Serum-free formulations for t-cell expansion

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 formulations for the expansion of T cells, in particular serum-free formulations for the expansion of T cells. [Background technology]

[0002] Cancer is extremely difficult to cure because it involves abnormal and uncontrollable cell growth, can invade other parts of the body, and can even metastasize. Common cancer treatments include surgery, chemotherapy, and radiation therapy. These treatments either physically remove cancerous tissue or use cytotoxic drugs or radiation, which can damage all cells, including cancer cells and normal cells, and inevitably damage many normal cells in the process of removing cancer cells. Furthermore, even if a patient undergoes treatment and the cancer remains undetected for some time, cancer recurrence is still possible and usually more difficult to deal with.

[0003] In pursuit of more effective and less damaging cancer treatments, cell therapy has been developed and introduced into cancer treatment, 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 are "naive CD4 + T cells" (helper T cells) and "naive CD8 + T cells (cytotoxic T cells and killer T cells) and naive CD4 + T cells and naive CD8 + Both T cells have the CD3 protein on their cell surface, and form a complex to recognize a target. In the present invention, naive T cells are preselected with an anti-CD3 antibody. + CD4 + T cells and CD3 + CD8 + The term "T cells" refers to activated naive T cells, and the cells that recognize and kill cancer cells in adoptive cell therapy are primarily CD3 + CD8 + T cells.

[0004] However, when performing adoptive cell therapy, the insufficient amount of T cells to be administered to patients is a serious problem, limiting the application of adoptive cell therapy. For example, typically, about 1 × 10 T cells are required for administration to patients in adoptive cell therapy. 9 T cells are required, but there are approximately 1 x 10 per milliliter (ml) in human peripheral blood. 6 There are only about 6 × 10 T cells in 60 ml of blood. 7 Therefore, to meet the required amount of T cells for adoptive cell therapy, at least approximately 16-17 fold expansion is still required. Therefore, how to effectively expand T cells is key to unlocking the potential of adoptive cell therapy.

[0005] To date, the most common method for expanding T cells is to culture them in an appropriate basal serum-free medium supplemented with animal serum and interleukin-2 (IL-2). Animal serum, such as fetal bovine serum (FBS), is commonly added to culture media to provide cells with several nutrients beneficial for cell growth. However, animal serum typically has the following drawbacks and risks: (1) The actual components of animal serum are numerous, complex, and unknown. (2) Different production lots of animal serum can vary significantly in properties, resulting in a lack of reasonable reproducibility. (3) The purification process of animal serum is complex, and quality testing of each production 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 xenogeneic materials such as animal serum is disadvantageous for adoptive cell therapy using autologous T cells for human patients.

[0006] Therefore, to avoid the uncertainties and risks associated with animal serum, there remains a need to develop methods that can expand T cells without using animal serum, thereby facilitating the advancement of adoptive cell therapy or immunotherapy. Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the problems of the prior art, one of the objects of the present invention is to provide a method by which T cells can be expanded under serum-free conditions.

[0008] Another object of the present invention is to + CD8 + To provide a method for selectively expanding T cells, which is + CD4 + CD3 to T cell ratio + CD8 + The percentage of T cells is shown to increase.

[0009] It is yet another object of the present invention to provide a method for the production of T cells, particularly CD3 T cells, under serum-free conditions, which results in a significant amount of expanded T cells compared to conventional methods of expanding T cells using animal serum. + CD8 + The object is to provide a method for expanding T cells. [Means for solving the problem]

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

[0011] The serum-free formulation for T cell proliferation of the present invention employs a combination of specific types and amounts of cytokines, which not only enables T cell proliferation without animal serum but also enhances CD3 + CD8+ T cells can be selectively expanded, resulting in the expansion of CD3 + CD8 + The proportion of T cells was clearly increased, and accordingly, CD3 + CD4 + CD3 to T cell ratio + CD8 + Therefore, the serum-free formulation for T cell expansion of the present invention can increase the percentage of T cells, particularly CD3 + CD8 + A method for expanding T cells is provided, thereby avoiding the uncertainties and risks associated with animal serum and facilitating the development of adoptive cell therapy or immunotherapy. DETAILED DESCRIPTION OF THE INVENTION

[0012] In some embodiments of the present invention, the serum-free formulation for T cell proliferation further comprises insulin, and the insulin content is 1 μg / ml to 10 μg / ml, based on the total volume of the basal serum-free medium. By further adding a specific content of insulin, the serum-free formulation for T cell proliferation of the present invention further has the effect of increasing the amount of T cells to a level close to that achieved when animal serum is used.

