated using global alignment. Unless otherwise specified, a test homology of a particular value or greater includes a value calculated by at least one of the above algorithms of a particular value or greater. Any of the above algorithms may be used with default settings.

[0098] In one embodiment of the present invention, the homology value of 90% or more may be, for example, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or may be any of these values ​​or more, or within a range of any two of these values. From the viewpoint of maintaining functional equivalence, this percentage is preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. In one embodiment of the present invention, the number of homologous sequences may be, for example, 15, 10, 8, 6, 5, 4, 3, 2, 1, or 0, or may be any of these values ​​or less, or within a range of any two of these values. From the viewpoint of maintaining functional equivalence, this number is preferably 15 or less, more preferably 10 or less, and particularly preferably 5 or less.

[0099] In one embodiment of the present invention, when substitutions are made to the original sequence, conservative amino acid substitutions (see, e.g., French et al., J. Mol. Evol. (1983) 19, 171-175) are preferred. In one embodiment of the present invention, conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a side chain with similar properties. Amino acid residues are classified into several families based on their side chains, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Conservative amino acid substitutions are preferably substitutions between amino acid residues within the same family.

[0100] In one embodiment of the present invention, the above-mentioned amino acid is a general term for organic compounds having an amino group and a carboxyl group. When a polypeptide according to an embodiment of the present invention comprises a "specific amino acid sequence," any of the amino acids in the amino acid sequence may be chemically modified. Furthermore, any of the amino acids in the amino acid sequence may form a salt or a solvate. Furthermore, any of the amino acids in the amino acid sequence may be in the L- or D-form. Even in such cases, the polypeptide according to an embodiment of the present invention can be said to comprise the above-mentioned "specific amino acid sequence." Examples of chemical modifications that amino acids contained in proteins undergo in vivo include N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, glycosylation, etc.).

[0101] In one embodiment of the present invention, the salt is not particularly limited and includes, for example, inorganic salts or organic salts (see, for example, "Bharate et al., Drug Discov Today. 2021 Feb;26(2):384-398" or "Berge et al., J Pharm Sci. 1977 Jan;66(1):1-19"). Salts include, for example, metal salts, ammonium salts, salts with organic bases, salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. Metal salts include, for example, alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, barium salts, etc.), aluminum salts, and the like. Salts with organic bases include, for example, salts with trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, and the like. Salts with inorganic acids include, for example, salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc. Salts with organic acids include, for example, salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, mesylic acid, tosylic acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Salts with basic amino acids include, for example, salts with arginine, lysine, ornithine, etc. Salts with acidic amino acids include, for example, salts with aspartic acid, glutamic acid, etc. Salts include pharmaceutically acceptable salts. In one embodiment of the present invention, pharmaceutically acceptable salts include forms that have reasonable benefits for pharmaceutical use. When used as medium components, these salts can exhibit effects similar to those of the free form.

[0102] All publications cited herein are incorporated by reference in their entirety. In this specification, "or" is used when "at least one or more" of the items listed in the text can be employed. The same applies to "alternative." In this specification, when it is stated that "within a range of two values," the range also includes the two values ​​themselves. In this specification, "A to B" includes A and B as well as values ​​between A and B.

