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

Abstract

The present invention relates to a method for mass proliferation and culture of iNKT cells, iNKT cells produced by the method, and uses thereof, the method comprising the steps of: expanding iNKT cells from peripheral blood mononuclear cells; and mass-proliferating and culturing the iNKT cells by treating same with an anti-CD28 antibody, the CD40 protein, and an anti-TCR Vα24-Jα18 antibody. Since the method for mass proliferation and culture of the present invention can produce iNKT cells while maintaining an excellent proliferation rate and purity without artificial antigen-presenting cells, the method can be effectively utilized in related technical fields such as treatment of cancer or inflammatory diseases using iNKT cells.

Description

In vitro mass proliferation and culture method for INKT cells without the need for artificial antigen-presenting cells

[0001] The present invention relates to a method for mass-proliferating and culturing iNKT cells, which exist in very small quantities in peripheral blood lymphocytes, in vitro, and relates to a method for mass-proliferating and culturing iNKT cells, iNKT cells produced by the method, and uses thereof.

[0002] Invariant Natural Killer T cells (iNKT cells) are activated by glycolipids presented by CD1d, a non-classical MHC class I molecule. They possess markers of both NK cells and T cells, and thus play an intermediate role between innate and adaptive immunity. The T cells that recognize antigens are known to express an invariant Vα24-Jα18 receptor, and are known as immune cells that can be activated by glycolipid antigens in an HLA-non-restricted manner.

[0003] In addition, iNKT cells are already known to have anticancer effects or anti-inflammatory effects because they express both Th1 / Th2 cytokines, so they can be used for anticancer purposes on their own or used as CAR-T to kill cancer cells by attaching CAR, and they are also used as cell therapy to suppress graft-versus-host disease (GVHD) due to their anti-inflammatory effects. However, iNKT cells are present in extremely small amounts, approximately 0.01 to 1%, in human peripheral blood mononuclear cells (PBMCs), which is considered a major drawback for use as cell therapy.

[0004] Meanwhile, it is known that the glycolipid antigen presented by CD1d of antigen-presenting cells (APC) is specifically recognized by the TCR (T cell receptor) of iNKT cells, resulting in the activation of iNKT cells. This can be utilized to activate iNKT cells by creating an environment in which CD1d presents glycolipids when culturing iNKT cells in vitro. In other words, a well-known method for obtaining iNKT cells is to treat dendritic cells, autologous peripheral blood mononuclear cells, or aAPC expressing CD1d with glycolipids to create an environment identical to that in which APCs present lipid antigens (Cancer Immunol Immunother. 2013 Apr;62(4):747-60). However, the possibility of residual cells added together with the final product, iNKT cells, poses a major obstacle to the development of cell therapy.

[0005] Against this backdrop, the inventors of the present invention have made extensive efforts to develop a technique for mass-cultivating iNKT cells with high purity in an environment containing CD1d or not treated with CD1d, and as a result, have confirmed that it is possible to mass-cultivate iNKT cells while maintaining their purity by culturing them with anti-CD28 antibody, CD40 protein, and anti-TCR Vα24-Jα18 antibody, thereby completing the present invention.

[0006] One object of the present invention is to provide a method for mass proliferation and culture of iNKT cells in a cell having CD1d or in an environment not treated with CD1d.

[0007] Another object of the present invention is to provide iNKT cells produced by the above mass proliferation culture method.

[0008] Another object of the present invention is to provide a medicinal use of the iNKT cells.

[0009] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. In other words, 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 described below.

[0010] Mass proliferation culture method for iNKT cells

[0011] The present inventors sought to develop a method for mass-proliferating iNKT cells under culture conditions without adding additional cells other than PBMCs. As previously mentioned, most iNKT cell cultures have involved creating artificial antigen-presenting cells that stably express CD1d, thereby inducing specific iNKT cell activity and proliferation. However, in the present invention, we have devised a method for mass-producing iNKT cells with high purity by treating them with anti-CD28 antibodies, CD40 protein, and anti-TCR Vα24-Jα18 antibodies.

[0012] To achieve the above purpose, the present invention provides a mass proliferation culture method for iNKT cells comprising the following steps:

[0013] (a) a step of expanding and culturing iNKT cells from peripheral blood mononuclear cells; and

[0014] (b) A step of culturing the cells of step (a) by treating them with anti-CD28 antibody, CD40 protein, and anti-TCR Vα24-Jα18 antibody to proliferate them.

[0015] In the present invention, "iNKT cells (invariant Natural Killer T cells)" are cells that share the characteristics of natural killer cells (NK cells), a new special type of T lymphocyte subpopulation that divides early in T lymphocyte development, and play an important role in both innate immunity and adaptive immunity.