[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 content of IL-15 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 types of leukocytes can be granulocytes (such as neutrophils, eosinophils, basophils), monocytes, and / or lymphocytes (such as T cells and B cells).

[0020] In some embodiments of the invention, the leukocytes comprise 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 In some embodiments of the present invention, the basal serum-free medium may be X-VIVO TM 15 medium.

[0023] In some embodiments of the present invention, the serum-free formulation for T cell expansion further comprises a T cell activator that activates naive T cells. Naive T cells are naive CD4 + T cells and naive CD8 + T cells. Activation of naive T cells involves the activation of naive CD4 + T cells and naive CD8 + T cells, activated CD4 + T cells (CD3 + CD4 + T cells) and activated CD8 + T cells (CD3 + CD8 + Those skilled in the art will know that the term "transformation" refers to the transformation of a cell into a T cell (labeled as a T cell).

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

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

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

[0027] Hereinafter, several examples and test examples will be given to illustrate the embodiments and effects of the present invention. Based on the contents of this specification, those skilled in the art can easily realize the effects and benefits of the present invention. Various modifications and variations can be made to implement or apply the present invention without departing from the spirit and scope of the present invention.

[0028] Example 1: Serum-free formulation for T cell expansion X-VIVO (Lonza) was used as the basal serum-free medium. TM A commercially available medium called X-VIVO 15 medium (item number 04-418Q) (hereinafter referred to as "X-VIVO 15") was used, and appropriate amounts of 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 basal serum-free medium to obtain the serum-free formulation for T cell proliferation of Example 1 (hereinafter referred to as "E1 medium"). Based on the total volume of 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, and the content of IL-15 was 70 ng / ml.

[0029] Example 2: Serum-free formulation (including insulin) for T cell expansion The preparation of Example 2 was the same as that of Example 1. Specifically, X-VIVO 15 was used as the basal serum-free medium, and appropriate amounts of cytokines IL-2, IL-4, IL-7, IL-10, and IL-15, as well as 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 of 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: Basal serum-free medium The basal serum-free medium X-VIVO 15 used in Examples 1 and 2 was used as Comparative Example 1 in the following test examples (hereinafter referred to as "CE1 medium").

[0031] Comparative Example 2: Formulation for T cell expansion containing animal serum Comparative Example 2 was prepared in the same manner as in Example 1. Specifically, X-VIVO 15 was used as the basal serum-free medium, and appropriate amounts of 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 a formulation for T cell proliferation in Comparative Example 2 (hereinafter referred to as "CE2 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, the IL-15 content was 70 ng / ml, and the FBS content was 10 vol% (volume percent).

[0032] Preparation example: CD3 + T cell selection 50 ml of human umbilical cord blood was collected and centrifuged at 2000 revolutions per minute (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) at a volume ratio of 1:1. The mixture was subjected to density centrifugation at 2000 rpm for 40 minutes to obtain a supernatant layer, a mononuclear cell layer, a Ficoll buffer layer, and an RBC layer. The mononuclear cell layer was then collected and centrifuged at 2000 rpm for 10 minutes. The supernatant was then 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, the CD3 in the mixture was separated by magnetic force using a VarioMACS separator (purchased from Miltenyi Biotec; item number: 130-090-282). + CD3 bound to microbeads + Screening T cells and CD3 for the following test examples + 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 E1 medium, E2 medium, CE1 medium, and CE2 medium in 24-well plates at 37°C under 5% CO2. + The original cell density of T cells was 1 × 10 5 The media volume in each well was 1 ml. Meanwhile, during T cell proliferation, CD3+ T cells were differentiated into CD3+CD4 + T cells and CD3+CD8 + CD3 / CD28 Dynabeads (Gibco) were used as T cell activators to transform T cells. TM CD3 / CD28 Dynabeads (purchased from Sigma-Aldrich Co., Ltd.; item number: 11161D) were also added to the medium, and the content of CD3 / CD28 Dynabeads 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 the cells of each group, i.e., the cells cultured in E1 medium, E2 medium, CE1 medium, and CE2 medium, and they were allowed to continue growing. After another 3.5 days of culture (7 days in total), the cells of each group were collected and subjected to the following analyses (1) to (3) to evaluate the T cell proliferation results between the different groups. After the above 7 days of culture, the cells were CD3 + T cells, CD3 + CD4 + T cells, CD3 + CD8 + It was found that T cells were simultaneously included in the T cells. In the following analyses (1) to (3), the group using E1 medium for T cell proliferation was designated E1, the group using E2 medium for T cell proliferation was designated E2, the group using CE1 medium for T cell proliferation was designated CE1, and the group using CE2 medium for T cell proliferation was designated CE2.

