Serum-free formulation for T cell proliferation

Abstract

To provide a serum-free formulation for T cell expansion.SOLUTION: A basal serum-free medium and a combination of cytokines, wherein the combination of cytokines comprises IL-2, IL-4, IL-7, IL-10, and IL-15, wherein, based on the total volume of the basal serum-free medium: A serum free formulation for T-cell expansion is provided wherein the amount of IL-2 is between 5ng / ml and 50ng / ml, the amount of IL-4 is between 5ng / ml and 200ng / ml, the amount of IL-7 is between 5ng / ml and 90ng / ml, the amount of IL-10 is between 5ng / ml and 50ng / ml, and the amount of IL-15 is between 5ng / ml and / ml. 200ng.EFFECT: The serum-free formulation for T-cell proliferation exerts an effect of proliferating T-cells under serum-free conditions, especially selectively proliferating CD3 + CD8 + T-cells, thereby avoiding uncertainties and risks derived from animal serum and facilitating the development of adoptive cell therapy or immunotherapy.SELECTED DRAWING: None

Description

[[TECHNICAL FIELD]]

[0001] The present invention relates to a formulation for T cell proliferation, and particularly to a serum-free formulation for T cell proliferation. [[BACKGROUND ART]]

[0002] Cancer is accompanied by abnormal and uncontrolled cell proliferation, can invade other parts of the human body and even metastasize, so it is very difficult to cure. Common cancer treatments include surgery, chemotherapy, radiation therapy and the like. These treatments inevitably damage many normal cells in the process of removing cancer cells by physically removing cancer tissues or using cytotoxic drugs or radiation that may damage all cells including cancer cells and normal cells. Furthermore, even if a patient receives treatment and no cancer is detected for a period of time, cancer recurrence is still possible and is usually more difficult to manage.

[0003] In order to pursue more effective cancer treatment with less damage to the human body, cell therapy has been developed and introduced into cancer treatment, which is called immunotherapy. Adoptive cell therapy (ACT) is an immunotherapy that mainly uses modified autologous T cells to specifically recognize and kill cancer cells. Naive T cells consist of "naive CD4 + T cells" (helper T cells) and "naive CD8 + T cells" (cytotoxic T cells or killer T cells), and naive CD4 + T cells and naive CD8 + T cells both have CD3 protein on their cell surfaces, and form a complex to recognize targets. In the present invention, naive T cells are pre-selected with an anti-CD3 antibody. Here, "CD3 + CD4 + T cells" and the expression "CD3 + CD8 + T cells" refers to activated naive T cells, and the cells that recognize and kill cancer cells in adoptive cell therapy are mainly CD3 + CD8 + T cells.

[0004] However, a significant problem in adoptive cell therapy is the insufficient amount of T cells that can be administered to the patient, which limits the applicability of adoptive cell therapy. For example, to administer a patient in adoptive cell therapy, typically about 1 × 10⁶ T cells are needed. 9 T cells are needed, but human peripheral blood contains approximately 1 × 10⁶ cells per milliliter (ml). 6 Only T cells are present, and even in 60 ml of blood, approximately 6 x 10 7 Only a small number of T cells are obtained, and at least 16 to 17 times proliferation is still needed to meet the required amount of T cells for adoptive cell therapy. Therefore, how to effectively proliferate T cells is key to unlocking the potential of adoptive cell therapy.

[0005] To date, the common method for proliferating T cells is to culture them in a suitable basal serum-free medium further supplemented with animal serum and interleukin-2 (IL-2). Animal serum, such as fetal bovine serum (FBS), is commonly added to the culture medium to provide cells with several nutrients beneficial for cell proliferation. However, animal serum usually has the following drawbacks and risks: (1) The actual components of animal serum are numerous, complex, and unknown. (2) Different manufacturing lots of animal serum can vary significantly in properties, lacking reasonable reproducibility. (3) The purification process for animal serum is complex, and quality testing of each manufacturing lot is time-consuming. (4) Animal serum is usually heterogeneous and may harbor pathogens, bacteria, or viruses that cause infection or inflammation. Furthermore, the use of heterogeneous materials such as animal serum is disadvantageous in adoptive cell therapy using autologous T cells in human patients.