[0016] iNKT cells possess a restricted T cell antigen receptor (TCR) that recognizes glycolipid antigens presented by the CD1d molecule. Following antigen stimulation, they rapidly respond and secrete numerous inflammatory cytokines, including IFN-γ and TNF-α. This initial response has a variety of effects on subsequent immune responses, and plays a particularly important role in the prevention or treatment of cancer.

[0017] In addition, iNKT cells can play a regulatory role in anti-inflammatory responses by secreting anti-inflammatory cytokines such as IL-4, IL-10, and TNF-β. Due to this regulatory function of iNKT cells, iNKT cells are involved in many pathological processes and play an important role in the prevention or treatment of inflammatory diseases.

[0018] In the present invention, "peripheral blood mononuclear cells (PBMC)" refers to a population of cells composed of lymphocytes such as T cells, B cells, and NK cells, and monocytes. The PBMC according to the present invention includes, without limitation, a blood-derived cell population containing iNKT cells, such as peripheral blood, umbilical cord blood, and bone marrow blood, and is not limited to human cells, but includes all animal-derived cells. In addition, the PBMC according to the present invention may also include, without limitation, a cell population containing iNKT cells that is 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, the culture medium used for PBMC culture refers to a medium for maintaining or culturing a cell population that contains nutrients that maintain cell viability and support proliferation. The culture medium used in the present invention includes a basic medium. The basic medium is any basic medium suitable for culturing animal or human cells, particularly iNKT cells.

[0020] The above-mentioned basic medium typically contains a number of components necessary to support the maintenance of cultured cells. Suitable combinations of these components can be readily formulated by skilled practitioners, taking into account the following. Furthermore, it includes a nutrient solution containing common standard cell culture components, such as amino acids, vitamins, lipid supplements, mineral salts, carbon energy sources, and buffers.

[0021] The above basic badges are commercially available, for example CTS TM OpTmizer TM The medium may be at least one selected from the group consisting of T Cell Expansion SFM, RPMI, DMEM (Dulbecco's Modified Eagles Media), MEM (Minimum Essential Media), KO-DMEM (KnockOut-DMEM), 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, and KnockOut Serum replacement XenoFree medium, but is not limited thereto.

[0022] Specifically, CTS TM OpTmizer TM T Cell Expansion It may be at least one selected from the group consisting of cell culture media such as SFM, RPMI, DMEM, and IMDM, and more specifically, CTS TM OpTmizer TM T Cell Expansion SFM may be, but is not limited to.

[0023] In the present invention, the step (a) is a step of expanding and culturing iNKT cells from peripheral blood mononuclear cells, and is a step of activating iNKT cells in a population of peripheral blood mononuclear cells to increase the proportion of iNKT cells in the population.

[0024] Any known method capable of increasing the proportion of iNKT cells in a PBMC population may be applied to step (a) above. For example, step (a) may be culturing peripheral blood mononuclear cells in a medium containing a glycolipid antigen and / or an iNKT activating cytokine.

[0025] In the present invention, “glycolipid antigen” refers to a glycolipid that is specifically recognized by the TCR of iNKT cells and induces activation of iNKT cells, and may include a glycolipid having α-linked galactose or glucose. For example, it may be at least one selected from the group consisting of α-galactosylceramide (αGalcer), α-galacturonosylceramide (αGalACer; Frontiers in Immunology. 2017 8:1858), α-monogalactosyldiacylglycerol (αMGalD; Christophe Ehret. Autre. Universitι de Strasbourg, 2012. Franηais), isoglobothrihexosylceramide (iGb3; RSC Adv. 2022, 12, 18493-18500), OCH (Nature 413, 531-534 (2001)), C20:2-αGalcer (J Biol Chem. 2012 Jan 6;287(2):1269-78) and analogs thereof, Specifically, it may be, but is not limited to, α-galactosylceramide (αGalcer).

[0026] In the present invention, “cytokine” refers to a variety of relatively small-sized proteins produced in cells and used for cell signal transmission, and is generally known to be related to immune responses to inflammation or infection.

[0027] In the present invention, "iNKT activating cytokine" is a cytokine that promotes the development and differentiation of iNKT cells, and can be understood to mean a cytokine that increases the proportion of iNKT cells in a PBMC population by inducing activation through interaction with iNKT cells in the present invention. The iNKT activating cytokine may be, for example, at least one selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, and GM-CSF, and may be specifically IL-2, but is not limited thereto.

