Method for producing car-t cells

Abstract

The present invention provides a method for producing CAR-T cells, the method comprising: a first step for stimulating a cell population containing unstimulated CAR-T cells under condition [A]; and a second step for stimulating a cell population containing CAR-T cells after the first step under condition [B]. [A] [Number of T cells (cells) in a cell population containing unstimulated CAR-T cells]:[Number of antigen recognition factors (pmol)]:[Number of costimulatory factors (pmol)] = 106:0.1-0.83:0.2-1.7; [B] [Number of T cells (cells) in a cell population containing CAR-T cells after first step]:[Number of antigen recognition factors (pmol)]:[Number of costimulatory factors (pmol)] = 106:0.05-0.17:0.05-0.28 (provided that the sum of the number of antigen recognition factors and the number of costimulatory factors in [B] is smaller than that in [A]).

Description

Method for producing CAR-T cells

[0001] The present invention relates to a method for producing CAR-T cells, and more particularly to a method for producing CAR-T cells, which comprises reacting an antigen recognition factor and a costimulatory factor at a specific ratio with a cell population containing CAR-T cells.

[0002] In recent years, CAR-T cell therapy has attracted attention as one type of cancer immunotherapy. In CAR-T cell therapy, a tumor-specific CAR is first introduced into T cells in the peripheral blood collected from a patient using genetic modification technology to produce CAR-T cells, which are then expanded ex vivo. The expanded CAR-T cells are then returned to the patient, where they directly attack cancer cells in the patient's body and activate the patient's immune system. As a result, the patient's cancer is treated.

[0003] Generally, the process for producing CAR-T cells consists of three steps: (1) obtaining T cells from a subject, (2) introducing a desired CAR into the obtained T cells, and (3) activating and expanding the T cells into which the desired CAR has been introduced (i.e., CAR-T cells). To obtain a large amount of CAR-T cells with a high anti-tumor effect, each of steps (1) to (3) is extremely important, and optimization of the design of each step is required. For example, Patent Document 1 discloses that CAR-T cells with high anti-tumor activity can be stably prepared by co-culturing the CAR-T cells produced in step (2) with cells expressing the target antigen of the CAR-T cells.

[0004] US2022-265714

[0005] To carry out CAR-T cell therapy, a large amount of high-quality CAR-T cells is required. However, for example, the method reported in Patent Document 1, while capable of stably preparing high-quality CAR-T cells, has the drawback of being inefficient due to the necessity of a step of co-culturing multiple types of cells, which inevitably makes the process complicated and expensive. Therefore, an object of the present invention is to provide a novel method that achieves the activation and proliferation of CAR-T cells in a simple, low-cost process, thereby enabling the easy and efficient preparation of CAR-T cells with a high anti-tumor effect.

[0006] As a result of intensive research into the above-mentioned problems, the present inventors have unexpectedly found that, when stimulating CAR-T cells with a CAR-specific antigen and a costimulatory factor for the purpose of activating and expanding CAR-T cells, performing the first stimulation at a level weaker than that conventionally considered appropriate, and then stimulating the CAR-T cells in the second stimulation at an even weaker level than the first stimulation level, and have carried out further research based on this finding, thereby completing the present invention. That is, the present invention is as follows.

[0007] [1] A method for producing CAR-T cells, comprising: a first step of stimulating a cell population containing unstimulated CAR-T cells under the following condition [A]; and a second step of stimulating the cell population containing CAR-T cells after the first step under the following condition [B]: [A] Number of T cells (cell) in the cell population containing unstimulated CAR-T cells: Number of antigen-recognition factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.1 to 0.83 : 0.2 to 1.7 [B] Number of T cells (cell) in the cell population containing CAR-T cells after the first step: Number of antigen-recognizing factors (pmol): Number of costimulatory factors (pmol) = 10 ６: 0.05 to 0.17: 0.05 to 0.28 (However, the sum of the number of antigen recognition factors and the number of costimulatory factors in [B] is smaller than that in [A].) [2] The method for producing CAR-T cells according to [1], further comprising a third step of stimulating the cell population containing CAR-T cells after the second step under the following condition [C]: [C] Number of T cells (cell) in the cell population containing CAR-T cells after the second step: number of antigen recognition factors (pmol): number of costimulatory factors (pmol) = 10 ６ : 0.4 to 2.0: 0.4 to 4.4 [3] The production method of [1] or [2], wherein the costimulatory factor is at least one selected from the group consisting of CD80, 4-1BBL, CD86, OX40L, ICOS-L, CD70, CD40L, CD270, ICAM-1, LFA-3, CD72, CD55, VCAM-1, MadCAM-1, CD111, CD112, CD155, CD153, PD-L2, PD-L1, Galectin-9, MHC, and CD113. [4] The production method of any of [1] to [3], wherein at least two costimulatory factors are used. [5] The production method of any of [1] to [4], wherein the costimulatory factors are CD80 and 4-1BBL. [6] In the first step, 10 T cells of a cell population containing unstimulated CAR-T cells are ６ [7] The method of producing CAR-T cells according to [5], characterized in that a cell population containing unstimulated CAR-T cells is stimulated under conditions in which the T cells of the cell population containing CAR-T cells after the first step are stimulated under the following conditions: 0.1 to 0.83 pmol of an antigen recognition factor, 0.2 to 0.85 pmol of CD80, and 0.2 to 0.85 pmol of 4-1BBL per cell. ６ The method of producing CAR-T cells according to [5] or [6], characterized in that the cell population containing CAR-T cells is stimulated under conditions in which the T cells of the cell population containing CAR-T cells after the second step are 0.05 to 0.17 pmol of an antigen recognition factor, 0.05 to 0.14 pmol of CD80, and 0.05 to 0.14 pmol of 4-1BBL per cell. ６

[0022] The method for producing CAR-T cells according to any one of [5] to [7], wherein a cell population containing CAR-T cells is stimulated under conditions in which 0.4 to 2.0 pmol of an antigen recognition factor, 0.2 to 2.2 pmol of CD80, and 0.2 to 2.2 pmol of 4-1BBL are present per cell. [9] The method for producing CAR-T cells according to any one of [1] to [8], wherein the antigen recognition factor and / or costimulatory factor are bound to the surface of a support.