[0103] One embodiment of the present invention may include, for example, the following numbered embodiments: 1. A method for detecting CD4+ in a cell population, comprising culturing a cell population containing human T cells in the presence of a human transforming growth factor β (human TGFβ) component to obtain a cultured cell population. + 2. The method according to claim 1, wherein the culture is carried out in a bovine serum-free environment. + T cells react with CD4 + 4. The method according to 1 or 2 above, wherein the cells are CD4 T cells by cell sorting. + 5. The method according to any one of 1 to 3 above, which does not include a step of separating T cells. 6. The method according to any one of 1 to 4 above, wherein the culture is carried out in the presence of 0.01 to 3 ng / mL of a human TGFβ component. + 7. The method according to any one of 1 to 5 above, wherein the cultured cell population is a cell population containing 40% or more T cells. + 8. The method according to any one of 1 to 6 above, wherein the proportion of T cells in the cell population is 60% or less. + 9. The method according to any one of 1 to 7 above, comprising the step of recovering a cell population in which the proportion of T cells is 40% or more. + 10. The method according to any one of 1 to 8 above, comprising the step of recovering a cell population in which the proportion of T cells is 60% or less. + CD8 - The proportion of T cells is CD4 - CD8 + 11. The method according to any one of 1 to 9 above, which comprises the step of recovering a cell population in an amount of 0.7 to 1.3 times the proportion of T cells. + CCR7 + 12. The method according to any one of 1 to 10 above, comprising the step of recovering a cell population in which the proportion of T cells is 30% or more. + T cell ratio ≥ 40%, CD8 + T cell percentage 60% or less and CD45RA + CCR7 + The method according to any one of claims 1 to 11 above, comprising the step of recovering a cell population in which the proportion of T cells is 30% or more. 13. The method according to any one of claims 1 to 12 above, wherein the cell population containing human T cells is a cell population containing CD3-positive T cells. 14. The method according to claim 13, wherein the proportion of CD3-positive T cells in the cell population containing human T cells is 90% or more. 15. The method according to any one of claims 1 to 14, wherein the cell population containing human T cells is a cell population containing αβ T cells. 16. The method according to claim 15, wherein the proportion of αβ T cells in the cell population containing human T cells is 90% or more. 17. The method according to any one of claims 1 to 16 above, comprising the step of producing a cell population enriched in CD3-positive T cells from human peripheral blood mononuclear cells (PBMCs). 18. The method according to any one of claims 1 to 17 above, comprising the step of producing a cell population enriched in αβ T cells from human PBMCs. 19. The method according to any one of 1 to 18 above, comprising a step of stimulating human PBMCs with an anti-CD3 antibody. 20. The method according to any one of 1 to 19 above, wherein the culturing is further carried out in the presence of ascorbic acid, a derivative thereof, or a salt thereof. 21. The method according to any one of 1 to 20 above, wherein the culturing is further carried out in the presence of platelet lysate. 22. The method according to any one of 1 to 21 above, wherein the culturing is carried out in the absence of an exogenous antibody. 23. The method according to any one of 1 to 22 above, wherein the human TGFβ component has TGFβ activity and is (a) a protein having an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3 or a biologically active fragment thereof, or (b) a protein having an amino acid sequence having 90% or more homology to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3 or a biologically active fragment thereof. 24. A cell population obtained by the method according to any one of 1 to 23 above. 25. A cell population containing CD3-positive T cells, wherein the CD3-positive T cells contained in the cell population are CD4 + CD8 - 26. A cell population containing 50% or more CD3-positive T cells. 27. A cell population containing 90% or more CD4+ T cells. -CD8 + 27. The cell population according to 25 or 26 above, wherein the proportion of T cells is 50% or less. 28. Among the CD3-positive T cells contained in the cell population, CD45RA + CCR7 + 29. The cell population according to any one of 25 to 28 above, which is not bound to an exogenous antibody. 30. A pharmaceutical composition comprising the cell population according to any one of 25 to 29 above. 31. The pharmaceutical composition according to 30 above, which is free of exogenous antibodies. 32. A method for producing a cell population containing human T cells, comprising culturing the cell population in the presence of a human TGFβ component, and immunizing the cell population with CD4 + 33. A method for producing a cell population, comprising the step of recovering a cultured cell population having a proportion of T cells of 40% or more. + 34. The method according to claim 32, wherein the cultured cell population contains 60% or less T cells. + CCR7 + 35. The method according to claim 33, wherein the proportion of T cells in the cell population is 30% or more. 36. A method according to claim 33, further comprising culturing a cell population containing human T cells in the presence of a human TGFβ component, and culturing the cell population containing human T cells in the presence of a human TGFβ component ... + CD8 - The proportion of T cells is CD4 - CD8 + 36. A method for producing a cell population, comprising the step of recovering a cultured cell population in which the proportion of CD4 in the cell population is 0.7 to 1.3 times the proportion of T cells. + T cells and CD8 + 37. A method for approximating the proportion of T cells, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + 38. A method for producing a cell population having an improved proportion of T cells. 39. A method for producing a cell population not bound to an exogenous antibody. 40. A method for producing a cell population having an improved proportion of CD4 T cells by cell sorting. + 41. The method for producing T cells according to any one of claims 37 to 39, which does not include a step of separating T cells. + CD8 - The proportion of T cells is CD4 - CD8 + 42. A method for producing a cell population containing human T cells in the presence of a human TGFβ component, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population containing CD4 + T cells and CD8 + 43. A method for producing a cell population with a similar proportion of T cells. + CD8 - The proportion of T cells is CD4 - CD8 + 43. A method for producing a cell population having an improved proportion of stem cell memory T cell marker-expressing cells in a cell population, the method comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. 44. A method for improving the proportion of cells expressing stem cell memory T cell markers in a cell population, the method comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. 45. A method for producing a cell population having an improved proportion of stem cell memory T cells in a cell population, the method comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. 46. A method for producing a cell population having an improved proportion of stem cell memory T cells in a cell population, the method comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + CCR7 + 47. A method for increasing the proportion of T cells, comprising: + CCR7 + The method according to claim 46, comprising the step of recovering a cell population in which the proportion of T cells is 30% or more. 48. The method according to claim 46 or 47, wherein the culturing is carried out in the presence of 0.01 to 3 ng / mL of a human TGFβ component. 49. The method according to any one of claims 46 to 48, wherein the culturing is carried out in a bovine serum-free environment. 50. The method according to any one of claims 46 to 49, wherein the proportion of CD3-positive T cells in the cell population containing human T cells is 90% or more. 51. The method according to any one of claims 46 to 50, comprising the step of stimulating human PBMCs with an anti-CD3 antibody. 52. A cell population obtained by the method according to any one of claims 32 to 51. 53. CD4 + A composition comprising a human TGFβ component for use in producing a cell population with an enhanced proportion of T cells. 54. CD4 + T cells and CD8 + 55. A composition comprising a human TGFβ component for use in producing a cell population with a similar proportion of T cells, the cell population comprising CD4 + CD8 - The proportion of T cells is CD4 - CD8 + The composition according to claim 54, which is a cell population with a proportion of 0.7 to 1.3 times the proportion of T cells. 56. A culture medium for T cells, comprising 0.01 to 3 ng / ml of a TGFβ component. 57. A culture medium for T cells according to claim 56, further comprising an ascorbic acid derivative. 58. A culture medium for T cells according to claim 56, further comprising an ascorbic acid derivative and a platelet lysate. 59. A method for culturing T cells, comprising a step of culturing T cells in the presence of 0.01 to 3 ng / ml of a TGFβ component. 60. The method for culturing T cells according to claim 59, wherein the culturing is performed in the presence of 0.01 to 3 ng / ml of a TGFβ component and in the presence of an ascorbic acid derivative. 61. The method for culturing T cells according to claim 59, wherein the culturing is performed in the presence of 0.01 to 3 ng / ml of a TGFβ component and in the presence of an ascorbic acid derivative and a platelet lysate. 62. The culture method according to any one of the above items 59 to 61, wherein the culture is carried out in a bovine serum-free environment. 63. The culture method according to any one of the above items 59 to 62, wherein the culture is a method for culturing T cells not bound to an exogenous antibody. 64. A method for culturing CD4 T cells by cell sorting.+ 64. The culture method according to any one of claims 59 to 63, which does not include a step of separating T cells.