[0028] In one specific embodiment, the concentration of the glycolipid antigen added to the PBMC culture medium of the present invention may be 1 to 1000 nM. More specifically, the concentration may be 10 to 500 nM, and even more specifically, 100 to 300 nM, but is not limited thereto.

[0029] In one specific embodiment, the iNKT activating cytokine may be added to the PBMC culture medium at intervals of 1 to 4 days, specifically at intervals of 2 to 3 days, but is not limited thereto.

[0030] In one specific embodiment, the concentration of the iNKT activating cytokine added to the PBMC culture medium of the present invention may be 1 to 1000 IU / mL. More specifically, the concentration may be 10 to 500 IU / mL, and even more specifically, 100 to 300 IU / mL, but is not limited thereto.

[0031] In the present invention, the step (a) may further include a step of isolating iNKT cells from expanded cultured cells.

[0032] In the present invention, the iNKT cell separation method may be FACS sorting and / or MACS sorting, but is not limited thereto.

[0033] In the present invention, the iNKT cells can be isolated by targeting one or more antigens selected from the group consisting of, for example, TCR Vα24-Jα18, CD3, CD4, CD8, CD25, CD28, CD40L, CD45RA, CD45RO, CD56, CD62L, CD69, CD161, HLA-DR, and Vβ11, and can be isolated by targeting TCR Vα24-Jα18 specifically, but is not limited thereto.

[0034] In one specific embodiment, step (a) may include culturing peripheral blood mononuclear cells for 6 to 18 days. More specifically, the culturing period may be 8 to 16 days, and more specifically, 10 to 14 days, but is not limited thereto.

[0035] The above step (b) refers to a step of treating a cell population including expanded iNKT cells with an anti-CD28 antibody, a CD40 protein, and an anti-TCR Vα24-Jα18 antibody, and can be understood as a step of mass-producing iNKT cells while maintaining a high purity of 90% or more. In the above step (b), the anti-CD28 antibody, CD40 protein, and anti-TCR Vα24-Jα18 antibody can be used as commercial products or manufactured directly, and the type can be appropriately selected by a person skilled in the art as long as the purpose of the present invention can be achieved through culturing after treatment with the anti-CD28 antibody, CD40 protein, and anti-TCR Vα24-Jα18 antibody.

[0036] In the present invention, the step of treating with an anti-CD28 antibody, CD40 protein, and anti-TCR Vα24-Jα18 antibody in step (b) may be performed by directly treating cells with the antibodies and proteins and culturing them, or by coating a culture vessel or bead with the anti-CD28 antibody, CD40 protein, and anti-TCR Vα24-Jα18 antibody and treating the cells. However, the method is not limited thereto as long as iNKT cells can be mass-produced with high purity.

[0037] In one specific embodiment, in step (b), the anti-CD28 antibody may be treated at a concentration of 100 to 5000 ng / mL. More specifically, the concentration may be 200 to 3000 ng / mL, and more specifically, 300 to 2000 ng / mL, but is not limited thereto.

[0038] In one specific embodiment, the concentration of the CD40 protein in step (b) may be 100 to 5000 ng / mL. More specifically, the concentration may be 200 to 3000 ng / mL, and more specifically, 300 to 2000 ng / mL, but is not limited thereto.

[0039] In one specific embodiment, the concentration of the anti-TCR Vα24-Jα18 antibody in step (b) may be 1 to 2000 ng / mL. More specifically, the concentration may be 10 to 1500 ng / mL, and even more specifically, 50 to 1000 ng / mL, but is not limited thereto.

[0040] In the present invention, the step of mass proliferation culture in step (b) may additionally add iNKT proliferative cytokines.

[0041] In the present invention, "iNKT proliferative cytokine" is a cytokine involved in the production, survival, proliferation, and homeostasis of iNKT cells, and can be understood to mean a cytokine that plays a role in inducing the proliferation of iNKT cells, particularly in the present invention. The iNKT proliferative cytokine may be, for example, at least one selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, and TGF-β, and specifically may be IL-2, IL-7, and / or IL-15, but is not limited thereto.

[0042] In one specific embodiment, the iNKT proliferative cytokine may be added at intervals of 1 to 4 days, specifically 2 to 3 days.

[0043] In one specific embodiment, the concentration of the iNKT proliferative cytokine may be 10 to 1000 IU / mL or 1 to 500 ng / mL. More specifically, the concentration may be, but is not limited to, 50 to 500 IU / mL or 10 to 200 ng / mL.

[0044] In one specific embodiment, step (b) may include culturing iNKT cells for 8 to 20 days. More specifically, the culturing period may be 10 to 18 days, and more specifically, 12 to 16 days, but is not limited thereto.