[10] The method for producing CAR-T cells according to [9], wherein the antigen recognition factor and costimulatory factor are bound to the surface of a support.

[11] The method for producing CAR-T cells according to [9] or

[10] , wherein the support is a bead.

[0008] According to the method of the present invention, activation and proliferation of CAR-T cells can be achieved in a simple and low-cost process, and as a result, CAR-T cells with a high anti-tumor effect can be prepared easily and efficiently.

[0009] The present invention will be described below.

[0010] Method for producing CAR-T cells The present invention provides a method for producing CAR-T cells (hereinafter, sometimes referred to as the "production method of the present invention"), which comprises a first step of stimulating a cell population containing unstimulated CAR-T cells under the following condition [A], and a second step of stimulating the cell population containing CAR-T cells after the first step under the following condition [B]: [A] Number of T cells (cell) in a cell population containing unstimulated CAR-T cells: Number of antigen-recognition factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.1 to 0.83 : 0.2 to 1.7 [B] Number of T cells (cell) in the cell population containing CAR-T cells after the first step: Number of antigen-recognizing factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.05 to 0.17: 0.05 to 0.28 (However, the sum of the number of antigen recognition factors and the number of costimulatory factors in [B] is smaller than that in [A]).

[0011] In the production method of the present invention, "CAR-T cells" means cells obtained by introducing a cancer-specific gene (so-called CAR gene) into T cells.

[0012] In the production method of the present invention, the origin of T cells for preparing CAR-T cells is not particularly limited. The T cells may be derived from a patient to be treated with CAR-T cell therapy, such as a cancer patient, or may be differentiated from stem cells such as iPS cells or hematopoietic stem cells. The T cells for preparing such CAR-T cells may be CD4-positive CD8-negative T cells, CD4-negative CD8-positive T cells, CD4-positive CD8-positive T cells, CD4-negative CD8-negative T cells, αβ-T cells, γδ-T cells, Treg cells, NK-like T cells, or NKT cells, but are not limited to these. Any T cells may be used. Furthermore, the T cells may be naive T cells, effector T cells, or memory T cells, or may be a subset thereof. In a preferred embodiment of the present invention, the T cells for preparing CAR-T cells are human peripheral blood mononuclear cells (PBMCs). Furthermore, T cells used to produce CAR-T cells may be autologous cells (i.e., cells derived from a subject to be treated with CAR-T cells produced by the production method of the present invention) or allogeneic cells. In a preferred embodiment, the T cells are autologous cells. In the production method of the present invention, a "cell population comprising CAR-T cells" refers to a cell population obtained via a step of introducing a CAR gene into a cell population containing T cells. That is, a "cell population comprising CAR-T cells" includes T cells into which a CAR gene has been introduced (i.e., CAR-T cells) and T cells into which a CAR gene has not been introduced. Furthermore, a cell population containing T cells, such as PBMCs obtained from a subject by apheresis or the like, may include cells other than T cells (hereinafter, cells other than T cells). When such a cell population is used in the present invention, the cell population containing CAR-T cells may include CAR-T cells, T cells into which a CAR gene has not been introduced, and cells other than T cells.In one embodiment of the production method of the present invention, the proportion of T cells (including both CAR-T cells and T cells into which the CAR gene has not been introduced) contained in the "cell population containing CAR-T cells" is not particularly limited, and can be typically 1% or more, preferably 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100%, but is not limited to these. Furthermore, the upper limit can be, but is not limited to, typically 100% or less, preferably 95% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. In one aspect, the percentage of T cells in a T cell population containing CAR-T cells can be, but is not limited to, typically 1 to 100%, preferably 5 to 95%, 10 to 95%, 20 to 95%, 30 to 95%, 40 to 95%, 50 to 95%, 60 to 95%, 70 to 95%, 80 to 95%, or 90 to 95%. Note that methods for enriching T cells in a cell population containing T cells are known, and a person skilled in the art would be able to adjust the proportion of T cells in a cell population containing CAR-T cells to an intended value.

[0013] In the production method of the present invention, the term "T cell population containing unstimulated CAR-T cells" refers to a T cell population containing CAR-T cells obtained by introducing a CAR gene into a population of T cells used to prepare the above-described CAR-T cells using a method known per se (such as a viral vector method or an electroporation method), and which has not been activated by an antigen recognition factor and / or a costimulatory factor.

[0014] Furthermore, in the production method of the present invention, "stimulating a cell population containing CAR-T cells" means bringing an antigen recognition factor and / or a costimulatory factor into contact with a cell population containing CAR-T cells, thereby activating and / or promoting the proliferation of CAR-T cells contained in the cell population.

[0015] In the production method of the present invention, the term "antigen recognition factor" refers to a substance that can activate CAR-T cells by specifically binding to a CAR (chimeric antigen receptor) present on the cell surface of CAR-T cells and can also confer antitumor properties to CAR-T cells. In other words, an antigen recognition factor refers to a substance that can achieve antigen stimulation of T cells. The antigen recognition factor is not particularly limited as long as it achieves the desired effect, and any substance may be used. Specifically, for example, in the case of CAR-T cells expressing an EPHB4-specific CAR, the full-length EPHB4 protein or a partial fragment thereof (limited to one that can bind to the EPHB4-specific CAR and stimulate the antigen) can be used as the antigen recognition factor. Note that the antigen recognition factor may be not only a protein or peptide fragment, but also a substance other than amino acids, such as a sugar chain, as long as it achieves the desired effect.