[0104] Although the embodiments of the present invention have been described above, these are merely examples of forms that may be included in the present invention, and the present invention is not limited to these, and various configurations other than those described above may also be adopted. Furthermore, the present invention may be adopted by combining or independently adopting each of the configurations or features described in the above embodiments.

[0105] The present invention will be further explained below with reference to examples, but is not limited to these.

[0106] Example 1 Experimental Procedure The experiment was carried out according to the following procedure. 1) On the day before the culture, OKT3 (anti-CD3 antibody, Thermo Fisher Scientific, Cat: 16-0037-85, hereinafter the same), an antibody that activates T cells, was immobilized on a 24-well plate at a concentration of 0.5 μg / ml. 2) Frozen PBMCs from healthy individuals (isolated from whole blood collected from a vein) were thawed, suspended in RPMI1640 (Nacalai Tesque, Cat: 30264-85, hereinafter the same), and centrifuged at 400 G for 5 minutes at room temperature. 3) After centrifugation, the supernatant was completely removed, and the sample was divided into 1 x 10 6 The cells were suspended in 1 ml of each of the following media 1 to 9 to obtain nine types of cell suspensions. 10 ng / ml IL-2 (Peprotech, Cat: AF-200-02, hereinafter the same) was added to each medium as a supplement for T cell proliferation. RPMI1640 was used as the basal medium for medium 1, and a different basal medium (commercially available serum-free medium) was used for media 2 to 9. The concentrations shown are final concentrations.

[0107] Medium 1: Basal medium containing 10% fetal bovine serum (GIBCO, Cat: 10437-028, hereinafter the same). Medium 2: Basal medium. Medium 3: Basal medium containing 0.2 ng / ml TGFβ3 (amino acid sequence: SEQ ID NO: 3, Peprotech, Cat: AF-100-36E, hereinafter the same). Medium 4: Basal medium containing 10 μM AA2G (Hayashibara, Cat: AG121, hereinafter the same). Medium 5: Basal medium containing 100 μM AA2G. Medium 6: Basal medium containing 1000 μM AA2G. Medium 7: Basal medium containing 10 μM AA2G and 0.2 ng / ml TGFβ3. Medium 8: Basal medium containing 100 μM AA2G and 0.2 ng / ml TGFβ3. Medium 9: Basal medium containing 1000 μM AA2G and 0.2 ng / ml TGFβ3.