[0006] Therefore, there remains a need to research and develop methods for growing T cells without using animal serum, in order to avoid the uncertainties and risks associated with animal serum, thereby accelerating advances in adoptive cell therapy or immunotherapy. [Overview of the project] [Problems that the invention aims to solve]

[0007] Considering the problems of the prior art, one of the objectives of the present invention is to provide a method for proliferating T cells under serum-free conditions.

[0008] Another object of the present invention is CD3 + CD8 + The objective is to provide a method for selectively proliferating T cells, which involves CD3 + CD4 + CD3 as a percentage of T cells + CD8 + This indicates an increase in the proportion of T cells.

[0009] Another object of the present invention is to provide a serum-free environment in which a considerable amount of proliferated T cells can be obtained compared to conventional methods of proliferating T cells using animal serum, particularly CD3 + CD8 + The objective is to provide a method for increasing the proliferation of T cells. [Means for solving the problem]

[0010] To achieve the above objective, the present invention provides a serum-free formulation for T cell proliferation comprising a basal serum-free medium and a combination of cytokines, wherein the cytokine combination comprises interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-10 (IL-10), and interleukin-15 (IL-15). Based on the total volume of the basal serum-free medium, the cytokine combination has an IL-2 content of 5 ng / ml to 50 ng / ml, an IL-4 content of 5 ng / ml to 200 ng / ml, an IL-7 content of 5 ng / ml to 90 ng / ml, an IL-10 content of 5 ng / ml to 50 ng / ml, and an IL-15 content of 5 ng / ml to 200 ng / ml.

[0011] The serum-free formulation for T cell proliferation of the present invention, by employing a specific combination of cytokines of certain types and contents, can not only proliferate T cells without using animal serum, but also CD3 + CD8+ It is possible to selectively proliferate T cells, and as a result, in the proliferated T cell population, CD3 + CD8 + The proportion of T cells clearly increased, and accordingly CD3 + CD4 + CD3 as a percentage of T cells + CD8 + The proportion of T cells increases. Therefore, the serum-free formulation for T cell proliferation of the present invention does not contain animal serum and promotes T cells, especially CD3 + CD8 + This invention provides a method for proliferating T cells, thereby avoiding the uncertainties and risks associated with animal serum and facilitating the development of adoptive cell therapy or immunotherapy. [Modes for carrying out the invention]

[0012] In some embodiments of the present invention, the serum-free formulation for T cell proliferation further comprises insulin, with an insulin content of 1 μg / ml to 10 μg / ml based on the total volume of the basal serum-free medium. The serum-free formulation for T cell proliferation of the present invention further has the effect of increasing the T cell count to a level close to that obtained when using animal serum by further adding a specific content of insulin.

[0013] In some embodiments of the present invention, the insulin content is 1 μg / ml to 9 μg / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the insulin content is 1 μg / ml to 8 μg / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the insulin content is 1 μg / ml to 7 μg / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the insulin content is 1 μg / ml to 6 μg / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the insulin content is 2 μg / ml to 6 μg / ml based on the total volume of the basal serum-free medium.

[0014] In some embodiments of the present invention, the IL-2 content is 10 ng / ml to 50 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-2 content is 15 ng / ml to 50 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-2 content is 20 ng / ml to 50 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-2 content is 20 ng / ml to 45 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-2 content is 20 ng / ml to 40 ng / ml based on the total volume of the basal serum-free medium.

[0015] In some embodiments of the present invention, the IL-4 content is 10 ng / ml to 150 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-4 content is 20 ng / ml to 120 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-4 content is 30 ng / ml to 120 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-4 content is 30 ng / ml to 100 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-4 content is 35 ng / ml to 100 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-4 content is 40 ng / ml to 100 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-4 content is 50 ng / ml to 80 ng / ml based on the total volume of the basal serum-free medium.