[0045] iNKT cells manufactured by mass proliferation culture method and their use

[0046] The present invention provides iNKT cells produced by a mass proliferation culture method of the above iNKT cells.

[0047] In the present invention, the iNKT cells may comprise a chimeric antigen receptor (CAR) construct.

[0048] In the present invention, a "chimeric antigen receptor (CAR)" encompasses an engineered receptor that implants artificial specificity onto specific immune effector cells. CARs confer monoclonal antibody specificity to T cells and can exhibit anticancer activity by targeting cancers expressing a specific antigen. Those skilled in the art can produce CAR-iNKT cells by introducing an appropriate CAR into iNKT cells produced using the mass proliferation culture method of the present invention, depending on the intended purpose.

[0049] The present invention provides a pharmaceutical use of iNKT cells produced by the mass proliferation culture method of the above iNKT cells.

[0050] One aspect of the present invention is a pharmaceutical composition for preventing or treating cancer or inflammatory disease, comprising the iNKT cells as an active ingredient.

[0051] Another aspect of the present invention is a method for treating or preventing cancer or an inflammatory disease, comprising administering a therapeutically effective amount of the iNKT cells to a subject in need thereof.

[0052] Another aspect of the present invention is the use of the iNKT cells for the manufacture of a medicament for use in the prevention or treatment of cancer or an inflammatory disease.

[0053] As for iNKT cells, it is as described above.

[0054] In the present invention, the cancer may include any cancer that can exhibit preventive or therapeutic efficacy through iNKT 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, colon cancer, chronic myeloid leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus 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, It may be at least one selected from the group consisting of malignant lymphoma, malignant mesothelioma, malignant melanoma, eye 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 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, blood cancer, and thymic cancer, and specifically, it may be ovarian cancer and pancreatic cancer, but is not limited thereto.

[0055] In the present invention, the inflammatory disease may include any inflammatory disease that can exhibit preventive or therapeutic efficacy mediated by iNKT cells. For example, it may be at least one selected from the group consisting of graft-versus-host disease, sepsis, septic shock, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, vasculitis, pleurisy, pericarditis, ischemia-related inflammation, inflammatory aneurysm, nephritis, hepatitis, chronic pulmonary inflammatory disease, rhinitis, gastritis, colitis, irritable bowel syndrome, fever or muscle pain due to infection, bronchitis, asthma, type 1 diabetes, inflammatory bowel disease, eosinophilic esophagitis, acute neutrophilic dermatitis, atopic dermatitis, seborrheic dermatitis, multiple sclerosis, and psoriasis, and specifically, it may be graft-versus-host disease, but is not limited thereto.

[0056] As used herein, the term "treatment" refers to intervention to alter the natural processes of an individual or cell with a disease, and this can be performed during the progression of a pathological condition or to prevent it. The desired therapeutic effect includes preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, alleviating or temporarily alleviating the disease state, and reversing or improving the prognosis. In particular, the present invention encompasses any action that improves the course of cancer or inflammatory disease by administering a composition containing iNKT cells as an active ingredient. In addition, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the iNKT cells. When the iNKT cells of the present invention are used for treatment or prevention purposes, they are administered to a subject in a therapeutically effective amount.

[0057] The term "therapeutically effective amount" as used herein refers to an effective amount of iNKT cells. Specifically, a "therapeutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the individual, age, sex, type of disease, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. It can be administered singly or in multiple doses. It is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art. The dosage of the pharmaceutical composition of the present invention can be determined by an expert according to 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 an effect of preventing, improving, or treating symptoms of cancer or inflammatory diseases.

[0058] The iNKT cells according to the present invention or a composition containing the same may be administered by oral administration, injection, intravenous administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, rectal administration, topical administration, intranasal administration, etc., but are not limited thereto.

[0059] The matters mentioned in the uses, compositions, and treatment methods of the present invention apply equally unless they are contradictory.

[0060] The mass proliferation culture method of the present invention can produce iNKT cells with excellent proliferation rate and purity without artificial antigen-presenting cells, and thus can be usefully utilized in related technical fields such as treatment of cancer or inflammatory diseases using iNKT cells.

[0061] Figure 1 is a schematic diagram of the in vitro mass proliferation culture process of iNKT cells.

[0062] Figure 2 shows the percentage of iNKT cells in peripheral blood mononuclear cells of three donors.

[0063] Figure 3 shows the percentage of iNKT cells in peripheral blood mononuclear cells from three donors after 12 days of expansion culture.

