Method for in vitro mass proliferation and culture of vδ2 t cells without need for artificial antigen-presenting cells

Abstract

The present invention relates to a method for mass proliferation and culture of Vδ2 T cells, Vδ2 T cells produced by the method, and uses thereof, the method comprising the steps of: activating lymphocytes in a biological sample containing the lymphocytes; and proliferating and culturing the activated cells in the activated cell population by treating same with an anti-CD28 antibody and CD70 recombinant protein (CD27 ligand). The method for mass proliferation and culture according to the present invention can produce Vδ2 T cells while maintaining an excellent proliferation rate and purity without artificial antigen-presenting cells and thus can be effectively utilized in related technical fields such as treatment of cancer using Vδ2 T cells.

Description

In vitro mass culture method of Vδ2 T cells that do not require artificial antigen-presenting cells

[0001] The present invention relates to a method for mass-producing and culturing Vδ2 T cells, which are present in small amounts in lymphocytes of peripheral blood, in vitro, specifically a method for mass-producing and culturing Vδ2 T cells, Vδ2 T cells produced by said method, and the use thereof for cancer prevention or treatment.

[0002] γδ T cells are known to generally constitute 1 to 10 percent of T cells in human peripheral blood. T cells are known to primarily express antigen-recognizing receptors in the form of Vδ2 T receptors and are known as immune cells that can be activated by non-peptide phosphoantigens in an HLA-free manner. Activated human γδ T cells express granzyme B and perforin, which can exhibit strong cytotoxic activity against tumor cells.

[0003] Generally, about 50 to 75% of γδ T lymphocytes in peripheral blood express the Vδ2 chain, and cells that express the Vγ9 chain together with it are called Vγ9Vδ2 T cells, which are found only in humans and non-human primates. Activated Vδ2 T cells express cell adhesion molecules such as CD86, CD80, and MHC-II, and have the characteristic of recognizing non-peptide phosphorylated antigens, and can perform very important functions in anti-infection, anti-viral, and anti-tumor.

[0004] However, since normal human γδ T cells exist in minute quantities in human peripheral blood, it is difficult to expect active anti-tumor functions. Therefore, the successful clinical application of γδ T cells faces the challenge of how to effectively activate and expand Vδ2 T cells (Ann Blood 2022;7:42), and accordingly, many protocols for the ex vivo expansion of human Vδ2 T cells are being developed in recent studies.

[0005] Most early studies attempted to activate Vδ2 T cells by directly administering synthetic phosphorylated antigens (such as BrHPP or 2M3B1PP) to patients being treated for solid tumors or hematological malignancies. However, while synthetic phosphorylated antigens had the advantage of directly activating Vδ2 T cells, they had the disadvantage of decreasing the number of Vδ2 T cells after long-term use. Subsequent studies demonstrated anti-tumor capabilities by utilizing low doses of IL-2 and aminobisphosphonates (such as pamidronate or zoledronic acid) to promote the activation of Vδ2 T cells and the apoptosis of sensitized cells, but these were insufficient to significantly improve the effect of anti-tumor activity (Cells. 2022 Nov 11;11(22):3572).

[0006] Against this backdrop, the inventors have made diligent efforts to develop a technology capable of mass-culturing Vδ2 T cells with high purity without artificial antigen-presenting cells. As a result, they confirmed that by treating Vδ2 T cells with an anti-CD28 antibody and a CD70 recombinant protein (CD27 ligand) and culturing them, they can mass-culture Vδ2 T cells with high purity while maintaining their characteristics, thereby completing the present invention.

[0007] One objective of the present invention is to provide a method for mass proliferation culture of Vδ2 T cells comprising the steps of activating lymphocytes from peripheral blood mononuclear cells and proliferating the activated cells by treating them with an anti-CD28 antibody and a CD70 recombinant protein.

[0008] Another objective of the present invention is to provide Vδ2 T cells produced by the above-described mass proliferation culture method.

[0009] Another objective of the present invention is to provide a medicinal use of the Vδ2 T cells for the prevention or treatment of cancer.

[0010] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below.