[0016] In some embodiments of the present invention, the IL-7 content is 10 ng / ml to 70 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-7 content is 20 ng / ml to 70 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-7 content is 20 ng / ml to 65 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-7 content is 25 ng / ml to 60 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-7 content is 30 ng / ml to 50 ng / ml based on the total volume of the basal serum-free medium.

[0017] In some embodiments of the present invention, the IL-10 content is 10 ng / ml to 40 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-10 content is 15 ng / ml to 40 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-10 content is 15 ng / ml to 35 ng / ml based on the total volume of the basal serum-free medium.

[0018] In some embodiments of the present invention, the IL-15 content is 10 ng / ml to 150 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-15 content is 10 ng / ml to 125 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-15 content is 10 ng / ml to 100 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-15 content is 20 ng / ml to 100 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-15 content is 30 ng / ml to 100 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-15 content is 40 ng / ml to 100 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-15 content is 50 ng / ml to 90 ng / ml based on the total volume of the basal serum-free medium. In some embodiments of the present invention, the IL-15 content is 60 ng / ml to 80 ng / ml based on the total volume of the basal serum-free medium.

[0019] In some embodiments of the present invention, the basal serum-free medium is a serum-free medium for culturing leukocytes. Those skilled in the art know that leukocytes are part of the body's immune system that helps the body fight infection or disease, and that the types of leukocytes may be granulocytes (neutrophils, eosinophils, basophils, etc.), monocytes and / or lymphocytes (T cells and B cells, etc.).

[0020] In some embodiments of the present invention, leukocytes include natural killer cells (NK cells), dendritic cells (DCs), macrophages, T cells, and / or B cells.

[0021] In some embodiments of the present invention, the basal serum-free medium comprises L-glutamine, human albumin, and human transferrin.

[0022] In some embodiments of the present invention, the basal serum-free medium is X-VIVOTM 15 medium or AIM-V TM It can be a culture medium. In some embodiments of the present invention, the basal serum-free medium is X-VIVO TM 15 is a possible culture medium.

[0023] In some embodiments of the present invention, the serum-free formulation for T cell proliferation further comprises a T cell activator that activates naive T cells. Naive T cells are naive CD4 + T cells and naive CD8 + Includes T cells. Activation of naive T cells is linked to naive CD4 + T cells and naive CD8 + T cells, each activated CD4 + T cells (CD3 + CD4 + (labeled as T cells) and activated CD8 + T cells (CD3 + CD8 + Those skilled in the art know that this means transforming into cells that are labeled as T cells.

[0024] In some embodiments of the present invention, the T cell activator comprises a peptide that activates the CD3 protein and a peptide that activates the CD28 protein. In some embodiments of the present invention, the T cell activator comprises an anti-CD3 antibody and an anti-CD28 antibody. In some embodiments of the present invention, the T cell activator may be magnetic microbeads coated with the anti-CD3 antibody and the anti-CD28 antibody. In some embodiments of the present invention, the T cell activator may be CD3 / CD28 Dynabeads.

[0025] In this specification, a range expressed as "lower limit ~ upper limit" indicates a value greater than or equal to the lower limit and less than or equal to the upper limit, unless otherwise specified. For example, "5 ng / ml ~ 50 ng / ml" indicates a range of "5 ng / ml or more and 50 ng / ml or less".

[0026] Other objectives, advantages, and novel features of the present invention will become more apparent from the following detailed description. [Examples]

[0027] To illustrate embodiments and effects of the present invention, several examples and test cases are provided below. Those skilled in the art will readily realize the effects and benefits of the present invention based on the descriptions herein. Various modifications and variations can be made to carry out or apply the present invention without departing from the spirit and scope of the invention.