[0016] In the production method of the present invention, the term "costimulatory factor" refers to a substance that promotes T cell activation by specifically binding to a costimulatory receptor present on the surface of T cells. The costimulatory factor used in the production method of the present invention is not particularly limited, and any substance may be used as long as it produces the desired effect. Examples of costimulatory factors include, but are not limited to, CD80, 4-1BBL, CD86, OX40L, ICOS-L, CD70, CD40L, CD270, ICAM-1, LFA-3, CD72, CD55, VCAM-1, MadCAM-1, CD111, CD112, CD155, CD153, PD-L2, PD-L1, Galectin-9, MHC, and CD113. Note that a single type of costimulatory factor may be used, or two or more types may be used in combination. In a preferred embodiment, the costimulatory factor may be, but is not limited to, CD80 and / or 4-1BBL.

[0017] The production method of the present invention is characterized by comprising two steps (step 1 and step 2) aimed at activating and proliferating CAR-T cells.

[0018] [Step 1] In the first step of the production method of the present invention, a cell population containing unstimulated CAR-T cells is stimulated under the conditions shown in [A] below:

[0019] [A] Number of T cells (cells) in a T cell population containing unstimulated CAR-T cells: number of antigen-recognizing factors (pmol): number of costimulatory factors (pmol) = 10 ６ : 0.1 to 0.83: 0.2 to 1.7

[0020] In a preferred embodiment, [A] can be any of the following: (A1) Number of T cells (cells) in a cell population containing unstimulated CAR-T cells: antigen recognition factor (pmol): costimulatory factor (pmol) = 10 ６ : 0.15 to 0.75: 0.4 to 1.6 (A2) Number of T cells (cell) in a cell population containing unstimulated CAR-T cells: antigen recognition factor (pmol): costimulatory factor (pmol) = 10 ６ : 0.2 to 0.65: 0.6 to 1.5 (A3) Number of T cells (cell) in a cell population containing unstimulated CAR-T cells: antigen recognition factor (pmol): costimulatory factor (pmol) = 10 ６ : 0.35 to 0.6: 0.8 to 1.4 (A4) Number of T cells (cell) in a cell population containing unstimulated CAR-T cells: antigen recognition factor (pmol): costimulatory factor (pmol) = 10 ６ : 0.45 to 0.55: 1.0 to 1.3

[0021] In another preferred embodiment, [A] can be any of the following: (A5) Number of T cells (cells) in a cell population containing unstimulated CAR-T cells: antigen recognition factor (pmol): costimulatory factor (pmol) = 10 ６ : 0.1 to 0.6: 0.2 to 1.2 (A6) Number of T cells (cell) in a cell population containing unstimulated CAR-T cells: antigen recognition factor (pmol): costimulatory factor (pmol) = 10 ６ : 0.1 to 0.5: 0.2 to 1.0 (A7) Number of T cells (cell) in a cell population containing unstimulated CAR-T cells: antigen recognition factor (pmol): costimulatory factor (pmol) = 10 ６ : 0.15 to 0.35: 0.3 to 0.7 (A8) Number of T cells (cell) in a cell population containing unstimulated CAR-T cells: antigen recognition factor (pmol): costimulatory factor (pmol) = 10 ６ : 0.15 to 0.25: 0.4 to 0.55

[0022] The medium in which the cell population containing CAR-T cells is cultured in the first step is not particularly limited, and any medium may be used as long as it allows T cells to survive and proliferate and is capable of activating T cells by adding an antigen recognition factor and a costimulatory factor. Such T cell culture media are well known to those skilled in the art, and commercially available media may be used. Examples of such commercially available media include, but are not limited to, RPMI 1640, MEM, X-VIVIO, IMDM, DMEM, DC medium, OptiMEM, TexMACS™ (Miltenyi Biotec), AIM V (registered trademark) (Thermo Fisher Scientific), and ALyS culture medium (Cell Science Institute, Inc.). The same applies to other conditions, such as temperature, and are not particularly limited as long as they allow T cells to survive and proliferate. For example, the temperature and oxygen / carbon dioxide concentration are 15 to 40°C (e.g., 37°C), CO ２ Examples of suitable medium replacement techniques include, but are not limited to, a pH value of 2 to 8% (e.g., 5%), an oxygen concentration of 0 to 20% (e.g., 1 to 5%), and the like. When the medium deteriorates due to the maintenance and proliferation of CAR-T cells, the medium can be replaced with a fresh medium at an appropriate timing. The timing of medium replacement can be appropriately determined by one skilled in the art based on the degree of medium deterioration, etc.

[0023] In one embodiment, additives for supporting the survival and proliferation of CAR-T cells may be further added to the medium. Examples of such additives include, but are not limited to, type 1 cytokine family members, type 2 cytokine family members, TNF superfamily cytokines, IL-1 family cytokines, and other cytokines (e.g., TNF-β, etc.). More specifically, examples include IL-1 to IL-41, etc., and preferred examples include IL-1, IL-2, IL-7, IL-15, and IL-21, etc. In a preferred embodiment, IL-7 and / or IL-15 can be added to the medium. Such additives can be prepared according to standard methods, or commercially available products may be used. Such additives may be derived from animal species other than humans, but are preferably derived from humans (which may be recombinant).

[0024] In another embodiment, the medium can be supplemented with at least one substance selected from the group consisting of SB431542, rapamycin, simvastatin, PD0325901, CD62L, GW9662, rosiglitazone, GW6471, Siglec-6, CD73, CXCR5, CCR7, and CCL5. In a preferred embodiment, the medium can be supplemented with at least one substance selected from the group consisting of SB431542, rapamycin, simvastatin, PD0325901, CD62L, GW9662, rosiglitazone, GW6471, Siglec-6, and CD73. In a more preferred embodiment, the medium can be supplemented with at least one substance selected from the group consisting of SB431542, rapamycin, simvastatin, PD0325901, CD62L, GW9662, rosiglitazone, and GW6471.