[0011] Mass proliferation culture method of Vδ2 T cells

[0012] The inventors have devised a method to mass proliferate Vδ2 T cells with high purity by first activating lymphocytes in a biological sample containing lymphocytes to induce proliferation of Vδ2 T cells, and then treating with an anti-CD28 antibody and a CD70 recombinant protein, unlike conventional mass proliferation methods.

[0013] One aspect of the present invention for achieving the above objective is,

[0014] (a) A step of activating lymphocytes from a biological sample containing lymphocytes;

[0015] (b) A method for mass proliferation culture of Vδ2 T cells comprising the step of treating the activated cells of step (a) with an anti-CD28 antibody and a CD70 recombinant protein to proliferate them.

[0016] In the present invention, "γδ T cells" refers to a subgroup of T cells that express a γδ TCR, which is a clearly distinct T-cell receptor (TCR) consisting of one γ-chain and one δ-chain on its surface. There are two main sub-types of γδ T cells, and the two sub-types can be defined by the type of δ and / or γ present in the cells. For example, γδ T cells that are dominant in peripheral blood are Vδ2 T cells that mainly express the delta variable 2 chain (Vδ2), and γδ T cells that are dominant in non-hematopoietic tissues (i.e., commensal to the tissue) are Vδ1 T cells that mainly express the delta variable 1 (Vδ1) chain. According to the culture method of the present invention, high-purity Vδ2 T cells can be obtained in large quantities from peripheral blood mononuclear cells.

[0017] Vδ2 T cells can recognize human leukocyte antigens (HLA) without restriction and, upon antigen stimulation, can secrete numerous inflammatory cytokines, such as INF-γ and TNF-α, as well as cytolytic molecules like granzyme B and perforin. These responses influence cell growth inhibition, immune system activation, and inflammatory reactions, playing a particularly important role in cancer prevention or treatment.

[0018] In the present invention, the term "biological sample" refers to a sample containing living cells for the purposes of the present invention, characterized in particular by containing lymphocytes. In the present invention, the biological sample containing lymphocytes includes, without limitation, blood-derived cell populations containing lymphocytes, such as peripheral blood mononuclear cells (PBMCs), umbilical cord blood, and bone marrow blood, and is not limited to human cells but includes all animal-derived cells. Furthermore, the biological sample containing lymphocytes according to the present invention may also include, without limitation, lymphocyte-containing cell populations that have been naturally or artificially differentiated from undifferentiated cells such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and hematopoietic stem cells (HSCs).

[0019] In the present invention, a culture medium used for culturing a biological sample refers to a medium for maintaining or culturing a cell population containing nutrients that maintain cell viability and support proliferation. The culture medium used in the present invention comprises a basic medium. The basic medium is any basic medium suitable for culturing animal or human cells, particularly Vδ2 T cells.

[0020] The above basic medium typically contains a number of components necessary to support the maintenance of cultured cells. A suitable combination of components can be easily formulated by a skilled person with consideration of the following. Additionally, it includes a nutrient solution containing common standard cell culture components, e.g., amino acids, vitamins, lipid supplements, inorganic salts, carbon energy sources, and buffers.

[0021] The above basic media may be commercially available and may be, but is not limited to, one or more selected from the group consisting of, for example, AIM-V, CTS™ OpTmizer™ T Cell Expansion SFM, Xuri™ T Cell Expansion Medium, ALyS505N, RPMI, DC Medium, DMEM (Dulbecco's Modified Eagles Media), MEM (Minimum Essential Media), KO-DMEM (KnockOut-DMEM), α-MEM, G-MEM (Glasgow's Minimum Essential Media), BME (Basal Medium Eagle), DMEM / Ham's F12, Advanced DMEM / Ham's F12, IMDM (Iscove's Modified Dulbecco's Media), Ham's F-10, Ham's F-12, Medium 199, X-VIVO™, and KnockOut Serum replacement XenoFree medium.

[0022] In the present invention, step (a) is a step of activating lymphocytes from a biological sample containing lymphocytes, wherein the activation of lymphocytes can be induced through interaction with an antigen in a cell population contained in the biological sample, or through stimulation by extracellular signaling molecules such as cytokines and growth factors, and means increasing the activity of a lymphocyte population containing Vδ2 T cells. When lymphocytes are activated, they rapidly divide, increasing the proportion of lymphocytes containing Vδ2 T cells within the population, and secrete cytokines that regulate or assist the immune response.