[0064] Figure 4 shows the iNKT % when iNKT cells in peripheral blood mononuclear cells of three donors were expanded and cultured for 12 days, then iNKT cells were isolated, stimulated with anti-CD28 antibody, CD40 protein, and anti-TCR Vα24-Jα18 antibody, and cultured for 7 days with IL-2, IL-7, and IL-15.

[0065] Figure 5 shows the iNKT % when iNKT cells in peripheral blood mononuclear cells of three donors were expanded and cultured for 12 days, then iNKT cells were isolated, stimulated with anti-CD28 antibody, CD40 protein, and anti-TCR Vα24-Jα18 antibody, and cultured for 14 days with IL-2, IL-7, and IL-15.

[0066] Figure 6 shows the purity and cell proliferation rate of iNKT cells from three donors during the mass proliferation culture process of the present invention.

[0067] Figure 7 shows the cancer cell killing effect of iNKT cells from three donors cultured in large-scale proliferation in OVCAR-3 and AsPC-1 cell lines.

[0068] Figure 8 shows the results of measuring cytokines secreted by iNKT cells cultured in large quantities in OVCAR-3 and AsPC-1 cell lines.

[0069] Hereinafter, the composition and effects of the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0070] The present invention relates to a method for mass-proliferation and culture of iNKT cells, comprising a step of treating iNKT cells with an anti-CD28 antibody, a CD40 protein, and an anti-TCR Vα24-Jα18 antibody to proliferate and culture them, and to a use of iNKT cells obtained thereby. The method for mass-proliferation and culture of the present invention can produce iNKT cells with excellent proliferation rate and purity without artificial antigen-presenting cells. As an exemplary embodiment, the entire process of the method for mass-proliferation and culture of iNKT cells of the present invention is illustrated in FIG. 1, and specific examples are as follows.

[0071] Example 1. Expansion and culture of iNKT cells from individual peripheral blood mononuclear cells.

[0072] Since the iNKT cell population in peripheral blood mononuclear cells is within 0.01 to 1% on average, an extended culture period was performed to obtain the maximum number of iNKT cells before isolation from PBMC, and the procedure was as follows.

[0073] Human peripheral blood mononuclear cells (PBMC, Cellular Technology Limited) stocks were thawed and added to RPMI (WELGENE) medium containing 10% FBS (Capricon) at a ratio of 1:9 and centrifuged at 300 g for 5 minutes. The cell pellet was lysed in 5% CTS. TMCTS containing Immune Cell SR (GIBCO), 0.1% gentamicin (GIBCO), 1 X MEM non-essential amino acid solution (WELGENE), 2 mM L-glutamine (WELGENE) TM OpTmizer TM T Cell Expansion SFM (GIBCO) was cultured with 200 nM α-galactosylceramide (αGalcer, Avanti) and 200 IU / mL IL-2 (PEPROTECH), with IL-2 added every 2 to 3 days of culture. Cells were harvested on day 0 (the day the stock was thawed) and after 12 days of culture and labeled 6B11. + The percentage of cells was measured using a flow cytometer and performed as follows.

[0074] The obtained cells were washed with FACS buffer (PBS containing 2% FBS), and then incubated with CD3-FITC antibody (Biolegend), 6B11-PE antibody (BD bioscience), CD4-APC antibody (Biolegend), CD8-percpCy5.5 antibody (Biolegend), CD40L-PE-Cy7 antibody (Biolegend), and Dead&Live-Pacific orange (ThermoFisher) dye for 30 minutes at 4°C. After washing with FACS buffer, the cells were suspended in 400 μL FACS buffer, and the fluorescence expressed on the cell surface was measured using a flow cytometer (Novocyte, Agilemt). 6B11 + The % of cells was measured and graphed in Fig. 2 for day 0 and in Fig. 3 for day 12 of culture. The results in Fig. 2 and Fig. 3 represent the results before and after expansion culture of iNKT cells in peripheral blood mononuclear cells of three donors (donors A, B, and C).

[0075] As a result, 6B11 was 0.30, 0.18, and 0.06% in natural peripheral blood mononuclear cells before expansion culture. +It was confirmed that the population (Fig. 2) increased to 28.19, 16.78, and 4.19%, respectively, after 12 days of expansion culture (Fig. 3).

[0076] From the above results, it can be seen that the added glycolipid antigen (e.g., αGalcer) during the culture of peripheral blood mononuclear cells specifically activates iNKT cells, which are present in small amounts of 0.06 to 0.30% in natural peripheral blood mononuclear cells, and that the proliferation of iNKT cells is induced by iNKT activating cytokines (e.g., IL-2).