[0025] In the production method of the present invention, the timing of performing the first step is not particularly limited as long as the desired effect is achieved, and the first step may be performed at any timing, but the timing may be appropriately set so that the ultimately obtained CAR-T cells are of high quality and can be obtained in large quantities. As an example of such timing, the first step can usually be performed immediately to 3 days after introduction of the CAR gene, preferably 1 to 2 days after introduction of the CAR gene, and more preferably 1 day after introduction of the CAR gene.

[0026] [Second Step] In the second step of the production method of the present invention, the cell population containing CAR-T cells after the first step is stimulated under the conditions shown in [B] below:

[0027] [B] Number of T cells (cells) in the cell population containing CAR-T cells after the first step: number of antigen-recognition factors (pmol): number of costimulatory factors (pmol) = 10 ６ : 0.05 to 0.17: 0.05 to 0.28

[0028] In a preferred embodiment, [B] can be any of the following: [B1] Number of T cells (cells) in the cell population containing CAR-T cells after the first step: Number of antigen-recognizing factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.05-0.14: 0.08-0.27 [B2] Number of T cells (cell) in the cell population containing CAR-T cells after the first step: number of antigen-recognition factors (pmol): number of costimulatory factors (pmol) = 10 ６ : 0.06-0.13: 0.11-0.26 [B3] Number of T cells (cell) in the T cell population containing CAR-T cells after the first step: Number of antigen-recognizing factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.07 to 0.12: 0.17 to 0.25

[0029] One of the features of the production method of the present invention is that the stimulation level used in step 2 is weaker than the stimulation level used in step 1. Therefore, the sum of the number of antigen-recognition factors and the number of costimulatory factors in [B] is smaller than that in [A].

[0030] The medium and conditions under which the cell population containing CAR-T cells is cultured when the second step is carried out are also not particularly limited, and any medium and conditions may be used, as long as they allow T cells to survive and / or proliferate, and the T cells can be activated by the addition of an antigen recognition factor and a costimulatory factor, similar to those described in the first step.

[0031] In the production method of the present invention, the timing of performing the second step is not particularly limited as long as the desired effect is achieved, and the second step may be performed at any timing. However, the timing may be appropriately selected so that the CAR-T cells ultimately obtained are of high quality and can be obtained in large quantities. One example of such timing is typically 3 to 14 days, preferably 4 to 12 days, 5 to 10 days, or 6 to 8 days (e.g., 7 days) after performing the first step. If the medium deteriorates due to the maintenance and proliferation of CAR-T cells, the medium may be replaced with fresh medium at an appropriate time. The timing of medium replacement can be appropriately determined by one skilled in the art based on the degree of medium deterioration, etc.

[0032] In one embodiment, the production method of the present invention includes a further stimulation step as a third step after the second step. In the third step, the cell population containing CAR-T cells after the second step is stimulated under the conditions shown in [C] below:

[0033] [C] Number of T cells (cell) in the cell population containing CAR-T cells after the second step: number of antigen-recognition factors (pmol): number of costimulatory factors (pmol) = 10 ６ : 0.4 to 2.0: 0.4 to 4.4

[0034] In a preferred embodiment, [C] can be any of the following: [C1] Number of T cells (cells) in the cell population containing CAR-T cells after the second step: Number of antigen-recognizing factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.4-1.6 : 0.7-3.5 [C2] Number of T cells (cell) in the cell population containing CAR-T cells after the second step: Number of antigen-recognizing factors (pmol): Number of costimulatory factors (pmol) = 10 ６: 0.5-1.4 : 1.3-3 [C3] Number of T cells (cell) in the cell population containing CAR-T cells after the second step: Number of antigen-recognizing factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.6-1.2 : 1.6-2.5 [C4] Number of T cells (cell) in the cell population containing CAR-T cells after the second step: Number of antigen-recognizing factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.7 to 1: 1.9 to 2.2

[0035] The medium and conditions under which the cell population containing CAR-T cells is cultured when the third step is carried out are also similar to those described in the first and second steps, and are not particularly limited as long as they are medium and conditions that allow T cells to survive and / or proliferate and that allow T cells to be activated by the addition of an antigen recognition factor and a costimulatory factor, and any medium may be used.

[0036] In the production method of the present invention, the timing of carrying out the third step is not particularly limited as long as the desired effect is achieved, and the third step may be carried out at any timing that can be appropriately determined by a person skilled in the art. As an example of such timing, the third step may be carried out usually 1 to 14 days, preferably 2 to 12 days, 2 to 10 days, or 2 to 8 days (e.g., 3 days or 6 days) after carrying out the second step.

[0037] In a preferred embodiment of the production method of the present invention, at least two types of costimulatory factors can be used to stimulate CAR-T cells. When at least two types of costimulatory factors are used, the same combination may be used in each step, or different combinations may be used. In one embodiment, the combination of at least two types of costimulatory factors used in each step may be the same. In a more preferred embodiment, a combination of CD80 and 4-1BBL can be used in all stimulation steps in the production method of the present invention.

[0038] When multiple costimulatory factors are used, the "number of costimulatory factors" in the above [A], [B], or [C] refers to the "sum" of the numbers of each costimulatory factor used. Specifically, when CD80 and 4-1BBL are used as costimulatory factors, the number of costimulatory factors is the sum of the number of CD80 molecules and the number of 4-1BBL molecules.

[0039] Furthermore, when multiple costimulatory factors are used, the ratio of the costimulatory factors used is not particularly limited as long as the desired effect is achieved, and any ratio may be used. For example, when two types of costimulatory factors (X and Y) are used, the molecular ratio may be, for example, X:Y = 1:1 to 100, or X:Y = 1 to 100:1.