[0108] 4) Each cell suspension from 3) above was seeded separately into the wells of the plate from 1) above, which had been washed three times with PBS. 5) After 3 days of culture, 1 ml of medium (the same medium as used in 3) above) was added to each well and pipetted, and the cells were expanded to two wells of a 24-well plate without OKT3 immobilization (Day 3). 6) After 5 days of culture, 1 ml of medium (the same medium as used in 3) above) was added to each well and pipetted, and the cells were expanded to two to four wells (Day 5). 7) After 7 days of culture, the cells cultured in four wells were harvested and counted and analyzed by FCM. Some cells were also expanded (Day 7).

[0109] Counting was performed using a DeNovix CellDrop FL. For FCM analysis, samples were suspended in PBS containing 1% FBS, Fc-blocked, and then stained with a combination of CD4 / CD8a / CD3 antibodies or CCR7 / CD45RA / CD3 antibodies. The same counting and FCM procedures were repeated on day 11 of culture.

[0110] Experimental Results Figures 1 to 3 show the analysis results of the CD4 / CD8 ratio when media 1 to 3 were used. Medium 1 is a serum-containing medium containing fetal bovine serum, and medium 2 is a serum-free medium. Medium 3 is a serum-free medium containing TGFβ3. When cultured in either medium 1 or 2, the proportion of CD4(-)CD8(+) T cells in the cell population was high, and the proportion of CD4(+)CD8(-) T cells was low. On the other hand, when cultured in medium 3, the proportion of CD4(-)CD8(+) T cells decreased, and the proportion of CD4(+)CD8(-) T cells increased.

[0111] Figures 4 to 9 show the analysis results of the CD4 / CD8 ratio when media 4 to 9 were used. Media 4 to 6 are serum-free media containing AA2G. Media 7 to 9 are serum-free media containing AA2G and TGFβ3. When cultured in media 4 to 6, no effect of AA2G concentration was observed. When cultured in media 7 to 9, the proportion of CD4(-)CD8(+) T cells decreased and the proportion of CD4(+)CD8(-) T cells increased in an AA2G concentration-dependent manner.

[0112] The analysis results of the CD45RA / CCR7 ratio when medium 1 to 3 were used are shown in Figures 10 to 12. CD45RA and CCR7 are stem cell memory T cell markers. When cultured in medium 3, the percentage of CD45RA(+)CCR7(-) T cells decreased, while the percentage of CD45RA(+)CCR7(+) T cells increased.

[0113] The analysis results of the CD45RA / CCR7 ratio when medium 4 to 9 were used are shown in Figures 13 to 18. When cultured in medium 4 to 9, no effect of AA2G concentration was observed. Note that Figures 1 to 18 show the results on Day 7, but a similar trend was observed on Day 11.

[0114] Example 2 Experimental Procedure The experiment was carried out according to the following procedure. 1) On the day before culturing, OKT3, an antibody that activates T cells, was immobilized on a 24-well plate at a concentration of 0.5 μg / ml. 2) Frozen PBMCs from healthy individuals (isolated from whole blood collected from a vein) were thawed, suspended in RPMI1640, and centrifuged at 400 G for 5 minutes at room temperature. 3) After centrifugation, the supernatant was completely removed, and the sample was divided into 1 x 10 6The cells were suspended in 1 ml of each of the following media 10 to 14 to obtain five types of cell suspensions. 10 ng / ml IL-2 was added to each medium as a supplement for T cell proliferation. RPMI1640 was used as the basal medium for medium 10, while a different basal medium (commercially available serum-free medium) was used for media 11 to 14. The concentrations shown are final concentrations.

[0115] Medium 10: Basal medium containing 0.2 ng / ml TGFβ3 Medium 11: Basal medium containing 0.2 ng / ml TGFβ3 Medium 12: Basal medium containing 0.1 w / v% PLT (Sartorius, Cat: PLTGOLD100R, the same applies below) and 0.2 ng / ml TGFβ3 Medium 13: Basal medium containing 0.3 w / v% PLT and 0.2 ng / ml TGFβ3 Medium 14: Basal medium containing 1 w / v% PLT and 0.2 ng / ml TGFβ3

[0116] 4) Each cell suspension from 3) above was seeded separately into the wells of the plate from 1) above, which had been washed three times with PBS. 5) After 3 days of culture, 1 ml of medium (the same medium as used in 3) above) was added to each well and pipetted, and the cells were expanded to two wells of a 24-well plate without OKT3 immobilization (Day 3). 6) After 5 days of culture, 1 ml of medium (the same medium as used in 3) above) was added to each well and pipetted, and the cells were expanded to two to four wells (Day 5). 7) After 7 days of culture, the cells cultured in four wells were harvested and counted and analyzed by FCM. Some cells were also expanded (Day 7).