[0028] Example 1: Serum-free formulation for T cell proliferation As a base serum-free medium, we purchased X-VIVO from Lonza. TM Using a commercially available medium called Medium 15 (item number 04-418Q) (hereinafter referred to as "X-VIVO 15"), appropriate amounts of the cytokines IL-2 (purchased from PeproTech; item number: 200-02), IL-4 (purchased from PeproTech; item number: 200-04), IL-7 (purchased from PeproTech; item number: 200-07), IL-10 (purchased from PeproTech; item number: 200-10), and IL-15 (purchased from PeproTech; item number: 200-15) were added to this basic serum-free medium to obtain the serum-free formulation for T cell proliferation in Example 1 (hereinafter referred to as "E1 medium"). Based on the total volume of the basal serum-free medium, the IL-2 content was 30.8 ng / ml, the IL-4 content was 67.3 ng / ml, the IL-7 content was 40.3 ng / ml, the IL-10 content was 26.8 ng / ml, and the IL-15 content was 70 ng / ml.

[0029] Example 2: Serum-free formulation for T cell proliferation (containing insulin) The preparation of Example 2 was the same as in Example 1. Specifically, X-VIVO 15 was used as the basal serum-free medium, and appropriate amounts of the cytokines IL-2, IL-4, IL-7, IL-10, and IL-15, along with an appropriate amount of insulin (purchased from Novo Nordisk; item number: A10AC01), were added to this basal serum-free medium to obtain the serum-free formulation for T cell proliferation in Example 2. Based on the total volume of the basal serum-free medium, the IL-2 content was 30.8 ng / ml, the IL-4 content was 67.3 ng / ml, the IL-7 content was 40.3 ng / ml, the IL-10 content was 26.8 ng / ml, the IL-15 content was 70 ng / ml, and the insulin content was 4.39 μg / ml.

[0030] Comparative Example 1: Basic serum-free medium The basic serum-free medium X-VIVO 15 used in Examples 1 and 2 was designated as Comparative Example 1 and used in the following test example (hereinafter referred to as "CE1 medium").

[0031] Comparative Example 2: Formula for T cell proliferation containing animal serum The preparation of Comparative Example 2 was carried out in the same manner as in Example 1. Specifically, X-VIVO 15 was used as the basal serum-free medium, and appropriate amounts of the cytokines IL-2, IL-4, IL-7, IL-10, and IL-15, as well as FBS (purchased from Cytiva; item number: SH30071.03), were added to this basal serum-free medium to obtain the formulation for T cell proliferation of Comparative Example 2 (hereinafter referred to as "CE2 medium"). Based on the total volume of the basal serum-free medium, the content of IL-2 was 30.8 ng / ml, the content of IL-4 was 67.3 ng / ml, the content of IL-7 was 40.3 ng / ml, the content of IL-10 was 26.8 ng / ml, the content of IL-15 was 70 ng / ml, and the content of FBS was 10 vol% (volume percent).

[0032] Preparation example: CD3 + T cell selection 50 ml of human umbilical cord blood was collected and then centrifuged at 2000 rpm for 20 minutes to separate the plasma layer, buffy coat cell layer, and red blood cell (RBC) layer. The buffy coat cell layer was then collected and added to Ficoll buffer (purchased from Cytiva; item number: 17144003) in a 1:1 volume ratio, and density centrifugation was performed at 2000 rpm for 40 minutes to obtain the supernatant, mononuclear cell layer, Ficoll buffer layer, and RBC layer. Next, the mononuclear cell layer was collected and centrifuged at 2000 rpm for 10 minutes, and the supernatant was collected and mixed with CD3 microbeads (purchased from Miltenyi Biotec; item number: 130-097-043), i.e., magnetic microbeads coated with anti-CD3 antibody, to obtain a mixture. Next, using a VarioMACS separator (purchased from Miltenyi Biotec; item number: 130-090-282), the CD3 in the mixture is separated by magnetic force. + CD3 bound to microbeads + Screen T cells and CD3 for the following test cases + T cells were obtained.