[0077] Example 2. Isolation of expanded iNKT cells

[0078] The iNKT cells expanded in Example 1 were isolated by positive selection using TCR Vα24-Jα18 micro beads (Miltenyi Biotec). After obtaining the results in Example 1, the cell number was measured and 10 8 The cell count was adjusted with cells / 400 μl buffer (PBS containing EDTA and BSA). TCR Vα24-Jα18 microbeads were added in proportion to the cell number and reacted at 4°C for 15 minutes. After the reaction, the cells were washed with 1 to 2 mL of buffer, suspended in 1 mL of buffer, and filtered through a mesh. The cells were loaded onto an LS column (Miltenyi Biotec) mounted on a magnet soaked with 3 mL of buffer, washed with 3 mL of buffer, and the LS column was separated from the magnet. After loading 3 mL of medium, the cells bound to the microbeads were collected by pushing with a plunger, and the cell number was measured. iNKT cell isolation was performed from peripheral blood mononuclear cells of three donors (donors A, B, and C).

[0079] Example 3. Mass proliferation culture after iNKT cell isolation

[0080] Purity 6B11 with micro beads +After obtaining iNKT cells with a purity of more than 90%, the cells were cultured in a large scale while maintaining purity by stimulating the iNKT cells with costimulatory factors and adding cytokines necessary for proliferation, as follows.

[0081] The iNKT cells obtained in Example 2 were cultured in wells coated with 1 μg / mL anti-CD28 antibody (Thermofisher), 1 μg / mL CD40 protein (Sigma), and 1 μg / mL anti-TCR Vα24-Jα18 antibody (Miltenyi Biotec) at 37°C for 4 hours, and 200 IU / mL IL-2, 50 ng / mL IL-7, and 100 ng / mL IL-15 (peprotech) were added, and IL-2, IL-7, and IL-15 were added every 2 to 3 days of culture. 6B11 after 7 and 14 days of proliferation culture + The percentage of cells was measured using a flow cytometer and the results are shown in Figures 4 and 5. The results in Figures 4 and 5 show the results after 7 days (total culture 19 days) and 14 days (total culture 26 days) of proliferation culture after expansion of peripheral blood mononuclear cells from three donors (donors A, B, and C) for 12 days and isolation of iNKT cells.

[0082] As a result, after isolating iNKT cells on the 12th day of expansion culture, the percentages were 99.74, 99.20, and 86.23% on the 7th day (Fig. 4), and 98.79, 99.48, and 92.23% on the 14th day (Fig. 5), respectively, 6B11 + It was confirmed that the cells maintained more than 90% viability after 26 days of total culture (12 days of expansion culture and 14 days of proliferation culture after iNKT cell isolation).

[0083] The overall iNKT cell % change and cell proliferation rate during the iNKT mass proliferation culture process of the present invention are shown in Figure 6.

[0084] iNKT cells initially started at 0.06 to 0.30% in peripheral blood mononuclear cells and showed a purity of more than 90% on day 26 when culture ended (left side of Fig. 6), and the cell proliferation rate was approximately 38,000, 16,000, and 29,000 times on day 26 of culture (right side of Fig. 6).

[0085] In conclusion, the present invention confirmed that the method is suitable for mass culturing iNKT cells that maintain the purity of iNKT cells over a long culture period while exhibiting a high proliferation rate by stimulating CD28, CD40L, and TCR Vα24-Jα18 of iNKT cells to induce activity by culturing them while treating them with anti-CD28 antibody, CD40 protein, and anti-TCR Vα24-Jα18 antibody after isolating iNKT cells.

[0086] Example 4. Measurement of cancer cell death by iNKT cells

[0087] The cancer cell killing ability of iNKT cells cultured in Example 3 was confirmed by the following method. As target cells, two cells, OVCAR-3 and AsPC-1, were used as examples, and the killing ability of the two cells was confirmed by the following method.

[0088] iNKT cells were treated with target cells (OVCAR-3) stained with Calcein-AM at effector (E):target (T) ratios of 20:1, 10:1, 5:1, and 2.5:1, and simultaneously treated with culture medium in the spontaneous wells and 2% Triton X-100 (Sigma) in the maximum wells. Meanwhile, target cells were cultured at 1 x 10 6 Cells were obtained at 1 mL / mL and treated with 5 μM Calcein-AM (BD Bioscience), then stained at 37°C for 1 hour. After staining, 5 × 10 were washed twice and counted. 5Target cells obtained at a density of 5 cells / mL were treated in 50 μl of V-bottom 96-well plates. After centrifugation at 1,500 rpm for 15 minutes, the cells were cultured in a CO2 incubator for 4 hours. After 4 hours, the plates were centrifuged at 1,500 rpm for 5 minutes. Next, 70 μl of the supernatant was transferred to a black 96-well plate and measured using a spectrofluorometer (480 / 530) (Biotek). The % lysis of cancer cells by iNKT cells from three donors (donors A, B, and C) was calculated and is shown on the left side of Figure 7.