[0035] Analysis (1):CD3 + CD4 + CD3 to T cell ratio + CD8 + T cell ratio After 7 days of culture, the number of cells in each group was counted using a hemocytometer, and the cell density was 1 × 10 5 Cells E1, E2, CE1, and CE2 were collected from each tube and centrifuged at 2000 rpm for 10 minutes. After removing excess medium, the cells were incubated with an anti-CD3 antibody (BD Pharmingen) bearing FITC. TM Anti-CD4 antibody with PerCP (purchased from BD Pharmingen; item number: 561807) TM Item number: 566924), and anti-CD8 antibody with PE (BD Pharmingen TM The E1, E2, CE1, and CE2 samples were analyzed by a flow cytometer (purchased from BD Biosciences; item number: 23-13347-00) to obtain the cell counts of different types of T cells, which were then used to measure CD3+ CD4 + CD3 to T cell ratio + CD8 + The ratio of the proportion of T cells (hereinafter referred to as "CD8 / CD4") was obtained. The average CD8 / CD4 results for E1, E2, CE1, and CE2 are shown in Table 1 below. The results shown in Table 1 are the average and standard deviation based on four repeated experiments (n=4).

[0036] [Table 1]

[0037] According to the results in Table 1, the average CD8 / CD4 ratios of E1 and E2 were approximately 0.76 and 0.73, respectively, while the average CD8 / CD4 ratios of CE1 and CE2 were both approximately 0.55 or less. Compared with 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), the CD3 / CD4 ratios in E1 and E2 were significantly higher. + CD8 + The proportion of T cells is CD3 + CD4 + Therefore, the serum-free formulation for T cell expansion of the present invention significantly increased the CD3 + CD8 + It can selectively expand T cells, thereby + CD8 + As T cells play a major role in these treatments, it has become clear that there is an interest in developing adoptive cellular or immunotherapies.

[0038] Analysis (2):CD3 + CD8 + T cell percentage Preparation of E1, E2, CE1, and CE2 samples was performed as in analysis (1). E1, E2, CE1, and CE2 samples were then analyzed by flow cytometry to obtain cell counts of different types of T cells, which were used to calculate total CD3 + CD3 in T cells + CD8 +The percentage of T cells was obtained. The results for E1, E2, CE1, and CE2 are shown in Table 2 below. In addition, the CD3 + CD8 + By normalizing the number of T cells to 100%, the CD3 + CD8 + The relative number of T cells was calculated and shown in Table 2. The results shown in Table 2 are the average 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 proportion of T cells is CD3 + About 50% of all T cells were CD3 + CD8 + The proportion of T cells is CD3 + Only about 40% of all T cells were CD3 T cells, and the E1 and E2 CD3 T cells were + CD8 + This was consistent with the effect of selectively expanding T cells.

[0041] Looking at the results for E1, E2, and CE1, the CD3 + CD8 + The relative numbers of T cells were 100% and approximately 131%, respectively, which were at least three times higher than that of CE1 (approximately 36%). Therefore, the serum-free formulation for T cell expansion of the present invention was able to effectively inhibit CD3 T cells in a serum-free environment. + CD8 + Not only can T cells be expanded, but they also produce much higher amounts of CD3 T cells than when using only basal serum-free T cell expansion medium. + CD8 + It was found that T cells could be obtained.

[0042] Furthermore, when the results of E2 (using a medium containing insulin) and CE2 (using a medium containing FBS) were compared, the CD3 + CD8 + The relative number of T cells was approximately 131%, and the CD3 + CD8 + The relative number of T cells was about 156%, with comparable results for E2 and CE2. Thus, the serum-free formulation for T cell expansion of the present invention produced comparable amounts of CD3 T cells compared to conventional methods using animal serum. + CD8 + It has been shown that T cells can be obtained but without the uncertainties and risks that come from animal serum.