[0033] Test example: Evaluation of T cell proliferation effect under serum-free conditions CD3 obtained in the preparation example + T cells were cultured in 24-well plates using E1 medium, E2 medium, CE1 medium, and CE2 medium, under 5% CO2 conditions at 37°C. CD3 + The original cell density of T cells is 1 × 10⁻⁶ 5 The cell volume was 1 ml per well, and the culture medium volume in each well was 1 ml. On the other hand, during T cell proliferation, CD3+ T cells were divided into CD3+CD4 cells. + T cells and CD3+CD8 + CD3 / CD28 Dynabeads (Gibco) are T cell activators that transform cells into T cells. TM Purchased from (item number: 11161D) and added to the culture medium, the CD3 / CD28 Dynabeads content was 2 μl / ml / 1 × 10 5 It was a cell.

[0034] After 3.5 days of culture, 1 ml of the corresponding fresh medium was added to each group of cells, i.e., cells cultured in E1 medium, E2 medium, CE1 medium, and CE2 medium, and growth was continued. After a further 3.5 days of culture (total of 7 days), cells from each group were collected and subjected to the following analyses (1) to (3) to evaluate the T cell proliferation results between different groups. The cells cultured for the above 7 days were CD3 + T cells, CD3 + CD4 + T cells, CD3 + CD8 + It was found that the T cells contained T cells simultaneously. In the following analyses (1) to (3), the group that used E1 medium for T cell proliferation was designated E1, the group that used E2 medium for T cell proliferation was designated E2, the group that used CE1 medium for T cell proliferation was designated CE1, and the group that used CE2 medium for T cell proliferation was designated CE2.

[0035] Analysis (1):CD3 + CD4 + CD3 as a percentage of T cells + CD8 + Ratio of T cell percentages After the 7-day culture period described above, the number of cells in each group was counted using a hemocytometer, and the cell density was determined to be 1 × 10⁶. 5 Cells were collected in cells / tubes as E1, E2, CE1, and CE2 and centrifuged at 2000 rpm for 10 minutes. Next, after removing excess medium, the cells were treated with an anti-CD3 antibody (BD Pharmingen) containing FITC. TM Purchased from the company; item number: 561807), anti-CD4 antibody with PerCP (BD Pharmingen TM Purchased from the company; item number: 566924), and anti-CD8 antibody with PE (BD Pharmingen TM The samples were mixed with (purchased from the company; item number: 555367) and reacted for 30 minutes. After the reaction, the solutions were centrifuged at 2000 rpm for 10 minutes to remove excess antibody and obtain samples ready for analysis. Samples E1, E2, CE1, and CE2 were analyzed using a flow cytometer (purchased from BD Bioscience; item number: 23-13347-00) to obtain the cell counts of different types of T cells, which were then used to analyze CD3+ CD4 + CD3 as a percentage of T cells + CD8 + We were able to obtain the ratio of T cells (hereinafter referred to as "CD8 / CD4"). The mean CD8 / CD4 results for E1, E2, CE1, and CE2 are shown in Table 1 below. Note that the results shown in Table 1 are the mean and standard deviation based on four replicate experiments (n=4).

[0036] [Table 1]

[0037] According to the results in Table 1, the average CD8 / CD4 ratios for E1 and E2 were approximately 0.76 and 0.73, respectively, while the average CD8 / CD4 ratios for CE1 and CE2 were both approximately 0.55 or less. Compared to CE1 (using only basal serum-free medium for T cell culture and proliferation) and CE2 (using medium containing animal serum for T cell culture and proliferation), CD3 in E1 and E2 + CD8 + The proportion of T cells is CD3 + CD4 + The proportion of T cells was clearly higher. Therefore, the serum-free formulation for T cell proliferation of the present invention is CD3 + CD8 + It is possible to selectively proliferate T cells, thereby CD3 + CD8 + Since T cells play a major role in these treatments, it has become clear that this will be beneficial for the development of adoptive cell therapy or immunotherapy.