[0040] As described above, in a preferred embodiment of the production method of the present invention, CD80 and 4-1BBL are used as costimulatory factors. Preferred embodiments of each step when using this combination are as follows: [Step 1] 10 T cells of a cell population containing unstimulated CAR-T cells are treated with 4-1BBL. ６ [Step 2] After Step 1, 10 T cells of the cell population containing CAR-T cells are subjected to the following steps: 0.1 to 0.83 pmol of an antigen recognition factor, 0.2 to 0.85 pmol of CD80, and 0.2 to 0.85 pmol of 4-1BBL per cell. ６ [Step 3] (only if performed) 10 T cells of the cell population containing CAR-T cells after Step 2 are added to 10 T cells of the cell population containing CAR-T cells. ６ Per cell, 0.4 to 2.0 pmol of antigen recognition factor, 0.2 to 2.2 pmol of CD80, and 0.2 to 2.2 pmol of 4-1BBL.

[0041] In the production method of the present invention, the antigen recognition factor and / or costimulatory factor used to stimulate CAR-T cells may be free molecules (i.e., not bound to any substance) or may be bound to the surface of a support. Only the antigen recognition factor may be bound to the surface of the support, and the costimulatory factor may be a free molecule; alternatively, the antigen recognition factor may be a free molecule, and only the costimulatory factor may be bound to the surface of the support; or both the antigen recognition factor and the costimulatory factor may be bound to the surface of the support. When multiple costimulatory factors are used, all of the costimulatory factors may be free molecules, all of the costimulatory factors may be bound to the surface of the support, or some of the costimulatory factors may be free molecules, and the remaining costimulatory factors may be bound to the surface of the support. In a preferred embodiment, both the antigen recognition factor and the costimulatory factor may be bound to the surface of the support. In this specification, the phrase "antigen recognition factor and / or costimulatory factor bound to the surface of the support" encompasses not only an embodiment in which the antigen recognition factor and / or costimulatory factor are directly bound to the surface of the support, but also an embodiment in which the antigen recognition factor and / or costimulatory factor are indirectly bound via some substance. Furthermore, the term "bond" is intended to encompass any type of bond, such as a covalent bond, a non-covalent bond, an electrostatic bond, or a hydrophobic bond.

[0042] Furthermore, when an antigen recognition factor and / or a costimulatory factor are bound to the surface of a support, the same (i.e., one type) support may be used for each factor, or two or more types of supports may be used. For example, the antigen recognition factor and the costimulatory factor may be bound to the surface of one type of support, or the antigen recognition factor may be bound to the surface of support A and the costimulatory factor may be bound to the surface of support B, which is a type different from support A. In a preferred embodiment, only one type of support may be used.

[0043] The support is not particularly limited as long as it can successfully activate and enhance the proliferation of T cells when an antigen recognition factor and / or a costimulatory factor is bound to its surface and brought into contact with T cells, and any known support can be used, including, but not limited to, magnetic substances, latex, agarose, glass, cellulose, sepharose, nitrocellulose, polystyrene, retronectin, collagen, etc.

[0044] The shape of the support is not particularly limited and may be any of a film, sponge, fiber, rod, bead, colloid (e.g., gel), nanomatrix structure, and the like. In a preferred embodiment, the support may be beads. The particle size of the beads may typically be about 10 nm to 500 μm, preferably about 100 nm to 100 μm, about 500 nm to 50 μm, about 1 μm to 30 μm, or about 1 μm to 15 μm, and more preferably about 1 μm to 5 μm or about 3 μm to 5 μm (e.g., 3.5 μm), but is not limited thereto. The support is preferably magnetic beads. This is because the use of magnetic beads enables the magnetic beads to be easily removed after stimulation of CAR-T cells by using a magnet without stressing the CAR-T cells.

[0045] The antigen recognition factor and / or costimulatory factor can be bound to the surface of the support by a method known per se. One example of such a method is to add biotin to an appropriate site (e.g., terminal) of the antigen recognition factor and / or costimulatory factor, coat the surface of the support with a monoclonal anti-biotin antibody, and bring them into contact with each other, thereby binding the antigen recognition factor and / or costimulatory factor to the surface of the support.

[0046] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0047] [Example 1] 1. Materials and Methods [Cell Preparation] T cells were isolated from an apheresis sample obtained from a healthy subject using an antibody and washed with D-PBS (Wako). The washed T cells were suspended in electroporation buffer (Miltenyi Biotec).

[0048] [CAR gene introduction] PB transposase plasmid and pIRII-EPHB4-28z plasmid were added to a T cell suspension. The suspension containing T cells and plasmid DNA was transferred to the EP-2 unit of a CliniMACS Prodigy (Miltenyi Biotec), and the plasmid DNA was introduced into the T cells by electroporation. Note that the proportion of T cells in the cell population containing CAR-T cells prepared in this example was approximately 90%.

[0049] [Expansion Culture] T cells after CAR gene introduction were collected and suspended in complete culture medium prepared by adding 5% artificial serum (Animal-free; Cell Science & Technology Institute), IL-7 (10 ng / mL; Miltenyi Biotec), and IL-15 (5 ng / mL; Miltenyi Biotec) to ALyS™705 Medium (Cell Science & Technology Institute), and then cultured for 14 days. Culture was performed in a 24-well cell bind plate or a T25 flask (CORNING). Complete culture medium was added every 2 to 3 days.

[0050] [Preparation of beads for stimulating CAR-T cells] Beads for stimulating CAR-T cells were prepared, with antigen recognition factors and costimulatory factors bound to their surfaces. Anti-Biotin MACSiBead Particles, cell culture grade (#130-092-357, Miltenyi Biotec) were used as the support. Biotinylated human EPHB4 protein, His-tag (Acro BIOSYSTEMS, #EP4-H8229, SEQ ID NO: 1) was used as the antigen recognition factor bound to the surface of the beads. The costimulatory factors bound to the surface of the beads were biotinylated human B7-1 / CD80 protein, Fc Avi-tag (Acro BIOSYSTEMS, #B71-H82F2, SEQ ID NO: 2) and biotinylated human 4-1BB Ligand / TNFSF9 protein, Fc Avi-tag (Acro BIOSYSTEMS, #41L-H82F9, SEQ ID NO: 3). These were mixed and reacted for 2 hours or more at 4°C while slowly rotating using a tube rotator to prepare beads for stimulating CAR-T cells. The quantities of each factor bound to the beads were as follows: EPHB4 (antigen recognition factor): 86.36 pmol / bead 1.0 x 10 ８ CD80 (costimulatory factor 1): 95.60 pmol / bead 1.0 x 10 ８ 4-1BBL (co-stimulatory factor 2): 99.60 pmol / bead 1.0×10 ８ pieces