[0023] Step (a) above may be applied using any known method capable of increasing the proportion of lymphocytes within the cell population of a biological sample. For example, Step (a) above may selectively proliferate Vδ2 T cells from a biological sample by culturing the biological sample in a medium containing aminobisphosphonate and / or Vδ2 T cell activating cytokines.

[0024] In the present invention, "aminobisphosphonate" is a γδ T cell agonist, and its activity depends on the presence of a phosphate moiety. As a derivative of pyrrolic acid, a nitrogen-containing bisphosphonate compound in which the oxygen atom of POP of the pyrrolic acid backbone is substituted with a carbon atom is referred to as aminobisphosphonate. Aminobisphosphonate can induce the expansion of γδ T cells by activating lymphocytes after bystander cells present in a PBMC culture release isopentenyl pyrophosphate (IPP) into the culture.

[0025] The above aminobisphosphonate may be, for example, one or more selected from the group consisting of alendronic acid, etidronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, and zoledronic acid, and specifically may be zoledronic acid, but is not limited thereto.

[0026] In the present invention, "cytokines" are various proteins of relatively small size produced in cells and used for cell signaling, and are generally known to be associated with immune responses to inflammation or infection.

[0027] In the present invention, "Vδ2 T cell activating cytokine" is understood to mean a cytokine that promotes the growth, proliferation, and differentiation of lymphocytes including Vδ2 T cells, and in the present invention, a cytokine that induces activation through interaction with peripheral blood lymphocytes including Vδ2 T cells to increase the proportion of Vδ2 T cells within a PBMC population. The Vδ2 T cell activating cytokine may be, for example, one or more selected from the group consisting of IL-1, IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, interferon (IFN)-γ, and IFN-α, and specifically may be IL-2, but is not limited thereto.

[0028] In one specific embodiment, step (a) may selectively proliferate Vδ2 T cells from a biological sample by culturing the biological sample containing lymphocytes in a medium containing zoledronic acid and / or IL-2, and may involve culturing by adding aminobisphosphonate and / or Vδ2 T cell activating cytokines as lymphocyte activating components.

[0029] In another specific embodiment, step (a) may involve culturing a biological sample containing lymphocytes in a medium that does not contain other aminobisphosphonates and Vδ2 T cell activating cytokines as lymphocyte-activating components other than zoledronic acid and IL-2.

[0030] In one specific embodiment, the concentration of aminobisphosphonate added to the biological sample culture medium of the present invention may be 1 to 10 μM. More specifically, the concentration may be 2 to 8 μM, and even more specifically, 3 to 7 μM, but is not limited thereto.

[0031] As a specific embodiment, the Vδ2 T cell activating cytokine may be added to the biological sample culture medium at intervals of 1 to 4 days, specifically at intervals of 2 to 3 days, but is not limited thereto.

[0032] In one specific embodiment, the concentration of Vδ2 T cell activating cytokine added to the biological sample culture medium of the present invention may be 100 to 5000 IU / ml. More specifically, it may be 300 to 3000 IU / ml, and even more specifically 500 to 2000 IU / ml, but is not limited thereto.

[0033] In one specific embodiment, the step (a) may include the step of culturing a biological sample for 5 to 15 days. More specifically, the culture period may be 6 to 14, more specifically 7 to 12 days, but is not limited thereto.

[0034] In the present invention, step (b) refers to a step of proliferating the activated cells of step (a) by treating them with an anti-CD28 antibody and a recombinant CD70 protein, and can be understood as a step of mass-producing Vδ2 T cells while maintaining a high purity of 90% or more, or selectively mass-producing Vδ2 T cells present in a low proportion within the cell population with a high purity of 90% or more. The anti-CD28 antibody and the recombinant CD70 protein may be commercially available products or manufactured directly, and the type thereof may be appropriately selected by a person skilled in the art as long as the objective of the present invention can be achieved through culture treated with the anti-CD28 antibody and the recombinant CD70 protein.