[0038] Analysis (2):CD3 + CD8 + Percentage of T cells The preparation of samples E1, E2, CE1, and CE2 was carried out in the same manner as in analysis (1). Subsequently, the samples E1, E2, CE1, and CE2 were analyzed by flow cytometry to obtain the cell counts of different types of T cells, and these were used to determine the total CD3 + CD3 in T cells + CD8 +The percentage of T cells was obtained. The results for E1, E2, CE1, and CE2 are as shown in Table 2 below. In addition, CD3 of E1 + CD8 + T cells, by normalization defining the cell count as 100%, CD3 + CD8 + The relative cell count of T cells was calculated and shown in Table 2 below. The results shown in Table 2 are the mean and standard deviation based on four repeated experiments (n=4).

[0039] [Table 2]

[0040] According to the results in Table 2, CD3 of E1 and E2 + CD8 + The percentage of T cells was approximately 50% of the total CD3 + T cells, whereas the percentage of CD3 + CD8 + T cells in CE1 and CE2 was only approximately 40% of the total CD3 + T cells, which was consistent with the effect of selectively proliferating CD3 + CD8 + T cells of E1 and E2 shown in Analysis (1).

[0041] Referring to the results of E1, E2 and CE1, the relative number of CD3 + CD8 + T cells of E1 and E2 was 100% and approximately 131%, respectively, which was at least about 3 times higher than that of CE1 (approximately 36%). Therefore, the serum-free formulation for T cell proliferation of the present invention can be used in a serum-free environment to produce CD3 + CD8 + T cells can not only be proliferated, but also a far larger amount of CD3 + CD8 + T cells can be obtained compared with the case of using only a basal serum-free medium for T cell proliferation.

[0042] Furthermore, referring to the results of E2 (using a medium containing insulin) and CE2 (using a medium containing FBS), the CD3 of E2 + CD8 + The relative number of T cells is approximately 131%, and CD3 in CE2. + CD8 + The relative number of T cells was approximately 156%, and the results for E2 and CE2 were similar. Therefore, the serum-free formulation for T cell proliferation of the present invention yields an equivalent amount of CD3 compared to conventional methods using animal serum. + CD8 + It was found that T cells could be obtained, while eliminating the uncertainties and risks associated with animal serum.

[0043] Analysis (3):CD3 + CD8 + T cell proliferation ratio The preparation of E2, CE1, and CE2 samples was carried out in the same manner as in analysis (1). Subsequently, the E2, CE1, and CE2 samples were analyzed by flow cytometry to obtain the cell counts of different types of T cells, and the total CD3 + CD3 in T cells + CD8 + The analysis focused particularly on the proportion of T cells. Furthermore, prior to the 7-day culture period, E2, CE1, and all CD3 of CE2 were analyzed. + CD3 in T cells + CD8 + The initial proportion of T cells was also analyzed by flow cytometry. Next, CD3 + CD8 + CD3 as the initial percentage of T cells (before the aforementioned 7-day culture) + CD8 + The final percentage of T cells (after the 7-day culture period mentioned above) can be obtained, and this is the CD3 ratio. + CD8 + This represents the proliferation ratio of T cells. The results for E2, CE1, and CE2 are shown in Table 3 below. Note that the results shown in Table 3 are the mean and standard deviation based on four replicated experiments (n=4).

[0044] [Table 3]

[0045] According to the results in Table 3, CD3 of E2 + CD8 + The T cell proliferation factor was approximately 5.6, which was significantly higher than that of CE1 (approximately 1.0). This indicates that E2 is far more effective at promoting CD3+CD8+ T cell proliferation than CE1, which is consistent with the results shown in analysis (2).

[0046] Furthermore, referring to the results for E2 and CE2, the CD3 values ​​for E2 (approximately 5.6) and CE2 (approximately 5.7) are as follows: + CD8 + The T cell proliferation ratios were almost the same, and E2 (using insulin-containing medium) showed a similar amount of CD3 compared to CE2 (using FBS-containing medium). + CD8 + This demonstrated that T cells could be obtained. This was consistent with the results shown in analysis (2).