[0051] [Stimulation of a T cell population containing CAR-T cells using beads] On days 1 and 8 after gene transfer into the T cells, beads for stimulating CAR-T cells were added to the medium, and the cell population containing CAR-T cells was stimulated twice. For samples No. 2 and 4, beads for stimulation were also added on day 11 after gene transfer, and the cell population containing CAR-T cells was stimulated three times. The proportions of each factor used to stimulate the cell population containing CAR-T cells are shown in Table 1 below. Then, the properties of the CAR-T cells on day 14 after CAR gene transfer were confirmed according to the analysis method described below in 2.

[0052]

[0053] Furthermore, sample No. 1 contained 10 T cells on day 14 after CAR gene transfer. ６ A third stimulation was performed with 0.96 pmol of EPHB4, 1.07 pmol of CD80, and 1.10 pmol of 4-1BBL per cell, and the properties of CAR-T cells on day 21 after CAR gene transfer were confirmed according to 2. Analysis method.

[0054] 2. Analysis Method [Measurement of Cell Count] The number of T cells was measured using an NC-3000 (MS Techno Systems) using the program Viability and Cell Count Assay, which can analyze the number of live cells, the number of dead cells, the viability, the cell diameter, and the aggregation rate.

[0055] [Flow cytometry (FCM) measurement] Cells were stained with anti-Ephrin B2 antibody (R&D), anti-CD3 antibody, anti-CD45RA antibody, and anti-CCR7 antibody (BioLegend), and analyzed using a MACSQuant analyzer 10 (Miltenyi Biotec).

[0056] Antibody staining was performed at 1x10 ６ The manufacturer's recommended per test volume was added to cells / 100 μL 4°C D-PBS and incubated at 4°C for 15 minutes. Primary staining was performed using anti-Ephrin-B2 antibody, secondary staining was performed using the secondary antibody used in the primary staining, and tertiary staining was performed using all remaining antibodies. Between each staining, cells were washed with 1 mL of D-PBS.

[0057] The CAR positivity rate was measured by staining dead cells with 7-AAD (Miltenyi Biotec) and gating on the 7-AAD-negative cell group and CD3-positive cell group. T subset analysis was performed by gating on the 7-AAD-negative, CD3-positive, and CAR-positive cell group, with CD45RA on the X-axis and CCR7 on the Y-axis. FCM results were analyzed using MACS Quantify analyzer software.

[0058] [Anti-cancer test] The test was carried out in vitro. The cancer cells used were Rh30 cells (ATCC), a human rhabdomyosarcoma cell line. 2 x 10 cancer cells were used per condition. ５ The cells were adjusted to 1 mL per well with complete medium, seeded onto a 24-well cell bind plate (CORNING), and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator under the conditions of for 2 to 24 hours as adherent culture.

[0059] Thereafter, CAR-T cells were added to the cell population containing CAR-T cells:cancer cells = 4:1, and the cells were incubated at 37°C, 5% CO ２ The cells were co-cultured for 3 days in an incubator under the conditions of

[0043] . The medium used during the co-culture was the complete medium for Rh30 cells (total 2 mL / well).

[0060] On day 3 of culture, the cells in the supernatant were collected in a 15 mL tube, and adherent cells were detached with Trypsin-EDTA (0.25%) (Thermo). Five volumes of complete medium were added to inactivate the Trypsin, and the cells were collected in the 15 mL tube (the tube from which the cells in the supernatant were collected). 100 μL of the collected cells were used to measure the cell count (described above).

[0061] FCM measurements were performed to determine the ratio of cell populations containing CAR-T cells to cancer cells.

[0062] The ratio was calculated using a MACSQuant analyzer 10 (Miltenyi Biotec) after staining cells with an anti-CD3 antibody for cell populations containing CAR-T cells and with an anti-CD221 antibody (BioLegend) for cancer cells.

[0063] Antibody staining was performed at 1x10 ６ To the cells / 100 μL 4° C. D-PBS, the per test amount specified by the manufacturer was added, and the mixture was allowed to react at 4° C. for 15 minutes.

[0064] Dead cells were stained with 7-AAD (Miltenyi Biotec), and the cells gated on 7-AAD-negative cells were plotted with CD3 on the X axis and CD221 on the Y axis. The FCM results were analyzed using MACS Quantify analyzer software.

[0065] 3. Results The cell count, CAR-positive rate, and T cell subset (Naive / Tscm) of the cell population containing CAR-T cells on day 14 after CAR gene transfer, as well as the results of the anti-cancer test (Killing Rh30), are shown in Table 2. The cell count of the cell population containing CAR-T cells is shown as the ratio (fold expansion) of the cell count on day 14 to the cell count immediately before CAR gene transfer, which is defined as 1.

[0066]

[0067] As shown in Table 2, when cell populations containing CAR-T cells were stimulated at a low level (No. 1 and 2), favorable results were obtained in the CAR-positive rate, the proportion of young, non-exhausted T cells, and the evaluation items of antitumor effect. Furthermore, although the fold expansion values ​​for No. 1 and No. 2 were smaller than those for the other samples, the cell populations containing CAR-T cells proliferated by more than 20-fold in the 14 days from immediately before gene transfer, demonstrating that even low-level stimulation can promote sufficient cell proliferation.

[0068] The cell count, CAR-positive rate, and T cell subset (Naive / Tscm) of the cell population containing CAR-T cells on day 21 after CAR gene transfer for Sample No. 1, as well as the results of the anti-cancer test (Killing Rh30), are shown in Table 3. The cell count of the T cell population containing CAR-T cells is shown as the ratio (fold expansion) of the cell count on day 21, where the cell count immediately before CAR gene transfer is defined as 1.