[0035] In the present invention, the step of treating the anti-CD28 antibody and CD70 recombinant protein in step (b) may involve directly treating the anti-CD28 antibody and CD70 recombinant protein into the cells for culture, or coating the anti-CD28 antibody and CD70 recombinant protein onto a culture vessel or beads and treating the cells, but is not limited to such a method as long as it enables the mass proliferation of Vδ2 T cells with high purity.

[0036] As a specific embodiment, in step (b) above, the anti-CD28 antibody and CD70 recombinant protein may be treated at a concentration of 10 to 5000 ng / ml. More specifically, it may be treated at a concentration of 100 to 3000 ng / ml. Even more specifically, it may be treated at a concentration of 500 to 2000 ng / ml, but is not limited thereto.

[0037] In the present invention, the step of proliferating culture in step (b) may additionally add a Vδ2 T cell proliferative cytokine.

[0038] In the present invention, "Vδ2 T cell proliferative cytokine" is understood to mean a cytokine involved in the growth, survival, proliferation, and homeostasis of Vδ2 T cells, and specifically, in the present invention, a cytokine that plays a role in inducing the proliferation of Vδ2 T cells. The Vδ2 T cell proliferative cytokine may be, for example, one or more selected from the group consisting of IL-2, IL-4, IL-9, IL-12, IL-15, IL-18, IL-21, and IL-33, and specifically may be IL-2, but is not limited thereto.

[0039] As a specific embodiment, the above Vδ2 T cell proliferative cytokine can be added at intervals of 1 to 4 days, specifically 2 to 3 days.

[0040] As a specific embodiment, the concentration of the Vδ2 T cell proliferative cytokine may be 10 to 1000 IU / ml. More specifically, it may be 30 to 500 IU / ml. Even more specifically, it may be 50 to 200 IU / ml, but is not limited thereto.

[0041] In one specific embodiment, step (b) may include the step of culturing Vδ2 T cells for 5 to 16 days. More specifically, the culture period may be 6 to 13 days, and even more specifically, 7 to 10 days, but is not limited thereto.

[0042] In the present invention, the Vδ2 T cells produced may be positive for TCRVδ2, γδ-TCR, CD3, CD40, NKG2D, NKp30, NKp44, 41BB, CD28, FASL, PD-1, TLR, and CD16 markers, and may be negative for TCRVδ1 marker; specifically, they may be positive for TCRVδ2 and CD3 markers and negative for TCRVδ1 marker, but are not limited thereto.

[0043] In the present invention, the Vδ2 T cells prepared above can recognize tumor cells and / or infected cells and secrete various cytokines and cell lysis molecules. For example, they can secrete IL-10, IL-6, IFN-γ, and TNF-α as cytokines, and secrete Granzyme A, Granzyme B, Perforin, and Granulysin as cell lysis molecules.

[0044] Vδ2 T cells and their uses

[0045] Another aspect of the present invention is a Vδ2 T cell produced by the mass proliferation culture method of the Vδ2 T cell.

[0046] Another aspect of the present invention is a pharmaceutical composition for the prevention or treatment of cancer, comprising the Vδ2 T cells as an active ingredient.

[0047] Another aspect of the present invention is a method for treating cancer, comprising the step of administering the composition to an individual in need thereof.

[0048] Another aspect of the present invention is a pharmaceutical composition for use in the prevention or treatment of cancer, or the use of said composition for the prevention or treatment of cancer.

[0049] Vδ2 T cells are as described above.

[0050] In the present invention, the cancer may include all cancers capable of exhibiting preventive or therapeutic efficacy mediated by Vδ2 T cells. For example, pseudomyxoma, intrahepatic cholangiocarcinoma, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, retinoblastoma, choroidal melanoma, ampullary cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, rhinosinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma, It may be one or more selected from the group consisting of malignant mesothelioma, malignant melanoma, ocular cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoid tumor, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid tumor, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, hematological cancer, and thymic cancer, and specifically may be ovarian cancer and pancreatic cancer, but is not limited thereto.