[0117] Counting was performed using a DeNovix CellDrop FL. For FCM analysis, samples were suspended in PBS containing 1% FBS, Fc-blocked, and then stained with a combination of CD4 / CD8a / CD3 antibodies or CCR7 / CD45RA / CD3 antibodies. The same counting and FCM procedures were repeated on day 11 of culture.

[0118] Experimental Results The analysis results of the CD4 / CD8 ratio when medium 10 and 11 were used are shown in Figures 19 and 20. Medium 10 and 11 are serum-free media containing TGFβ3. When cultured in either medium 10 or 11, the proportion of CD4(-)CD8(+) T cells in the cell population was low, and the proportion of CD4(+)CD8(-) T cells was high.

[0119] The analysis results of the CD4 / CD8 ratio when medium 12 to 14 were used are shown in Figures 21 to 23. Medium 12 to 14 are serum-free media containing PLT and TGFβ3. When cultured in medium 12 to 14, the percentage of CD4(-)CD8(+) T cells decreased and the percentage of CD4(+)CD8(-) T cells increased in a PLT concentration-dependent manner.

[0120] The analysis results of the CD45RA / CCR7 ratio when medium 10 to 14 were used are shown in Figures 24 to 28. When cultured in medium 12, the proportion of CD45RA(+)CCR7(+) T cells tended to be highest. Note that Figures 19 to 28 show the results on Day 7, but a similar trend was observed on Day 11.

[0121] Example 3 Experimental Procedure The experiment was carried out according to the following procedure. 1) On the day before culturing, OKT3, an antibody that activates T cells, was immobilized on a 24-well plate at a concentration of 0.5 μg / ml. 2) Frozen PBMCs from healthy individuals (isolated from whole blood collected from a vein) were thawed, suspended in RPMI1640 containing no supplements, and centrifuged at 400 G for 5 minutes at room temperature. 3) After centrifugation, the supernatant was completely removed, and the sample was divided into 1 x 10 6 Cells were suspended in 1 ml of each of the following media 16 to 22 to obtain eight types of cell suspensions. Each medium was supplemented with 10 ng / ml IL-2 as a supplement for T cell proliferation. RPMI1640 was used as the basal medium for media 15 to 18, while a different basal medium (commercially available serum-free medium) was used for media 19 to 22. The concentrations shown are final concentrations.

[0122] Medium 15: Basal medium containing 10% fetal bovine serum Medium 16: Basal medium containing 0.2 ng / ml of TGFβ3 and 10% fetal bovine serum Medium 17: Basal medium containing 0.6 ng / ml of TGFβ3 and 10% fetal bovine serum Medium 18: Basal medium containing 2 ng / ml of TGFβ3 and 10% fetal bovine serum Medium 19: Basal medium Medium 20: Basal medium containing 0.2 ng / ml of TGFβ3 Medium 21: Basal medium containing 0.6 ng / ml of TGFβ3 Medium 22: Basal medium containing 2 ng / ml of TGFβ3

[0123] 4) Each cell suspension from 3) above was seeded separately into the wells of the plate from 1) above, which had been washed three times with PBS. 5) After 3 days of culture, 1 ml of medium (the same medium as used in 3) above) was added to each well and pipetted, and the cells were expanded to two wells of a 24-well plate without OKT3 immobilization (Day 3). 6) After 5 days of culture, 1 ml of medium (the same medium as used in 3) above) was added to each well and pipetted, and the cells were expanded to two to four wells (Day 5). 7) After 11 days of culture, the cells were expanded, harvested, counted, and analyzed by FCM (Day 11).

[0124] Counting was performed using a CellDrop FL (DeNovix). For FCM analysis, samples were suspended in PBS containing 1% FBS, Fc-blocked, and then stained with a combination of CD4 / CD8a / CD3 antibodies or CCR7 / CD45RA / CD3 antibodies.