[0047] Therefore, according to the results of the above analyses (1) to (3), the serum-free formulation for T cell proliferation of the present invention is effective even under serum-free conditions, and CD3 + CD8 + In addition to increasing T cells, CD3 + CD8 + Since T cells can be selectively proliferated, CD3 in the proliferated T cell population + CD8 + It became clear that the proportion of T cells increased significantly.

[0048] In summary, the present invention employs specific types and content of cytokines, so the serum-free formulation for T cell proliferation of the present invention does not contain animal serum and promotes T cell proliferation, particularly CD3 + CD8 + This invention provides a method for proliferating T cells, thereby avoiding the uncertainties and risks associated with animal serum and facilitating the development of adoptive cell therapy or immunotherapy.

[0049] While the above description outlines numerous features and advantages of the present invention, along with details of its structure and features, this disclosure is illustrative only. Modifications can be made within the scope of the principles of the present invention, to the maximum extent indicated by the broad general meaning of the terms used in the appended claims, particularly with respect to the details, especially the shape, size, and arrangement of the components.

Claims

1. A serum-free formulation for T cell proliferation comprising a basal serum-free medium and a combination of cytokines, wherein the combination of cytokines comprises interleukin-2, interleukin-4, interleukin-7, interleukin-10, and interleukin-15, wherein, based on the total volume of the basal serum-free medium, the content of interleukin-2 is 5 ng / ml to 50 ng / ml, the content of interleukin-4 is 5 ng / ml to 200 ng / ml, the content of interleukin-7 is 5 ng / ml to 90 ng / ml, the content of interleukin-10 is 5 ng / ml to 50 ng / ml, and the content of interleukin-15 is 5 ng / ml to 200 ng / ml.

2. The serum-free medium composition for T cell proliferation according to claim 1, further comprising insulin, wherein the content of the insulin is 1 μg / ml to 10 μg / ml based on the total volume of the basal serum-free medium.

3. The serum-free medium composition for T cell proliferation according to claim 1 or 2, wherein the interleukin-2 content is 10 ng / ml to 50 ng / ml based on the total volume of the aforementioned serum-free medium.

4. A serum-free medium composition for T cell proliferation according to claim 1 or 2, wherein the interleukin-4 content is 10 ng / ml to 150 ng / ml based on the total volume of the aforementioned serum-free medium.

5. A serum-free serum-free medium composition for T cell proliferation according to claim 1 or 2, wherein the interleukin-7 content is 10 ng / ml to 70 ng / ml based on the total volume of the aforementioned serum-free medium.

6. The serum-free medium composition for T cell proliferation according to claim 1 or 2, wherein the interleukin-10 content is 10 ng / ml to 40 ng / ml based on the total volume of the aforementioned serum-free medium.

7. A serum-free medium composition for T cell proliferation according to claim 1 or 2, wherein the interleukin-15 content is 10 ng / ml to 150 ng / ml based on the total volume of the aforementioned serum-free medium.

8. The serum-free medium composition for T cell proliferation according to claim 1, wherein the base serum-free medium is a serum-free medium for culturing leukocytes.

9. The serum-free medium composition for T cell proliferation according to claim 8, wherein the leukocytes include natural killer cells, dendritic cells, macrophages, T cells, or B cells.

10. The serum-free medium composition for T cell proliferation according to claim 1, wherein the basal serum-free medium comprises L-glutamine, human albumin, and human transferrin.

11. The serum-free medium composition for T cell proliferation according to claim 1, further comprising a T cell activator that activates naive T cells.

12. The serum-free medium composition for T cell proliferation according to claim 11, wherein the T cell activator comprises a peptide that activates the CD3 protein and a peptide that activates the CD28 protein.

13. The serum-free medium composition for T cell proliferation according to claim 11, wherein the T cell activator is a magnetic microbead coated with an anti-CD3 antibody and an anti-CD28 antibody.

Images