[0051] In the present invention, the term "treatment" refers to intervention to alter the natural processes of an individual or cell having a disease, which may be performed while the pathological condition is progressing. The intended therapeutic effects include alleviating the symptoms of the disease, reducing all direct or indirect pathological consequences associated with the disease, preventing metastasis, reducing the rate of disease progression, alleviating or temporarily resolving the disease state, reversing the condition, or improving the prognosis. In particular, the present invention includes all actions that improve the course of cancer by administering a composition containing Vδ2 T cells as an active ingredient. Furthermore, the term "prevention" refers to all actions that suppress or delay the onset or recurrence of the disease by administering the said Vδ2 T cells. When the Vδ2 T cells of the present invention are used for therapeutic or preventive purposes, they are administered to the individual in a therapeutically effective amount.

[0052] The term "therapeutically effective amount" as used in the present invention refers to an effective amount of Vδ2 T cells. Specifically, "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including individual type and severity, age, sex, type of disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with commercially available therapeutic agents. It may also be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above factors, and this can be easily determined by a person skilled in the art. The dosage of the pharmaceutical composition of the present invention may be determined by an expert based on various factors such as the patient's condition, age, sex, and complications. The therapeutic composition according to the present invention may be administered in combination with a known compound having the effect of preventing, improving, or treating symptoms of cancer.

[0053] The Vδ2 T cells according to the present invention or a composition containing the same may be administered intravenously, intramuscularly, subcutaneously, intraperitoneally, rectally, locally, or intranasally, but is not limited thereto.

[0054] Since the mass proliferation culture method of the present invention can produce Vδ2 T cells while maintaining excellent proliferation rate and purity even without artificial antigen-presenting cells, it can be usefully applied in related technical fields such as cancer treatment using Vδ2 T cells.

[0055] Figure 1 illustrates the in vitro proliferation culture process of Vδ2 T cells.

[0056] Figure 2 shows the results of FACS analysis of peripheral blood mononuclear cells.

[0057] Figure 3 shows the results of FACS analysis when γδT cells from peripheral blood mononuclear cells were activated with zoledronic acid and IL-2 for 10 days.

[0058] Figure 4 shows the FACS analysis results when Vδ2 T cells were cultured for activation and proliferation in peripheral blood mononuclear cells for 17 days.

[0059] Figure 5 shows the FACS analysis results when Vδ2 T cells were cultured for activation and proliferation in peripheral blood mononuclear cells for 20 days.

[0060] Figure 6 shows the purity and cell proliferation rate of Vδ2 T cells during the mass proliferation culture process of the present invention.

[0061] Figure 7 shows the results of an in vitro cytotoxicity analysis of Vδ2 T cells cultured in large quantities in an OVCAR-3 cell line.

[0062] Figure 8 shows the results of measuring the cytokines secreted by Vδ2 T cells cultured in large-scale proliferation in the OVCAR-3 cell line.

[0063] The structure and effects of the present invention will be explained in more detail below through examples. These examples are solely for the purpose of illustrating the present invention, and the scope of the present invention is not limited by these examples.

[0064] The present invention relates to a method for mass proliferation culture of Vδ2 T cells comprising the step of treating γδ T cells activated from a biological sample containing lymphocytes with an anti-CD28 antibody and a recombinant CD70 protein and culturing them for proliferation, and to the use of Vδ2 T cells obtained thereby. The mass proliferation culture method of the present invention enables the mass production of Vδ2 T cells while maintaining excellent proliferation rates and purity, even without artificial antigen-presenting cells. As one exemplary embodiment, the entire process of the method for mass proliferation culture of Vδ2 T cells according to the present invention is illustrated in FIG. 1, and specific examples are as follows.