[0125] Experimental Results: The analysis results of the CD4 / CD8 ratio when using media 15-22 are shown in Figures 29-36. Media 15-18 are serum-free media containing fetal bovine serum and containing 0, 0.2, 0.6, or 2 ng / ml of TGFβ3, respectively. All media resulted in a high percentage of CD4(-)CD8(+) T cells and a low percentage of CD4(+)CD8(-) T cells in the cell population. Media 19-22 are serum-free media containing 0, 0.2, 0.6, or 2 ng / ml of TGFβ3, respectively. Compared to media 19, media 20-22 showed an improved percentage of CD4(+)CD8(-) T cells. The percentage of CD4(+)CD8(-) T cells was highest in media 21 (0.6 ng / ml), followed by media 22 (2 ng / ml) and media 20 (0.2 ng / ml).

[0126] The analysis results of the CD45RA / CCR7 ratio when medium 15-22 was used are shown in Figures 37-44. CD45RA and CCR7 are stem cell memory T cell markers. When cultured in medium 16-18 and 20-22, the percentage of CD45RA(+)CCR7(-) T cells decreased, while the percentage of CD45RA(+)CCR7(+) T cells increased. In medium 16-18, the percentage of CD45RA(+)CCR7(+) T cells was highest in medium 17 (0.6ng / ml), medium 18 (2ng / ml), and medium 16 (0.2ng / ml). In medium 20-22, the percentage of CD45RA(+)CCR7(+) T cells was highest in medium 21 (0.6ng / ml), medium 22 (2ng / ml), and medium 20 (0.2ng / ml).

[0127] The results of analyzing the proportion of CD3-positive T cells when media 15 to 22 were used are shown in Figures 45 to 52. All media were shown to have a high proportion of CD3-positive T cells in the cell population.

[0128] Example 4 Experimental Procedure The experiment was carried out according to the following procedure. 1) On the day before culturing, OKT3, an antibody that activates T cells, was immobilized on a 24-well plate at a concentration of 0.5 μg / ml. 2) Frozen PBMCs from healthy individuals (isolated from whole blood collected from a vein) were thawed, suspended in RPMI1640 containing no supplements, and centrifuged at 400 G for 5 minutes at room temperature. 3) After centrifugation, the supernatant was completely removed, and the sample was divided into 1 x 10 6 The cells were suspended in 1 ml of each of the following media 23 to 26 to obtain four types of cell suspensions. 10 ng / ml IL-2 was added to each medium as a supplement for T cell proliferation. Media 23 to 26 contained OpTmizer (Gibco) as the basal medium. TM CTS TM OpTmizer TM T Cell Expansion SFM, bottle format (Brand: Gibco TM A1048501) was used. The concentrations shown are final concentrations.

[0129] Medium 23: Basal medium Medium 24: Basal medium containing TGFβ3 0.2ng / ml Medium 25: Basal medium containing TGFβ3 0.6ng / ml Medium 26: Basal medium containing TGFβ3 2ng / ml

[0130] 4) Each cell suspension from 3) above was seeded separately into the wells of the plate from 1) above, which had been washed three times with PBS. 5) After 3 days of culture, 1 ml of medium (the same medium as used in 3) above) was added to each well and pipetted, and the cells were expanded to two wells of a 24-well plate without OKT3 immobilization (Day 3). 6) After 5 days of culture, 1 ml of medium (the same medium as used in 3) above) was added to each well and pipetted, and the cells were expanded to two to four wells (Day 5). 7) After 11 days of culture, the cells were expanded, harvested, counted, and analyzed by FCM (Day 11).

[0131] Counting was performed using a CellDrop FL (DeNovix). For FCM analysis, samples were suspended in PBS containing 1% FBS, Fc blocked, and then stained with a combination of CD4, CD8a, and CD3 antibodies.

[0132] Experimental Results Figure 53 shows the analysis results of the CD4 / CD8 ratio when medium 23-26 was used. Medium 23-26 is a serum-free medium containing 0, 0.2, 0.6, or 2 ng / ml of TGFβ3, respectively. Compared to medium 23, medium 24-26 showed an improved percentage of CD4(+)CD8(-) T cells. The percentage of CD4(+)CD8(-) T cells was highest in medium 25 (0.6 ng / ml), medium 26 (2 ng / ml), and medium 24 (0.2 ng / ml).

[0133] The results of Examples 1 to 4 showed that (i) the addition of TGFβ3 to the basal medium improves the proportion of CD4(+)CD8(-) T cells in the cell population, (ii) the improvement in the proportion of CD4(+)CD8(-) T cells is further increased by adding additional components such as AA2G, (iii) the addition of TGFβ3 to the basal medium improves the proportion of CD45RA(+)CCR7(+) T cells in the cell population, (iv) the proportion of CD4(+)CD8(-) T cells or the proportion of CD45RA(+)CCR7(+) T cells is particularly increased under bovine serum-free conditions, and (v) the proportion of CD4(+)CD8(-) T cells or the proportion of CD45RA(+)CCR7(+) T cells is particularly increased within a specific concentration range of TGFβ3.