[0089] The following calculation method was used.

[0090] (Sample-Natural Occurrence) / (Maximum-Natural Occurrence) X 100

[0091] As a result of confirming the OVCAR-3 killing ability of iNKT cells, in the group treated with iNKT cells 20 times the cancer cell volume, donors A, B, and C showed 18%, 4%, and 9% cancer cell killing ability, respectively. This was confirmed to decrease to 11%, 8%, and 4% in donor A, 3%, 2%, and 1% in donor B, and 6%, 4%, and 2% in donor C as the ratio with cancer cells decreased to 10:1, 5:1, and 2.5:1, respectively (Fig. 7, left). Therefore, it was confirmed that the iNKT cells expanded and proliferated through the present invention have the killing ability against OVCAR-3.

[0092] Next, the apoptotic capacity of iNKT cells was tested against AsPC-1 MSLN-GFP+ It was confirmed using the following method using cells.

[0093] AsPC-1 MSLN-GFP+After 24 hours of seeding in a 96-well plate, iNKT cells from three donors (donors A, B, and C) were treated with the same number of E:T ratios of 20:1, 10:1, and 5:1, and simultaneously, the spontaneous wells were treated with culture medium, and the maximum wells were treated with 2% Triton X-100 (Sigma). After 24 hours of iNKT cell treatment, the area of ​​cells expressing GFP was measured under a fluorescence microscope (Biotek), and the % cancer cell lysis was calculated as (sample-max) / (spontaneous-max) X 100, and is shown on the right side of Fig. 7.

[0094] As a result, 24 hours after iNKT cell treatment, the apoptosis of AsPC-1 decreased to about 46%, 37%, and 21% for donor A, 29%, 16%, and 10% for donor B, and 39%, 20%, and 11% for donor C, as the ratio with cancer cells decreased to 20:1, 10:1, and 5:1, respectively (Fig. 7, right). Therefore, it was confirmed that the iNKT cells expanded and cultured through the present invention have apoptotic ability against AsPC-1.

[0095] Example 5. Measurement of iNKT cell-secreted cytokines

[0096] To measure cytokines secreted by iNKT cells cultured in mass production in Example 3, target cancer cells (OVCAR-3, AsPC-1) and iNKT cells were co-cultured at a ratio of 1:3 for 24 hours, respectively. Thereafter, the supernatant was obtained and cytokines were measured using an ELISA kit (Biolegend), and the method was as follows.

[0097] To the obtained culture medium or 25 μl of standard, 25 μl of assay buffer and 25 μl of beads were added, and the mixture was 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, the mixture was resuspended in washing buffer, and the values ​​measured using a flow cytometer were entered into Legendplex data analysis software (Biolegend) and analyzed. The results of three donors (donors A, B, and C) were combined and presented in a graph in Figure 8.

[0098] As a result, it was confirmed that iNKT cells co-cultured with OVCAR-3 or AsPC-1 secrete various cytokines and cytolytic molecules in response to cancer cells. In particular, it was confirmed that cytolytic molecules such as Granzyme A, Granzyme B, Perforin, and Granulysin were highly increased. Therefore, it was found that iNKT cells expanded and cultured through Examples 1 to 3 of the present invention have the ability to kill cancer cells by secreting cytolytic molecules capable of killing cancer cells.

[0099] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. (a) a step of expanding and culturing iNKT cells from peripheral blood mononuclear cells; and (b) A method for mass proliferation of iNKT cells, comprising a step of treating the cells of step (a) with an anti-CD28 antibody, a CD40 protein, and an anti-TCR Vα24-Jα18 antibody and culturing them for proliferation.

2. In paragraph 1, The step (a) above is a mass proliferation culture method in which peripheral blood mononuclear cells are cultured in a medium containing a glycolipid antigen or an iNKT activating cytokine.

3. In paragraph 2, A mass proliferation culture method, wherein the above-mentioned glycolipid antigen is at least one selected from the group consisting of α-galactosylceramide (αGalcer), α-galacturonosylceramide (αGalACer), α-monogalactosyldiacylglycerol (αMGalD), isoglobinoside 3 (iGb3), OCH, and C20:2-αGalcer.

4. In paragraph 2, A mass proliferation culture method, wherein the above iNKT activating cytokine is at least one selected from the group consisting of IL-2, IL-4, IL-7, IL-15, IL-21, and GM-CSF.