[0065] Example 1. Mass proliferation culture of Vδ2 T cells

[0066] Step 1. γδT cell activation from biological samples containing lymphocytes

[0067] Frozen vials of peripheral blood mononuclear cells (PBMC, Cellular Technology Limited) from healthy donors were thawed in a 37°C water bath for 3 to 5 minutes. 1 ml of the thawed cells was added to 9 ml of RPMI (Welgene) medium containing 10% FBS (Capricon), washed, and the cells were counted using a hemocytometer. After counting, 5 x 10⁶ 6After centrifuging the lymphocytes, the cells suspended in a culture medium (T cell media) supplemented with CTS Optimizer T cell Expansion Basal Medium (Thermo), CTS Immune Cell SR (Thermo), Optimizer T cell Expansion Supplement (Thermo), 2 mM L-glutamine (Welgene), non-essential amino acids (Welgene), and gentamicin (Thermo) were transferred to a 6-well plate (Nunc). Then, 5 μM zoledronic acid (Sigma) and 1000 IU / ml IL-2 (peprotech) were added, and the cells were incubated for 10 days at 37°C and 5% CO2. IL-2 alone was added at a concentration of 1000 IU / ml at intervals of 2 to 3 days for 10 days.

[0068] Cells were obtained on day 0, the day the vial was thawed, and on day 10 of culture, respectively, and the percentage of Vδ2 T cells was measured using a flow cytometer as follows.

[0069] The obtained cells were washed with FACS buffer (PBS containing 2% FBS) and then stained with anti-TCRCD3-PE (Biolegend), anti-TCRVδ1-FITC (Biolegend), anti-TCRVδ2-percp cy5.5 (Biolegend), and anti-NKG2D-APC (Biolegend). Subsequently, the cells were incubated at 4°C for 30 minutes. After washing with FACS buffer and resuspending the cells in 400 μl of FACS buffer, the fluorescence expressed on the cell surface was measured using a flow cytometer. A Live / Dead fixable yellow dead kit (Thermo) was used to exclude dead cells during cell analysis. Characterization of the cell population was confirmed, and graphs showing day 0 are presented in Figure 2, and after 10 days of culture are presented in Figure 3. The results in Figures 2 and 3 represent the lymphocyte activation results in peripheral blood mononuclear cells from two donors before and after.

[0070] As a result, it was confirmed that the proportion of TCRVδ2 / CD3 cells in the entire cell population increased due to 10 days of activation, although the degree varied depending on the donor (Figs. 2 and 3).

[0071] Step 2. Culture of Vδ2 T cell proliferation after γδT cell activation

[0072] After coating 1000 ng / ml anti-CD28 antibody (Thermo) and CD70 recombinant protein (peprotech) onto a 24-well plate at 37°C for 4 hours, 2 x 10 γδ T cells activated in Example 1 6After transferring the cells to a coated 24-well plate (Nunc), they were incubated for 10 days at 37°C and 5% CO2 in a culture composition supplemented with 100 IU / ml IL-2. Growing Vδ2 T cells were harvested and counted at intervals of 2 to 3 days, re-plated, and IL-2 was added. Cell characteristics were measured using a flow cytometer as in Step 1 after 7 and 10 days of proliferation culture, and the results are shown in Figures 4 and 5.

[0073] As a result, when Vδ2 T cells were proliferated in cell populations isolated from each donor (day 17 of culture, day 7 of proliferation culture), Vδ2 + T cells showed 96.72% (Donor A) and 94.28% (Donor B), confirming that the proportion of TCRVδ2 / CD3 cells had significantly increased compared to day 10 of culture, when lymphocyte activation was performed (Fig. 4). This was further confirmed by showing 96.54% (Donor A) and 94.92% (Donor B) on day 20 of culture (day 10 of proliferation culture), indicating that high purity was maintained up to a total of 20 days of culture (Fig. 5). In each cell population, TCRVδ1 / CD3 cells were found to be less than 3%.

[0074] Meanwhile, during the mass proliferation culture process of Vδ2 T cells of the present invention, the overall Vδ2 + Changes in T cell % and cell proliferation rates are shown in Fig. 6. According to the mass proliferation culture method of Vδ2 T cells of the present invention, Vδ2 + T cells were initially present in trace amounts of 1.89% to 4.19% within peripheral blood mononuclear cells, but by day 20, when culture ended, they all showed a purity of over 90% regardless of the donor (left side of Fig. 6), and the cell proliferation rate was found to have increased by at least about 3,500 times by day 20 (right side of Fig. 6).

[0075] Example 2. Measurement of cancer cell death in Vδ2 T cells

[0076] The cancer cell killing ability of Vδ2 T cells obtained in Example 1 was confirmed by the following method. The target cells were, for example, OVCAR-3 cells.