[0134] The present invention has been described above based on the embodiments. However, these embodiments are merely illustrative, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

Claims

1. A method for detecting CD4 in a cell population, comprising culturing a cell population containing human T cells in the presence of a human transforming growth factor β (human TGFβ) component to obtain a cultured cell population. + How to improve T cell percentage.

2. The method according to claim 1, wherein the culture is carried out in a bovine serum-free environment.

3. CD4 + T cells react with CD4 + The method of claim 2, wherein the cell is a T cell.

4. Cell sorting to isolate CD4 + The method according to claim 2, which does not include a step of separating T cells.

5. A method according to any one of claims 1 to 4, wherein the culturing is carried out in the presence of 0.01 to 3 ng / mL of human TGFβ component.

6. The cultured cell population is a CD4 + The method according to any one of claims 1 to 5, wherein the proportion of T cells in the cell population is 40% or more.

7. The cultured cell population is CD8 + The method according to any one of claims 1 to 6, wherein the proportion of T cells in the cell population is 60% or less.

8. The process comprises: + The method according to any one of claims 1 to 7, comprising the step of recovering a cell population in which the proportion of T cells is 40% or more.

9. The process comprises the steps of: + The method according to any one of claims 1 to 8, comprising the step of recovering a cell population in which the proportion of T cells is 60% or less.

10. The process comprises: + CD8 - The proportion of T cells is CD4 - CD8 + The method according to any one of claims 1 to 9, comprising a step of recovering a cell population that is 0.7 to 1.3 times the proportion of T cells.

11. The process comprises: + CCR7 + The method according to any one of claims 1 to 10, comprising the step of recovering a cell population in which the proportion of T cells is 30% or more.

12. The process comprises: + T cell ratio ≥ 40%, CD8 + T cell percentage 60% or less and CD45RA + CCR7 + The method according to any one of claims 1 to 11, comprising the step of recovering a cell population in which the proportion of T cells is 30% or more.

13. The method according to any one of claims 1 to 12, wherein the cell population containing human T cells is a cell population containing CD3-positive T cells.

14. The method according to claim 13, wherein the cell population containing human T cells has a percentage of CD3-positive T cells in the cell population of 90% or more.

15. The method according to any one of claims 1 to 14, wherein the cell population containing human T cells is a cell population containing αβ type T cells.

16. The method according to claim 15, wherein the cell population containing human T cells has a proportion of αβ T cells in the cell population of 90% or more.

17. A method according to any one of claims 1 to 16, comprising producing a cell population enriched in CD3-positive T cells from human peripheral blood mononuclear cells (PBMCs).

18. A method according to any one of claims 1 to 17, comprising the step of producing a cell population enriched in αβ T cells from human PBMCs.

19. A method according to any one of claims 1 to 18, comprising stimulating human PBMCs with an anti-CD3 antibody.

20. The method according to any one of claims 1 to 19, wherein the culturing is further carried out in the presence of ascorbic acid, a derivative thereof, or a salt thereof.

21. The method of any one of claims 1 to 20, wherein the culturing is further carried out in the presence of a platelet lysate.

22. The method of any one of claims 1 to 21, carried out in the absence of exogenous antibodies.

23. A method according to any one of claims 1 to 22, wherein the human TGFβ component has TGFβ activity and is (a) a protein having an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a biologically active fragment thereof, or (b) a protein having an amino acid sequence having 90% or more homology to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 3, or a biologically active fragment thereof.

24. A cell population obtained by the method according to any one of claims 1 to 23.

25. A cell population containing CD3 positive T cells, wherein the CD3 positive T cells contained in the cell population are CD4 + CD8 - A cell population that is 50% or more of T cells.

26. The cell population described in claim 25, wherein the percentage of CD3-positive T cells in the cell population is 90% or more.

27. Among the CD3 positive T cells in the cell population, CD4 - CD8 + 27. The cell population of claim 25 or 26, wherein the proportion of T cells is 50% or less.

28. Among the CD3 positive T cells in the cell population, CD45RA + CCR7 + The cell population of claim 27, wherein the percentage of cells is 35% or more.

29. The cell population of any one of claims 25 to 28, which is free of exogenous antibodies.

30. A pharmaceutical composition comprising a cell population according to any one of claims 25 to 29.

31. The pharmaceutical composition of claim 30, which does not contain a foreign antibody.

32. Culturing a cell population containing human T cells in the presence of a human TGFβ component, and detecting CD4 + A method for producing a cell population, comprising a step of recovering a cultured cell population having a T cell ratio of 40% or more.