[0069]

[0070] As shown in Table 3, when a cell population containing CAR-T cells was stimulated at a low level and then stimulated a third time, favorable results were obtained in the CAR-positive rate, the proportion of young, non-exhausted T cells, and the evaluation items of antitumor effect.

[0071] [Example 2] 1. Materials and Methods [Cell Preparation] Cryopreserved peripheral blood mononuclear cells (PBMC; HEM) were added to ALySTM705 Medium (Cell Science & Technology Institute) with 5% artificial serum (Animal-free; Cell). Science & Technology Institute), IL-7 (10ng / mL; Miltenyi Biotec), IL-15 (5ng / mL; Miltenyi Biotec), T cell TransAct (10μL / 1x10 ６ The cells were suspended in complete medium supplemented with 1000kJ / ml PBS (Miltenyi Biotec) and cultured for 7 days. Culture was carried out in a 24-well cell bind plate or a T25 flask (CORNING). Complete medium was added every 2 to 3 days.

[0072] [CAR gene transduction] T cells obtained after 7 days of culture were infected with CD19-CAR lentivirus and then suspended in a complete culture medium containing ALyS™705 Medium (Cell Science & Technology Institute) supplemented with 5% artificial serum (Animal-free; Cell Science & Technology Institute), IL-7 (10 ng / mL; Miltenyi Biotec), and IL-15 (5 ng / mL; Miltenyi Biotec). The cells were then incubated at 37°C and 5% CO ２ The cells were cultured in an incubator. The proportion of T cells in the cell population containing CAR-T cells prepared in this example was approximately 98%.

[0073] [Expansion Culture] The CAR gene-transduced T cells were cultured for an additional 10 days. Culture was performed in a 24-well cell bind plate or a T25 flask (CORNING). Complete culture medium was added every 2 to 3 days.

[0074] [Preparation of beads for stimulating CAR-T cells] Beads for stimulating CAR-T cells were prepared in the same manner as described above in [Preparation of beads for stimulating CAR-T cells]. In this test, CD19 (Acro BIOSYSTEMS, #CD9-H82E9, SEQ ID NO: 4) was used as the recognition protein instead of EPHB4.

[0075] [Stimulation of a T cell population containing CAR-T cells using beads] On days 1 and 7 after gene transfer into the T cells, beads for stimulating CAR-T cells were added to the medium to stimulate the cell population containing CAR-T cells twice. The proportions of each factor used to stimulate the cell population containing CAR-T cells are shown in Table 4 below. The properties of the CAR-T cells on day 10 after CAR gene transfer were then confirmed according to the analysis method described below in 2.

[0076]

[0077] 2. Analysis Method [Measurement of Cell Count] The number of T cells was measured using an NC-3000 (MS Techno Systems) using the program Viability and Cell Count Assay, which can analyze the number of live cells, the number of dead cells, the viability, the cell diameter, and the aggregation rate.

[0078] [Flow cytometry (FCM) measurement] Cells were stained with anti-CD19 antibody (R&D), anti-CD3 antibody, anti-CD45RA antibody, and anti-CCR7 antibody (BioLegend), and the analysis was carried out using a MACSQuant analyzer 10 (Miltenyi Biotec).

[0079] Antibody staining was performed at 1x10 ６ The manufacturer's recommended per test volume was added to 100 μL of cells / 4°C D-PBS and incubated at 4°C for 15 minutes. Primary staining was performed using anti-CD19 antibody, secondary staining using the secondary antibody from the primary staining, and tertiary staining using all remaining antibodies. Between each staining, the cells were washed with 1 mL of D-PBS.

[0080] The CAR positivity rate was measured by staining dead cells with 7-AAD (Miltenyi Biotec) and gating on the 7-AAD-negative cell group and CD3-positive cell group. T subset analysis was performed by gating on the 7-AAD-negative, CD3-positive, and CAR-positive cell group, with CD45RA on the X-axis and CCR7 on the Y-axis. FCM results were analyzed using MACS Quantify analyzer software.

[0081] [Anti-cancer test] The test was carried out in vitro. NALM6 cells (ATCC), a human lymphoblastic leukemia cell line, were used as cancer cells. 2 x 10 cancer cells were used per condition. ５ The cells were adjusted to 1 mL per well with complete medium, seeded onto a 24-well cell bind plate (CORNING), and incubated at 37°C, 5% CO ２ The cells were cultured in an incubator under the conditions of

[0082] Thereafter, CAR-T cells were added to the cell population containing CAR-T cells:cancer cells = 2:1, and the cells were incubated at 37°C, 5% CO ２ The cells were co-cultured for 3 days in an incubator under the conditions of

[0043] . The medium used during the co-culture was the complete medium for NALM6 cells (total 2 mL / well).

[0083] On the third day of culture, the cells were harvested, and 100 μL of the cells was used to measure the cell number (as described above).

[0084] FCM measurements were performed to determine the ratio of cell populations containing CAR-T cells to cancer cells.

[0085] The ratio was calculated using a MACSQuant analyzer 10 (Miltenyi Biotec) after staining cells with an anti-CD3 antibody for cell populations containing CAR-T cells and with an anti-CD19 antibody (BioLegend) for cancer cells.

[0086] Antibody staining was performed at 1x10 ６ To the cells / 100 μL 4° C. D-PBS, the per test amount specified by the manufacturer was added, and the mixture was allowed to react at 4° C. for 15 minutes.

[0087] Dead cells were stained with 7-AAD (Miltenyi Biotec), and the cells gated on 7-AAD-negative cells were plotted with CD3 on the X axis and CD19 on the Y axis. The FCM results were analyzed using MACS Quantify analyzer software.

[0088] T subset analysis was performed by gating on 7-AAD-negative, CD3-positive, and CAR-positive cell groups, with CD45RA on the X-axis and CCR7 on the Y-axis. FCM results were analyzed using MACS Quantify analyzer software.