[0077] 5 μM Calcein-AM (BD) at 1 x 10 6 OVCAR-3 cells were treated and stained after incubation for 1 hour at 37°C and 5% CO2. Vδ2 T effector cells and Calcein-AM-stained target cells were placed in 96-well V-bottom plates with effector (E):target (T) ratios of 20:1, 10:1, 5:1, and 2.5:1, and then incubated at 37°C for 4 hours. After 4 hours, cells and debris were removed by centrifugation, and the supernatant (70 μl) was harvested to measure fluorescence using a spectrofluorometer (480 / 530) (Biotek). The specific lysis rate was calculated as follows and is shown in Figure 7.

[0078] 100 X [(Experimental Emission - Spontaneous Emission) / (Maximum Emission - Spontaneous Emission)]

[0079] Spontaneous release and maximum release were measured in the presence of medium and 2% Triton X-100 (Sigma), respectively.

[0080] As a result, the group treated with Vδ2 T cells at 20 times the number of cancer cells showed an average cancer cell killing ability of approximately 88%, which was confirmed to decrease to 64%, 41%, and 22% as the ratio to cancer cells decreased to 10:1, 5:1, and 2.5:1. Therefore, it was confirmed that Vδ2 T cells cultured through the present invention have the ability to kill OVCAR-3 cells (Fig. 7).

[0081] Example 3. Measurement of Vδ2 T cell secreted cytokines

[0082] To measure the cytokines secreted by Vδ2 T cells mass-produced in Example 1, Vδ2 T cells were co-cultured with OVCAR-3 cell lines at a ratio of 3:1 at 37°C for 24 hours. After removing cell debris by centrifugation, the supernatant was collected and stored at -20°C until the experiment. Subsequently, cytokines were measured using an ELISA kit (Biolegend), and the procedure was carried out as follows.

[0083] 25 μl of assay buffer and 25 μl of beads were added to 25 μl of the acquired culture medium or standard and shaken at room temperature for 2 hours. Then, the mixture was washed with washing buffer, 25 μl of detection antibody was added, and the mixture was shaken at room temperature. After 1 hour, 25 μl of SA-PE was added and the mixture was shaken at room temperature for 30 minutes. After washing with washing buffer and resuspending in washing buffer, the values ​​measured by a flow cytometer were entered into Legendplex data analysis software (Biolegend) for analysis. The results were graphed and are shown in Figure 8.

[0084] As a result, it was confirmed that Vδ2 T cells co-cultured with OVCAR-3 secreted various cytokines and cytolytic molecules in response to cancer cells (Fig. 8).

[0085] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. (a) A step of activating lymphocytes from a biological sample containing lymphocytes; (b) a step of proliferating the activated cells of step (a) by treating them with an anti-CD28 antibody and a CD70 recombinant protein, comprising Method for mass proliferation culture of Vδ2 T cells.

2. In Paragraph 1, The above step (a) is a mass proliferation culture method in which a biological sample containing lymphocytes is cultured in a medium containing aminobisphosphonate or Vδ2 T cell activating cytokine.

3. In Paragraph 2, A mass proliferation culture method in which the above aminobisphosphonate is one or more selected from the group consisting of alendronic acid, etidronic acid, ibandronic acid, pamidronic acid, risedronic acid, minodronic acid, and zoledronic acid.

4. In Paragraph 2, A mass proliferation culture method in which the above-mentioned Vδ2 T cell activating cytokines are one or more selected from the group consisting of IL-1, IL-2, IL-4, IL-7, IL-12, IL-15, IL-18, IL-21, interferon (IFN)-γ, and IFN-α.

5. In Paragraph 2, A mass proliferation culture method in which the concentration of the aminobisphosphonate is 1 to 10 μM.

6. In Paragraph 2, A mass proliferation culture method in which the above Vδ2 T cell activating cytokine is added at intervals of 1 to 4 days.

7. In Paragraph 2, A mass proliferation culture method in which the concentration of the above Vδ2 T cell activating cytokine is 100 to 5000 IU / ml.