5. In paragraph 3, A mass proliferation culture method wherein the concentration of the above-mentioned glycolipid antigen is 1 to 1000 nM.

6. In paragraph 4, A mass proliferation culture method wherein the above iNKT activating cytokine is added at intervals of 1 to 4 days.

7. In paragraph 4, A mass proliferation culture method wherein the concentration of the above iNKT activating cytokine is 1 to 1000 IU / mL.

8. In paragraph 1, A mass proliferation culture method, wherein step (a) further comprises a step of isolating iNKT cells from expanded cultured cells.

9. In paragraph 8, The above iNKT cell separation is a mass proliferation culture method targeting at least one antigen selected from the group consisting of TCR Vα24-Jα18, CD3, CD4, CD8, CD25, CD28, CD40L, CD45RA, CD45RO, CD56, CD62L, CD69, CD161, HLA-DR and Vβ11.

10. In paragraph 1, A mass proliferation culture method, wherein step (a) comprises a step of culturing peripheral blood mononuclear cells for 6 to 18 days.

11. In paragraph 1, A mass proliferation culture method, wherein the step of treating with an anti-CD28 antibody, a CD40 protein, and an anti-TCR Vα24-Jα18 antibody in the step (b) above comprises a step of treating cells directly with the anti-CD28 antibody, the CD40 protein, and the anti-TCR Vα24-Jα18 antibody, or treating cells by coating the anti-CD28 antibody, the CD40 protein, and the anti-TCR Vα24-Jα18 antibody on a culture vessel or beads.

12. In paragraph 1, A mass proliferation culture method, wherein in the step (b) above, the anti-CD28 antibody is treated at a concentration of 100 to 5000 ng / mL.

13. In paragraph 1, A mass proliferation culture method, wherein in the step (b) above, CD40 protein is treated at a concentration of 100 to 5000 ng / mL.

14. In paragraph 1, A mass proliferation culture method, wherein in the step (b) above, the anti-TCR Vα24-Jα18 antibody is treated at a concentration of 1 to 2000 ng / mL.

15. In paragraph 1, A mass proliferation culture method in which the step of mass proliferation culture in the above step (b) is performed by adding iNKT proliferative cytokines.

16. In paragraph 15, A mass proliferation culture method, wherein the iNKT proliferative cytokine is at least one selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, and TGF-β.

17. In paragraph 15, A mass proliferation culture method in which the above iNKT proliferative cytokine is added at intervals of 1 to 4 days.

18. In paragraph 15, A mass proliferation culture method wherein the concentration of the above iNKT proliferative cytokine is 10 to 1000 IU / mL or 1 to 500 ng / mL.

19. In paragraph 1, A mass proliferation culture method, wherein the step (b) comprises a step of culturing for 8 to 20 days.

20. An iNKT cell produced by a mass proliferation culture method of any one of claims 1 to 19.

21. In paragraph 20, The above iNKT cells are iNKT cells comprising a chimeric antigen receptor (CAR) construct.

22. A pharmaceutical composition for preventing or treating cancer or inflammatory disease, comprising the iNKT cells of Article 20 as an active ingredient.

23. In paragraph 22, the cancer is 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, colon cancer, chronic myeloid leukemia, retinoblastoma, choroidal melanoma, ampulla of Vater cancer, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus 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 A pharmaceutical composition comprising at least one selected from the group consisting of soft tissue cancer, malignant bone cancer, malignant lymphoma, malignant mesothelioma, malignant melanoma, eye 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 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, blood cancer, and thymic cancer.

24. A pharmaceutical composition according to claim 22, wherein the inflammatory disease is at least one selected from the group consisting of graft-versus-host disease, sepsis, septic shock, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, vasculitis, pleurisy, pericarditis, ischemia-related inflammation, inflammatory aneurysm, nephritis, hepatitis, chronic pulmonary inflammatory disease, rhinitis, gastritis, colitis, irritable bowel syndrome, fever or muscle pain due to infection, bronchitis, asthma, type 1 diabetes, inflammatory bowel disease, eosinophilic esophagitis, acute neutrophilic dermatitis, atopic dermatitis, seborrheic dermatitis, multiple sclerosis, and psoriasis.

25. A pharmaceutical composition comprising iNKT cells of claim 20 and a pharmaceutically acceptable additive.

26. Use of iNKT cells of claim 20 for use in the manufacture of a medicament for use in the treatment or prevention of cancer or inflammatory diseases.

27. A method for treating or preventing cancer or an inflammatory disease, comprising administering a therapeutically effective amount of iNKT cells of claim 20 to a subject in need thereof.

Images