[0089] 3. Results The cell count, CAR positivity rate, and T cell subset (Naive / Tscm) of the cell population containing CAR-T cells on day 10 after CAR gene transfer, as well as the results of the anticancer test (Killing NALM6 and Killing Naive / Tscm) are shown in Table 5. The cell count of the cell population containing CAR-T cells is shown as the ratio (fold expansion) of the cell count on day 10 after infection, where the cell count at the start of culture is defined as 1.

[0090]

[0091] As shown in Table 5, when a cell population containing CAR-T cells was stimulated at a weak level (No. 1), and when a cell population containing CAR-T cells was stimulated at a strong level (No. 2), CAR-T cells with sufficient anti-cancer activity were produced. However, in No. 2, the proportion of young T cells significantly decreased after co-culture with NALM6 cancer cells, indicating that stimulation at a weak level promotes sustained anti-tumor activity.

[0092] [Example 3] In order to confirm the importance of the amount of stimulatory protein, stimulatory beads were prepared with a quarter of the amount of protein (i.e., CD19, CD80, 4-1BBL) bound to the stimulatory beads (referred to as stimulatory beads in this example). Details of the stimulatory beads used in Example 2 and the stimulatory beads in this example are as follows.

[0093] Stimulation beads used in Example 2: 1E8 Maxibeads to which 5 μg of each of three proteins was bound. CD19 (antigen recognition factor): 148.40 pmol / bead 1.0×10 ８ CD80 (costimulatory factor 1): 95.60 pmol / bead 1.0 x 10 ８ 4-1BBL (costimulatory factor 2): 99.60 pmol / bead 1.0×10 ８ pieces

[0094] Stimulation beads of this example: 1.25 μg of each of three proteins bound to 1E8 Maxi Beads (1 / 4 the amount of bound protein compared to the beads of Example 2). CD19 (antigen recognition factor): 37.10 pmol / bead 1.0×10 ８ CD80 (costimulatory factor 1): 23.90 pmol / bead 1.0 x 10 ８ 4-1BBL (co-stimulatory factor 2): 24.90 pmol / bead 1.0×10 ８ pieces

[0095] CAR-T cells were produced and their characteristics were analyzed under the same conditions as in Example 2, except that the CAR-T cells were stimulated using the stimulatory beads of this example in an amount four times that of the stimulatory beads used in Example 2.

[0096] Even under these conditions, a sufficient amount of high-quality CAR-T cells could be produced, as in Example 2. Therefore, it was demonstrated that in the present invention, the amount of protein used to stimulate CAR-T cells is important, regardless of the amount of beads used.

[0097] According to the present invention, it is possible to achieve activation and proliferation of CAR-T cells in a simple and low-cost process, thereby providing a novel method for easily and efficiently preparing CAR-T cells with a high anti-tumor effect. Thus, the present invention is extremely useful in the field of cancer treatment, etc.

[0098] This application is based on patent application No. 2024-109169 filed in Japan (filing date: July 5, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing CAR-T cells, comprising: a first step of stimulating a cell population containing unstimulated CAR-T cells under the following condition [A]; and a second step of stimulating the cell population containing CAR-T cells after the first step under the following condition [B]: [A] Number of T cells (cell) in a cell population containing unstimulated CAR-T cells: Number of antigen-recognition factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.1 to 0.83 : 0.2 to 1.7 [B] Number of T cells (cell) in the cell population containing CAR-T cells after the first step: Number of antigen-recognizing factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.05 to 0.17: 0.05 to 0.28 (However, the sum of the number of antigen recognition factors and the number of costimulatory factors in [B] is smaller than that in [A]).

2. The method for producing CAR-T cells according to claim 1, further comprising a third step of stimulating a cell population containing CAR-T cells after the second step under the following condition [C]: [C] Number of T cells (cell) in the cell population containing CAR-T cells after the second step: Number of antigen-recognition factors (pmol): Number of costimulatory factors (pmol) = 10 ６ : 0.4 to 2.0: 0.4 to 4.4 3. The production method according to claim 1, wherein the costimulatory factor is at least one selected from the group consisting of CD80, 4-1BBL, CD86, OX40L, ICOS-L, CD70, CD40L, CD270, ICAM-1, LFA-3, CD72, CD55, VCAM-1, MadCAM-1, CD111, CD112, CD155, CD153, PD-L2, PD-L1, Galectin-9, MHC, and CD113.

4. The method of claim 1, wherein at least two types of costimulatory factors are used.

5. The method of claim 1, wherein the costimulatory factors are CD80 and 4-1BBL.

6. In the first step, 10 T cells of a cell population containing unstimulated CAR-T cells are ６ The production method according to claim 5, wherein a cell population containing unstimulated CAR-T cells is stimulated under conditions of 0.1 to 0.83 pmol of an antigen recognition factor, 0.2 to 0.85 pmol of CD80, and 0.2 to 0.85 pmol of 4-1BBL per cell.

7. In the second step, 10 T cells of the cell population containing CAR-T cells after the first step are ６ The production method according to claim 5, wherein the cell population containing CAR-T cells is stimulated under conditions of 0.05 to 0.17 pmol of an antigen recognition factor, 0.05 to 0.14 pmol of CD80, and 0.05 to 0.14 pmol of 4-1BBL per cell.

8. In the third step, 10 T cells of the cell population containing CAR-T cells after the second step are ６ The production method according to claim 2, wherein the cell population containing CAR-T cells is stimulated under conditions of 0.4 to 2.0 pmol of an antigen recognition factor, 0.2 to 2.2 pmol of CD80, and 0.2 to 2.2 pmol of 4-1BBL per cell.

9. The method according to claim 1, wherein the antigen recognition factor and / or the costimulatory factor is bound to the surface of the support.

10. The method of claim 9, wherein the antigen recognition factor and the costimulatory factor are bound to the surface of the support.

11. The method of claim 9, wherein the support is a bead.

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