8. In Paragraph 1, A mass proliferation culture method in which the above step (a) is performed for 5 to 15 days.

9. In Paragraph 1, A mass proliferation culture method in which the step of treating the anti-CD28 antibody and CD70 recombinant protein in step (b) above involves directly treating the anti-CD28 antibody and CD70 recombinant protein, or treating the cells by coating the anti-CD28 antibody and CD70 recombinant protein onto a culture vessel or beads.

10. In Paragraph 1, A mass proliferation culture method in which, in step (b) above, the anti-CD28 antibody and CD70 recombinant protein are treated at a concentration of 10 to 5000 ng / ml.

11. In Paragraph 1, The step of proliferating culture in step (b) above is a mass proliferation culture method in which Vδ2 T cell proliferation cytokines are added to culture.

12. In Paragraph 11, A mass proliferation culture method wherein the above Vδ2 T cell proliferative cytokine is one or more selected from the group consisting of IL-2, IL-4, IL-9, IL-12, IL-15, IL-18, IL-21, and IL-33.

13. In Paragraph 11, A mass proliferation culture method in which the above Vδ2 T cell proliferative cytokine is added at intervals of 1 to 4 days.

14. In Paragraph 11, A mass proliferation culture method in which the concentration of the above Vδ2 T cell proliferative cytokine is 10 to 1000 IU / ml.

15. In Paragraph 1, A mass proliferation culture method in which step (b) above is cultured for 5 to 16 days.

16. In Paragraph 1, A mass proliferation culture method in which the above-mentioned Vδ2 T cells are positive for TCRVδ2 and CD3, and negative for TCRVδ1.

17. In Paragraph 1, A mass proliferation culture method in which the above-described Vδ2 T cells secrete one or more cytokines or cell lysis molecules selected from the group consisting of IL-10, IL-6, IFN-γ, TNF-α, Granzyme A, Granzyme B, Perforin, and Granulysin.

18. Vδ2 T cells produced by the mass proliferation culture method of any one of claims 1 to 17.

19. A pharmaceutical composition for the prevention or treatment of cancer comprising Vδ2 T cells of claim 18 as an active ingredient.

20. In Paragraph 19, The above cancers include pseudomyxoma, intrahepatic cholangiocarcinoma, hepatoblastoma, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, mycosis fungoides, acute myeloid leukemia, acute lymphoblastic leukemia, basal cell carcinoma, ovarian cancer, male breast cancer, brain cancer, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colorectal cancer, chronic myeloid leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, rhinosinus cancer, non-small cell lung cancer, tongue cancer, astrocytoma, small cell lung cancer, pediatric brain cancer, pediatric lymphoma, pediatric leukemia, small intestine cancer, meningioma, esophageal cancer, glioma, renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, malignant soft tissue cancer, malignant bone cancer, malignant lymphoma. A pharmaceutical composition comprising one or more selected from the group consisting of malignant mesothelioma, malignant melanoma, ocular cancer, vulvar cancer, ureteral cancer, urethral cancer, cancer of unknown primary site, gastric lymphoma, gastric cancer, gastric carcinoid tumor, gastrointestinal stromal cancer, Wilms' cancer, breast cancer, sarcoma, penile cancer, pharyngeal cancer, gestational trophoblastic disease, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, metastatic bone cancer, metastatic brain cancer, mediastinal cancer, rectal cancer, rectal carcinoid tumor, vaginal cancer, spinal cord cancer, acoustic neuroma, pancreatic cancer, salivary gland cancer, Kaposi's sarcoma, Paget's disease, tonsil cancer, squamous cell carcinoma, lung adenocarcinoma, lung cancer, lung squamous cell carcinoma, skin cancer, anal cancer, rhabdomyosarcoma, laryngeal cancer, pleural cancer, hematological cancer, and thymic cancer.

21. A pharmaceutical composition comprising the Vδ2 T cells of claim 18 and pharmaceutically acceptable additives.

22. Use of Vδ2 T cells of claim 18 for use in the manufacture of drugs for the treatment or prevention of cancer.

23. A method for treating or preventing cancer, comprising the step of administering a therapeutically effective amount of Vδ2 T cells of claim 18 to a subject in need thereof.

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