33. The cultured cell population is CD8 + The method of claim 32, wherein the proportion of T cells is 60% or less of the cell population.

34. The cultured cell population is a CD45RA + CCR7 + The method of claim 33, wherein the proportion of T cells is 30% or more of the cell population.

35. Culturing a cell population containing human T cells in the presence of a human TGFβ component, and detecting CD4 + CD8 - The proportion of T cells is CD4 - CD8 + A method for producing a cell population, comprising the step of recovering a cultured cell population in an amount that is 0.7 to 1.3 times the proportion of T cells.

36. A method for detecting CD4 in a cell population, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + T cells and CD8 + How to get the T cell ratio closer.

37. A method for producing a CD4 T cell-containing cell population, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + A method for producing a cell population having an enhanced proportion of T cells.

38. The production method according to claim 37, wherein the culture is carried out in a bovine serum-free environment.

39. The method of claim 38, which is a method for producing a cell population in which the exogenous antibody is not bound.

40. Cell sorting to identify CD4 + The method of claim 38, which does not include a step of separating T cells.

41. The cultured cell population is a CD4 + CD8 - The proportion of T cells is CD4 - CD8 + The method of any one of claims 37 to 40, wherein the proportion of cells expressing stem cell memory T cell markers is 0.7 times or more higher than the proportion of T cells and the proportion of cells expressing stem cell memory T cell markers is 35% or more.

42. A method for producing a CD4 T cell-containing cell population, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + T cells and CD8 + A method for producing cell populations with similar proportions of T cells.

43. The cultured cell population is a CD4 + CD8 - The proportion of T cells is CD4 - CD8 + The method of producing the cell population according to claim 42, wherein the proportion of cells expressing stem cell memory T cell markers is 0.7 to 1.3 times higher than that of T cells and the proportion of cells expressing stem cell memory T cell markers is 35% or more.

44. A method for improving the proportion of cells expressing stem cell memory T cell markers in a cell population, comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population.

45. A method for producing a cell population having an improved proportion of stem cell memory T cells, comprising the step of culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population.

46. ​​A method for detecting CD45RA in a cell population, comprising culturing a cell population containing human T cells in the presence of a human TGFβ component to obtain a cultured cell population. + CCR7 + How to improve T cell percentage.

47. The process comprises: + CCR7 + The method of claim 46, comprising recovering a cell population having a T cell percentage of 30% or more.

48. The method of claim 46 or 47, wherein the culturing is carried out in the presence of 0.01 to 3 ng / mL of human TGFβ component.

49. The method according to any one of claims 46 to 48, wherein the culture is carried out in a bovine serum-free environment.

50. A method according to any one of claims 46 to 49, wherein the cell population containing human T cells has a proportion of CD3-positive T cells in the cell population of 90% or more.

51. The method of any one of claims 46 to 50, comprising stimulating human PBMCs with an anti-CD3 antibody.

52. A cell population obtained by the method according to any one of claims 32 to 51.

53. CD4 + A composition comprising a human TGFβ component for use in producing a cell population having an enhanced proportion of T cells.

54. CD4 + T cells and CD8 + A composition comprising a human TGFβ component for use in producing a cell population having a similar proportion of T cells.

55. The cell population is CD4 + CD8 - The proportion of T cells is CD4 - CD8 + The composition of claim 54, which is a cell population that is 0.7 to 1.3 times the proportion of T cells.

56. Culture medium for T cells containing 0.01-3ng / ml of TGFβ component.

57. The culture medium for T cells described in claim 56, further comprising an ascorbic acid derivative.

58. The culture medium for T cells described in claim 56, further comprising an ascorbic acid derivative and a platelet lysate.

59. A method for culturing T cells, comprising culturing T cells in the presence of 0.01 to 3 ng / ml of a TGFβ component.

60. A method for culturing T cells according to claim 59, wherein the culturing is carried out in the presence of 0.01 to 3 ng / ml of TGFβ component and in the presence of an ascorbic acid derivative.

61. A method for culturing T cells according to claim 59, wherein the culturing is carried out in the presence of 0.01 to 3 ng / ml of TGFβ component, and in the presence of an ascorbic acid derivative and a platelet lysate.

62. The culture method according to any one of claims 59 to 61, wherein the culture is carried out in a bovine serum-free environment.

63. The method of culturing T cells according to claim 62, which is a method of culturing T cells that are not bound to an exogenous antibody.

64. Cell sorting to identify CD4 + The culture method according to claim 62, which does not include a step of separating T cells.

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