[0043] Analysis (3):CD3 + CD8 + T cell fold expansion Preparation of E2, CE1, and CE2 samples was performed as in analysis (1). Then, E2, CE1, and CE2 samples were analyzed by flow cytometry to obtain cell counts of different types of T cells and total CD3 + CD3 in T cells + CD8 + The analysis focused on the proportion of T cells. Furthermore, before the 7-day culture, the total CD3 + CD3 in T cells + CD8 + The initial proportion of T cells was also analyzed by flow cytometry. + CD8 + CD3 to T cell initial percentage (before the 7-day culture) + CD8 + The final percentage of T cells (after 7 days of culture) was obtained, which was the CD3 + CD8 + The results for E2, CE1, and CE2 are shown in Table 3 below. The results shown in Table 3 are the average and standard deviation based on four repeated experiments (n=4).

[0044] [Table 3]

[0045] According to the results in Table 3, E2 CD3 + CD8 + The T cell proliferation fold increase was approximately 5.6, which was significantly higher than that of CE1 (approximately 1.0), indicating that E2 was far more effective in expanding CD3+CD8+ T cells than CE1, consistent with the results shown in analysis (2).

[0046] Also, looking at the results for E2 and CE2, the CD3 of E2 (approximately 5.6) and CE2 (approximately 5.7) + CD8 + T cell proliferation was similar, and E2 (using insulin-containing medium) produced comparable amounts of CD3 T cells compared with CE2 (using FBS-containing medium). + CD8 + 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 can promote CD3 + CD8 + Not only does it increase T cell proliferation, but it also stimulates CD3 + CD8 + It is possible to selectively expand T cells, and CD3 in the expanded T cell population + CD8 + It was revealed that the proportion of T cells increased significantly.

[0048] In summary, because the present invention employs specific types and amounts of cytokines, the serum-free formulation for T cell expansion of the present invention allows the proliferation of T cells, particularly CD3 + CD8 + A method for expanding T cells is provided, thereby avoiding the uncertainties and risks associated with animal serum and facilitating the development of adoptive cell therapy or immunotherapy.

[0049] Although the foregoing description sets forth numerous features and advantages of the present invention, together with details of its structure and features, the disclosure is illustrative only, and changes may be made in details, particularly as to the shape, size, and arrangement of parts, within the scope of the principles of the invention, to the fullest extent indicated by the broad and general meaning of the terms expressed in the appended claims.

Claims

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

2. 2. The serum-free formulation for T cell proliferation according to claim 1, wherein the serum-free formulation for T cell proliferation further comprises insulin, and 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. 3. The serum-free formulation for T cell proliferation according to claim 1, wherein the content of interleukin-2 is 10 ng / ml to 50 ng / ml based on the total volume of the basal serum-free medium.

4. 3. The serum-free formulation for T cell proliferation according to claim 1, wherein the content of interleukin-4 is 10 ng / ml to 150 ng / ml based on the total volume of the basal serum-free medium.

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

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

7. 3. The serum-free formulation for T cell proliferation according to claim 1, wherein the content of interleukin-15 is 10 ng / ml to 150 ng / ml based on the total volume of the basal serum-free medium.

8. 2. The serum-free formulation for T cell expansion of claim 1, wherein the basal serum-free medium is a serum-free medium for culturing leukocytes.

9. 9. The serum-free formulation for T cell expansion of claim 8, wherein the leukocytes comprise natural killer cells, dendritic cells, macrophages, T cells, or B cells.

10. 2. The serum-free formulation for T cell expansion of claim 1, wherein the basal serum-free medium comprises L-glutamine, human albumin, and human transferrin.

11. 2. The serum-free formulation for T cell expansion of claim 1, further comprising a T cell activator that activates naive T cells.

12. 12. The serum-free formulation for T cell expansion of claim 11, wherein the T cell activator comprises a peptide that activates the CD3 protein and a peptide that activates the CD28 protein.

13. 12. The serum-free formulation for T cell expansion according to claim 11, wherein said T cell activator is CD3 / CD28 Dynabeads.

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